Article

The chemical and energetic properties of muscles poisoned with fluorodinitrobenzene

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Abstract

1. The heat production and mechanical responses of frogs' sartorii have been recorded at 0 degrees C after immersion in normal Ringer solution, and also after poisoning with 1-fluoro-2, 4-dinitrobenzene (FDNB) and nitrogen. The muscles were later analysed chemically for changes in ATP, phosphocreatine (PC), inorganic phosphate (P(i)), lactate, total adenine nucleotide and total inosine nucleotide.2. Analysis of paired resting muscles established that the resting levels of these substances found in our experiments were similar to those reported elsewhere.3. Resting muscles that had been poisoned with FDNB and N(2) contained significantly more PC and less ATP than unpoisoned controls. Moreover, some of their adenine had been deaminated to inosine.4. In a normal muscle in oxygen, the PC that breaks down as a result of a 30 sec tetanus is restored with a roughly exponential time course whose half-time is about 10 min. Thus at least 40 min rest must be allowed between the different stages of an experiment.5. Isometric twitches of the poisoned muscle rapidly decline in size, but small twitches continue to be produced for a very long time. If stimulation is discontinued, substantial recovery takes place. The total tension development is equivalent to at least thirty normal twitches, and correspondingly, the total heat production is greater than could be accounted for even by complete break-down of the ATP in the muscle. In fact, the ATP break-down, though highly significant, is not nearly complete.6. In short series of isometric twitches there is significant break-down of ATP and, less consistently, of PC; also a significant increase in P(i) but no additional deamination of adenine. The rate of development of isometric tension is slightly decreased.7. The results described under (6) are definitely due to the presence of FDNB. In similar experiments with the muscles in N(2) but not otherwise poisoned the only significant change is a break-down of PC.8. In a long series of isotonic twitches the chemical effects are similar to those described under (5) and inosine formation is clearly demonstrable. The poisoned muscles show force-velocity curves of normal shape, the Fenn effect and the presence of shortening heat. Thus their contractions are normal or nearly so.9. The complexity of the chemical reactions that continue in the poisoned muscle makes it impossible to draw up an accurate energetic balance sheet.10. A new hypothesis is suggested to explain the energetic importance of inosine formation.

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... A portion of this investigation involved the use of the metabolic inhibitors 1- fluoro-2,4-dinitrobenzene (FDNB) and iodoacetic acid (IAA) to resolve the events responsible for the observed pHi shifts. The former compound blocks the Lohman reaction at the level of creatine phosphotransferase (Cain and Davies, 1962; Dydynska and Wilkie, 1966), while the latter compound blocks glycolysis at the level of glyceraldehyde phosphate dehydrogenase (cf. J6bsis, 1964). ...
... A set of 40 stimuli was again delivered (seeFig. 4D), and there was a marked sequential d~crease in twitch amplitude which is characteristic of FDNB-poisoned muscles (Dydynska and Wilkie, 1966). However, the slow alkalinization observed in the normal case is still present, as is the slower reacidification.Fig. ...
Article
The spectral characteristics of the pH-sensitive dyes neutral red (NR) and bromcresol purple (BCP) were utilized for studies of the changing intracellular pH (pHi) of sartorius muscles from Rana pipiens, both during the course of an isometric twitch and during recovery metabolism subsequent to a train of twitches. The information from the two dissimilar dyes correlated to confirm the methodology. Neither the fast realkalinization observed during a twitch nor the slow alkalizing phase of recovery metabolism was affected in an obvious manner when phosphocreatine (PC) hydrolysis was blocked by 1-fluoro-2,4-dinitrobenzene (FDNB). Iodoacetic acid (IAA) did inhibit the slow acidic phase of recovery metabolism. The conclusion is made that alkalizing reactions other than PC breakdown must be considered as operative at these levels of activity. Hypertonic solutions altered twitch tension and time course without altering the pHi shifts observed until approximately 75% of the twitch amplitude was abolished. Multiple effects of hypertonic solutions as the muscle approach tonic equilibrium are proposed.
... This is roughly equal to the difference in (external+ internal) work production in the two cases. Moreover, other experiments have shown that muscles poisoned with IAA, like those poisoned with fluorodinitrobenzene (FDNB) (Dydynska & Wilkie, 1966) do show a normal Fenn effect. 4. Stretches applied to the active muscle: negative work In past work on the heat production of muscles subjected to stretches, two rather different techniques have been employed. ...
... Then heat produced before FDNB _AH for PCheat produced after FDNB AH for ATP' However, after poisoning the contractions are often not so vigorous as they were before and a direct comparison of this kind has not yet been made. The fact that the ratio of heat to tension is not appreciably altered by FDNB (Dydynska & Wilkie, 1966, p. 763) makes it likely that similar contractions would have similar heat production and thus that AH (ATP) AH (PC) = -11*0 kcal/mole. No doubt experiments aimed specifically at this point, rather than the incidental observation alluded to, could produce more accurate information . ...
Article
1. A new instrument, the integrating thermopile, is described for measuring the total quantity of heat produced during muscular contraction.2. This instrument has been used to investigate the relation between change of enthalpy (- (heat produced + work produced)) and break-down of phosphorylcreatine (DeltaPC) in iodoacetate-poisoned frog sartorii at 0 degrees C. In a variety of different types of contraction-series of isometric twitches, isometric tetani, contractions with positive and with negative work-the relation between enthalpy change and DeltaPC was always the same, and corresponded to an in vivo molar enthalpy change (DeltaH) of -11.0 +/- 0.23 (S.E.; n = 52) kcal/mole.3. This value of DeltaH is used to estimate the in vivo DeltaH for ATP splitting and also the number of rephosphorylations to be expected per hexose unit oxidized by normal unpoisoned muscle.
... [ADP]/[ATP] 6 x 10-3. We had no direct measurements of the resting [Pi] for the experiments of series 2 so we have taken the value 2'75 mmol/ kg from Dydyn'ska & Wilkie (1966). From Table 1 and the mean value of Ct in series 2 (22.91 utmol/(g muscle)) we may estimate 6 from Table 2 to be 1-06 gmol/g (experimental estimate) or P16 ('best' estimate). ...
Article
1. The production of heat (h) and work (w) and the changes in phosphocreatine (PCr) and ATP have been measured on tetanized isolated frog muscles (unpoisoned and in oxygen at 0 degrees C) during shortening at constant velocity and during isometric contraction (both without relaxation). The former type of contraction was designed to maximize the fraction w/(h + w); the latter to minimize it.2. The duration of the isometric contraction was made considerably longer than that of the isovelocity contraction so that the (h + w) productions during the two contractions were approximately equal.3. The PCr break-down during the working contraction was considerably greater than that during the isometric contraction.4. No detectable ATP changes occurred.5. The break-down of PCr is sufficient to account for the work evolved: there is no reason to suppose that the work comes from an unidentified source.6. In both types of contraction extra energy is evolved that cannot be accounted for by concurrent splitting of PCr. The time course of evolution of this extra energy is similar in all types of contraction, suggesting that it may arise from a process other than cross-bridge interaction.7. The results are discussed in terms of current cross-bridge theory and muscle kinetics. The mean cycle times of a cross-bridge during working and isometric contractions are 0.12 sec and 0.34 sec respectively. During the working contraction cross-bridges spend about one quarter of the time attached to actin filaments.
... The magnitude of the change in phosphofructokinase activity will depend on the magnitude of the change in concentration of the regulator, AMP, and because of the reversibility of the adenylate kinase reaction the latter change is limited (see Newsholme & Start, 1973a). The conversion of AMP into NH4+ and IMP is irreversible (see Dydynska & Wilkie, 1966;Sugden, 1973), so that the change in NH4+ concentration is not necessarily proportional to that of AMP. Indeed the increases in NH4+ concentration that have been reported in muscle and brain (see above) are greater than the increases that have been reported in the concentrations of AMP. ...
Article
1. The effect of NH4+, Pi and K+ on phosphofructokinase from muscle and nervous tissues of a large number of animals was investigated. The activation of the enzyme from lobster abdominal muscle by NH4+ was increased synergistically by the presence of Pi or SO4(2-). In the absence of K+, NH4+ plus Pi markedly activated phosphofructokinase from all tissues studied. In the presence of 100 mM-K+, NH4+ plus Pi activated phosphofructokinase from nervous tissue and muscle of invertebrates and the enzyme from brain of vertebrates, but there was no effect of NH4+ plus Pi on the enzyme from the muscles of vertebrates. Nonetheless, NH4+ plus Pi increased the activity of vertebrate muscle phosphofructokinase in the presence of 50 mM-K+ at inhibitory concentrations of ATP, i.e. these ions de-inhibited the enzyme. In the absence of NH4+ plus Pi, K+ activated phosphofructokinase from vertebrate tissues at non-inhibitory ATP concentrations, but the effect was less marked with the enzyme from invertebrate tissues. Indeed, high concentrations of K+ (greater than 50 mM) caused inhibition of invertebrate tissue phosphofructokinase. Of the other alkali-metal ions tested, only Rb+ activated phosphofructokinase from lobster abdominal muscle and rat heart muscle. 2. The properties of lobster abdominal-muscle phosphofructokinase were studied in detail. This muscle was chosen as representative of invertebrate muscle because large quantities of tissue could be obtained from one animal and the enzyme was considerably more stable in tissue extracts than in extracts of insect flight muscle. In general, the properties of the enzyme from this tissue were similar to those of the enzyme from many other tissues: ATP concentrations above an optimum value inhibited the enzyme and this inhibition was decreased by raising the fructose 6-phosphate or the AMP concentration. In particular, NH4+ plus Pi activated the enzyme at noninhibitory concentrations of ATP and they also relieved ATP inhibition (see above). 3. It is suggested that increases in the concentration of NH4+ and Pi, under conditions of increased ATP utilization in certain muscles and/or nervous tissue, may play a part in the stimulation of glycolysis through the effects on phosphofructokinase (the effect may be a direct activation and/or a relief of ATP inhibition). Changes in the concentration of NH4+ and Pi are consistent with this theory in nervous tissue and the anaerobic type of muscles. The role of AMP deaminase in production of NH4+ from AMP in these tissues is discussed in relation to the control of glycolysis.
... A preliminary experiment was done to determine whether glycolytic reactions would be an important factor in the energy balance. In addition to the usual measurements of phosphocreatine, ATP and inorganic phosphate, the levels of ADP, AMP, and IMP were measured since it has been shown that these change in DNFB-treated muscles (Dydynska & Wilkie, 1966; Kushmerick & Davies, 1969). ...
Article
1. Heat production and chemical changes were measured in untreated and dinitrofluorobenzene (DNFB)-treated muscles during isometric tetani. Levels of total creatine (Ct), free creatine, ATP, ADP, AMP, inorganic phosphate, glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, pyruvate, phosphoenolpyruvate, and lactate were measured. Changes in inosinic acid (IMP) were also measured. 2. DNFB effectively inhibited the creatine kinase reaction (Lohmann reaction). 3. Our major finding is that even after effective treatment with DNFB the observed heat plus work after 2 sec and 5 sec of stimulation is significantly greater than the enthalpy change produced by the measured chemical changes. This confirms that an unidentified exothermic process occurs during muscle contraction; this conclusion was reached previously from studies of untreated muscle. 4. The unexplained heat plus work is unlikely to be derived from glycolytic reactions since under anaerobic conditions no formation of lactate, pyruvate, phosphoenolpyruvate or fructose-1,6-diphosphate could be detected in either untreated or DNFB-treated muscles even 34 sec after a series of three 5 sec isometric tetani. 5. In the first 2 sec of stimulation the unexplained heat plus work is less in DNFB-treated muscles than in untreated muscles. However from 2 to 5 sec of stimulation the unexplained heat plus work is the same in treated and untreated muscles.
... D. CARLSON et al., 1963;F. D. CARLSON & SIGER, 1959DYDYNSKA & WILKIE, 1966;HOMSHER et al., 1972 and many others). These authors based their analyses of PCr on A.H. ENNOR (1957) and some added that "internal standard runs showed no interfering substances present for any of the analyses nor did the perchloric acid extraction procedure result in the loss of any PCr, TCr or ATP", but none of them explained how this accurate number of "10.8%" had been obtained, although they gave extended analyses on the best methods to obtain the "size" of muscles, or the calibrations of tension or heat measurements. ...
Book
Full-text available
In the early nineteen-sixties, a decisive breakthrough occurred in our understanding of muscle energetics. The author is in the fortunate position of being able to present these findings from the perspective of his own experimental experience, because he himself has worked out a large part of the necessary methodological effort to allow the exact confrontation of chemical changes with mechanical and thermal events, obtained reliably with the help of statistical analysis and planning of experiments. Since then, they have been confirmed, deepened and extended up to now by many other researchers His early report written in 1964 is updated by many notes and references up to 2018; many hyperlinks allow easy and quick navigation throughout the text of the book.
... The valve behavior may be attributed to regulation by calcium, which in this interpretation controls the rate of a process "in series" with the mechanochemically coupled set of reactions. The constant-velocity pump is less readily identified, although Dydyn'ska and Wilkie have claimed that fluorodinitrobenzene-poisoned muscles do not merely draw on their store of ATP to meet their energetic requirements, but continue to support complicated chemical changes, including some which can regenerate ATP (47). A more satisfactory analog is obtained by dispensing with the constant-velocity pump and introducing the additional high-level "primary" reservoir. ...
Article
All discussions of muscle energetics concern themselves with the Hill force-velocity relation, which is also the general output relation of a class of self-regulated energy converters and as such contains only a single adjustable parameter —the degree of coupling. It is therefore important to see whether in principle muscle can be included in this class. One requirement is that the muscle should possess a working element characterized by a dissipation function of two terms: mechanical output and chemical input. This has been established by considering the initial steady phase of isotonic and isometric tetanic contraction to represent a stationary state of the fibrils (a considerable body of evidence supports this). Further requirements, which can be justified for the working element, are linearity and incomplete coupling. Thus the chemical input of the muscle may be expected to follow the inverse Hill equation (see Part I). The relatively large changes in activities of reactants which the equation demands could only be controlled by local operation of the regulator, and a scheme is outlined to show how such control may be achieved. Objections to this view recently raised by Wilkie and Woledge rest on at least two important assumptions, the validity of which is questioned: (a) that heat production by processes other than the immediate driving reaction is negligible, which disregards the regulatory mechanism (possibly this involves the calcium pump), and (b) that the affinity of the immediate driving reaction is determined by over-all concentrations. The division of heat production into “shortening heat” and “maintenance heat” or “activation heat” is found to be arbitrary.
Article
1. Measurements have been made of heat production and changes in levels of phosphorylcreatine (PC), ATP and lactic acid resulting from contraction of tortoise muscle under anaerobic conditions.2. The only significant chemical change found was a break-down of PC.3. The amount of heat produced per mole of PC split (-DeltaH) was 13.18 +/- 1.04 kcal/mole (mean and S.E. from thirty-five observations).4. (-DeltaH) is probably rather greater in tortoise than in frog muscle. The value of (-DeltaH) cannot easily be accounted for by the known processes accompanying PC splitting in either type of muscle.
Measurements have been made of the changes in adenosine triphosphate and inorganic phosphate associated with activation and shortening in isolated sartorius muscles of Rana pipiens at 0 degrees C. The muscles were pretreated with 2,4-dinitrofluorobenzene to inhibit adenosine triphosphate: creatine phosphotransferase. When unloaded muscles were stimulated electrically, allowed to shorten fully and stay shortened during further stimulation, it was found that the usage of adenosine triphosphate (and production of inorganic phosphate) per pulse became less and less at greater rates of stimulation. The adenosine triphosphate usage per second reached a plateau and continued for a short while after the last pulse. This adenosine triphosphate is presumably used almost entirely for pumping calcium. Very little adenosine triphosphate was used per pulse by muscles in hypertonic solutions. These muscles passed normal action potentials but did not shorten or develop tension. Muscles which were re-extended after each of a series of almost completely unloaded shortenings had been completed used only the small amount of extra adenosine triphosphate needed for the little extra work done. Even the total breakdown of adenosine triphosphate was much too small to account for the heat of shortening, if this heat is assumed to be degraded free energy from adenosine triphosphate. Experiments were carried out according to Professor A. V. Hill's 'Further challenge to biochemists'. They showed that less adenosine triphosphate was used in the first 200 ms by a lightly loaded isotonic muscle than by an isometric muscle. Thus the heat of shortening, which is observed only while the muscle is shortening but not in the whole contraction-relaxation cycle, cannot be degraded free energy from adenosine triphosphate.
Article
Strophanthidin-sensitive and insensitive unidirectional fluxes of Na were measured in fog sartorius muscles whose internal Na levels were elevated by overnight storage in the cold. ATP levels were lowered, and ADP levels raised, by metabolic poisoning with either 2,4-dinitrofluorobenzene or iodoacetamide. Strophanthidin-sensitive Na efflux and influx both increased after poisoning, while strophanthidin-insensitives fluxes did not. The increase in efflux did not require the presence of external K but was greatly attenuated when Li replaced Na as the major external cation. Membrane potential was not markedly altered by 2,4-dinitrofluorobenzene. These observations indicate that the sodium pump of frog skeletal muscle resembles that of squid giant axon and human erythrocyte in its ability to catalyze Na-Na exchange to an extent determined by intracellular ATP/ADP levels.
Article
Methods are described whereby initial processes of muscular contraction may be investigated in a mammalian preparation, the soleus muscle of the rat. Conditions are chosen so that recovery is avoided. An isometric tetanus is investigated and an energy balance sheet is drawn up. It is found that there is more heat evolved than can be accounted for in terms of measured chemical reaction. This discrepancy is discussed with reference to the similar results that have been obtained using frog muscle.
Article
In this chapter fundamental energetic properties of skeletal muscles as elucidated from isolated muscle preparations are described. Implications of these intrinsic properties for the energetic characterization of different fiber types and for the understanding of locomotion have been considered. Emphasis was placed on the myriad of physical and chemical techniques that can be employed to understand muscle energetics and on the interrelationship of results from different techniques. The anaerobic initial processes which liberate energy during contraction and relaxation are discussed in detail. The high-energy phosphate (approximately P) utilized during contraction and relaxation can be distributed between actomyosin ATPase or cross-bridge cycling (70%) and the Ca2+ ATPase of the sacroplasmic reticulum (30%). Muscle shortening increases the rate of approximately P hydrolysis, and stretching a muscle during contraction suppresses the rate of approximately P hydrolysis. The economy of an isometric contraction is defined as the ratio of isometric mechanical response to energetic cost and is shown to be a fundamental intrinsic parameter describing muscle energetics. Economy of contraction varies across the animal kingdom by over three orders of magnitude and is different in different mammalian fiber types. In mammalian skeletal muscles differences in economy of contraction can be attributed mainly to differences in the specific actomyosin and Ca2+ ATPase of muscles. Furthermore, there is an inverse relationship between economy of contraction and maximum velocity of muscle shortening (Vmax) and maximum power output. This is a fundamental relationship. Muscles cannot be economical at developing and maintaining force and also exhibit rapid shortening. Interestingly, there appears to be a subtle system of unknown nature that modulates the Vmax and economy of contraction. Efficiency of a work-producing contraction is defined and contrasted to the economy of contraction. Unlike economy, maximum efficiency of work production varies little across the animal kingdom. There are difficulties associated with the measurement of maximum efficiency of contraction, and it has yet to be determined unequivocally if the maximum efficiency of contraction varies in different fiber types. The intrinsic properties of force per cross-sectional area, economy, and Vmax determine the basic energetic properties of skeletal muscles. Nonetheless, the mechanics and energetics of skeletal muscles in the body are profoundly influenced by muscle architecture, attachment of muscles to the skeleton, and motor unit organization.(ABSTRACT TRUNCATED AT 400 WORDS)
Article
A previously developed animal model of exercise-induced muscle contractures, which utilized intra-aortic injection of iodoacetate (IOA) to inhibit the second stage glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, showed histological evidence of selective type II muscle fiber involvement with sparing of the type I muscle fibers. A new model has been developed using dinitrofluorobenzene (DNFB) as a selective inhibitor of creatine phosphokinase in a similar, but slightly modified distal aortic injection protocol. Two hours after the injection of a dinitrofluorobenzene solution of 2.22 mg/kg body weight, spontaneous electrically-silent contracture developed in the injected lower extremity, involving principally the soleus muscle. Histologically, selective damage was apparent in the type I muscle fibers, with sparing of the type II muscle fibers. The contrast in findings associated with iodoacetate inhibition of glycolysis or with DNFB inhibition of the phosphocreatine shuttle suggests that type I and type II fibers have markedly different usable pools of readily available ATP: type II fibers must rely on the minute-by-minute replenishment of the usable pool of ATP from glycolysis, while type I fibers must regenerate the usable pool of ATP from phosphocreatine through a creatine phosphokinase-mediated process.
Article
1. Contraction and recovery of bull-frog skeletal muscles were studied using 31P nuclear magnetic resonance (NMR), with a time resolution of 16 s, at 4 degrees C. The muscles were stimulated tetanically for various periods (0.2-10 s) at a sarcomere length of 2.4 microns. Changes in the concentrations of inorganic phosphate (Pi), phosphocreatine (PCr) and other metabolites were studied for repeated cycles of contraction and recovery. 2. In resting muscles, bathed in a solution gassed with 95% O2 and 5% CO2, the concentration of Pi was 1.15 +/- 0.21 mmol kg-1 wet weight (mean +/- one S.D., n = 12), that of ATP was 3.32 +/- 0.15 mmol kg-1 (mean +/- one S.D., n = 12) and that of sugar phosphates was less than 0.5 mmol kg-1. The intracellular pH (pHi) was 7.22 +/- 0.01 (mean +/- one S.D., n = 12). These results are averages for fibres which probably have different values. 3. On stimulating the muscles pHi shifted in the alkaline direction and subsequently recovered. The extent of the alkaline shift was linearly related to the contraction duration (0.2-10 s) with a rate of 0.01 pH unit s-1. 4. The increase in Pi with stimulus duration was biphasic, consisting of an early burst, 0.38 +/- 0.10 mmol kg-1 (+/- 1 S.D., n = 5), complete within about 0.2-0.5 s, followed by a slower steady-state increase. The steady-state rate of Pi increase was 0.33 +/- 0.02 mmol kg-1 s-1 (+/- 1 S.D., n = 5) in agreement with the results of previous studies involving chemical analyses. 5. The time course of Pi recovery was well described by a single exponential. Intensities of ATP, sugar phosphates and the peaks in the phosphodiester region did not change during a 2 s tetanus. 6. The amount of PCr hydrolysed (-delta PCr), associated with contractions of various durations, coincided well with Pi increase (delta Pi). This was found during the whole recovery period except for the initial few minutes following relaxation when -delta PCr was significantly smaller than delta Pi. The difference was 0.35 +/- 0.03 mmol kg-1 (mean +/- 1 S.D., n = 3) immediately after a 2 s tetanus. 7. In contractions of 2 s or longer, the extent of the temporal separation between delta Pi and -delta PCr was almost the same, but in contractions of less than 2 s it was significantly reduced.(ABSTRACT TRUNCATED AT 400 WORDS)
Article
This review summarizes a variety of estimates for the concentrations of the principal cytosolic constituents in frog skeletal muscle. From these estimates (listed in the APPENDIX), we chose representative values and used electroneutrality and osmotic considerations to ensure that all major constituents have been considered. Given total cytosolic concentrations of these constituents from the literature, we employed a computer program to calculate the concentrations of all the major ionic species in the cytosol. In relaxed muscle, electroneutrality and osmotic constraints are fulfilled if, in addition to diffusible species, the charge contribution of the myofilaments is considered. Mean buffer power of the diffusible cytosolic species is calculated to be less than one-third of that experimentally determined for whole muscle. Computations indicate that recent estimates of intracellular free magnesium concentration approximately 1 mM are likely to be correct.
Article
The use of glycerol extracted muscle allows mechanical studies on the contractile mechanism to be carried out under defined biochemical conditions. It is important to know whether enzymes, other than the myofibrillar ATPase, which act on adenine nucleotides are present in this preparation. Therefore, the effects of glycerol extraction were studied on adenylate kinase and arginine kinase activities in insect fibrillar muscle and on adenylate kinase, creatine kinase and AMP deaminase activities in rabbit psoas muscle. All these activities decreased during glycerol extraction. A substantial part of the activity was lost in the first day of glycerol extraction, after which a very slow rate of loss occurred. There was some indication of a preferential loss from the soluble fraction of the fibres but this was insufficient to explain the biphasic loss. Measurements of the total activity of adenylate kinase in the fibres plus the glycerol medium suggested that some inactivation had occurred. It is also possible that the outer fibres in a bundle are extracted more rapidly than are the inner fibres. All three effects are probably playing a part but we are unable to offer a complete quantitative explanation of the results.
Article
All discussions of muscle energetics concern themselves with the Hill force-velocity relation, which is also the general output relation of a class of self-regulated energy converters and as such contains only a single adjustable parameter -the degree of coupling. It is therefore important to see whether in principle muscle can be included in this class. One requirement is that the muscle should possess a working element characterized by a dissipation function of two terms: mechanical output and chemical input. This has been established by considering the initial steady phase of isotonic and isometric tetanic contraction to represent a stationary state of the fibrils (a considerable body of evidence supports this). Further requirements, which can be justified for the working element, are linearity and incomplete coupling. Thus the chemical input of the muscle may be expected to follow the inverse Hill equation (see Part I). The relatively large changes in activities of reactants which the equation demands could only be controlled by local operation of the regulator, and a scheme is outlined to show how such control may be achieved. Objections to this view recently raised by Wilkie and Woledge rest on at least two important assumptions, the validity of which is questioned: (a) that heat production by processes other than the immediate driving reaction is negligible, which disregards the regulatory mechanism (possibly this involves the calcium pump), and (b) that the affinity of the immediate driving reaction is determined by over-all concentrations. The division of heat production into "shortening heat" and "maintenance heat" or "activation heat" is found to be arbitrary.
Article
1-Fluoro-2,4-dinitrobenzene (FDNB) caused the cessation of beating of heart cells in tissue culture. The beating stopped after 5 to 10 min in 0.19 mm FDNB. The cessation of beating in FDNB-treated cells correlated with a decrease in ATP to 70–80% of the control level, without any change in phosphocreatine concentration. FDNB appeared therefore to inhibit ATP:creatine phosphotransferase.In a time period equivalent to that used in the FDNB experiments (5–10 min), cells incubated with 2-deoxyglucose (2-DG) + oligomycin continued to beat, and the change in ATP concentration was very small. The total high energy phosphate was greatly depleted and reflected in phosphocreatine decrease.The total change in high-energy phosphate was the same in the presence of oligomycin + 2-DG as in FDNB + oligomycin + 2-DG. In the former case the energy utilization was reflected in a phosphocreatine depletion, while in the latter in the ATP level. It was concluded that the spontaneous contractions of heart cells utilize ATP directly and are independent of the phosphocreatine level as long as the ATP level is essentially undiminished. Possible effects of FDNB on metabolism, contractile proteins, and 45Ca uptake and washout were investigated.
Article
1. Frog semitendinosus muscles were stretched to various lengths beyond the rest length (l(0)) and their initial heat and isometric tension production were measured.2. As the overlap between the thick and thin filaments is reduced, the initial twitch heat and tension decline in a linear manner. At a point at which the twitch tension approaches zero, the initial heat is 30% of that seen at l(0). It is concluded that this heat is the activation heat and reflects the energetics of calcium release and reaccumulation. The initial heat at shorter sarcomere lengths appears to be the sum of the activation heat plus a heat production associated with the interaction of the thick and thin filaments.3. A similar relationship between heat and tension production is seen in tetanic contractions.4. The time course of activation heat production in a twitch can be resolved into two phases: a temperature insensitive (Q(10) < 1.3) ;fast' phase (with a time constant of 45 msec) and a temperature sensitive (Q(10) = 2.8) ;slow' phase (with a time constant of 330 msec at 0 degrees C).5. Measurements of the creatine phosphate (PC) hydrolysis by muscles contracting isometrically at various muscle lengths at and beyond l(0), indicate an enthalpy change of -11.2 kcal/mole PC hydrolysed. The enthalpy change for the ATP hydrolysis by muscles stretched so that little or no tension was produced with stimulation was -9.9 kcal/mole ATP hydrolysed. It is concluded that the net activation heat is produced by the hydrolysis of PC or ATP.
Article
Zusammenfassung Nach 1-Fluor-2,4-dinitrobenzol-Vergiftung von perfundierten Kaninchenherzen waren bei vollständiger Insuffizienz der ATP-Gehalt sehr signifikant und der Kreatinphosphatgehalt weniger stark vermindert. Glykogen-, Glucose- und Lactatgehalt unterschieden sich von den Kontrollwerten nicht.
Article
1. Changes in the levels of phosphocreatine (PCr) and ATP during relaxation after tetanic contraction have been observed and compared with the heat + work production during the same period.2. The results were the same using 2 sec or 15 sec tetani and using 15 or 30 Hz stimulation.3. During relaxation the mean chemical change (of ATP + PCr) was -0.105 +/- 0.078 mumol/g muscle (n = 53). This change is less than would be required to explain the heat + work produced.4. The PCr split during both 2 sec and 15 sec tetani (up to the last stimulus) was less than would be required to explain the heat + work produced. This confirms previous experiments.5. It is concluded that the discrepancy between chemical change and heat + work production does not diminish during relaxation but actually increases.
Article
Full-text available
Review on the the heat and work production during contractions of isolated muscles and the corresponding expenditures of chemical energy, Shortcomings of the classical theory are thoroughly discussed,
Article
For small changes in ion concentration within the physiological range the membrane potential transients can be explained in terms of two linear models both for passive and active transport. Using frog sartorius muscle as a suitable model system the ion pump is considered to work within the steepest range of the flux-concentration characteristic. Further for the small perturbations the equations describing passive ion transport can be safely linearized. The conclusion appears inescapable that for the muscle membrane the intracellular ion concentration adjusts itself in some optimal manner to the level of the extracellular ions. The active ion transport represents a control parameter for the membrane potential. The model structure corresponds to a dynamic system, the control processes of which are optimized with respect to a quadratic integral-criterion function. Here, both the performance index of the control sequence in the membrane processes and the energy consumed by the ion fluxes have been considered for small perturbations of Na+, K+, and Cl- in the neighbourhood of the physiological working point. As it is, the control system governing the active and passive ion transport processes is essentially optimized with respect to a minimal energy usage. The amount of energy consumed during the transients predicted by the model has been calculated.
Article
1. The production of heat and (internal) work and the changes in the amount of phosphocreatine (PCr), ATP, inorganic phosphate (Pi) and sometimes lactate have been measured from moment to moment during and after tetanic isometric contractions of isolated frog muscles at 0 degrees C.2. Heat production was measured by thermopiles and a novel apparatus was employed for freezing the muscles rapidly at a chosen instant so as to halt the chemical processes before analysis.3. Using unpoisoned muscles in oxygen, it was shown that neither oxidative recovery processes nor glycolytic ones led to appreciable restitution of PCr or ATP during 15 sec of contraction. However, clear signs of recovery processes could be seen within a minute. In our preparations artificial ;ageing' by storage at low temperature did not interfere with the capacity for glycolysis.4. Our clearest result was that the break-down of PCr was not nearly large enough to account for the rapid heat production during the first few sec of contraction. By the end of a 15 sec tetanus as much as 10 mcal/g remained unaccounted for.5. The source of this heat is not clear. At no time is there any sign of net break-down of ATP; indeed there appears to be a slight increase of ATP in the stimulated muscle.6. Break-down of PCr continues both during relaxation and during the minute following, while the muscle is at rest. Thus during contraction there is heat production without PCr break-down, while subsequently there is PCr break-down without heat production.
Article
1. Basal and recovery O2 consumption, delatO2, in frog sartorius muscles at 0 degrees C were measured with a polarographic electrode. Reproducible observations were made with the same muscle over many hours. 2. The experimental records had an exponential form except for the early phases of recovery following a single isometric tetanus. Diffusion of O2 within the muscle was adequate to account for this deviation from an exponential time course of recovery. The time constant of the recovery O2 consumption increased with the duration of tetanic stimulation from 5 to 20 sec. 3. Lactate synthesis was measureable in unstimulated aerobic muscles and increased in proportion to total O2 consumption as long as the muscle did not lack O2. The contribution of glycolysis to the total chemical energy production during recovery was 6-9%; for hypoxic muscles it was greater. 4. The resynthesis of phosphorylcreatine and the decrease in inorganic phosphate and free creatine following a tetanus showed an exponential time course similar to recovery O2. Initial concentrations were re-attained within 60 min following a 20 sec tetanus. 5. We conclude that recovery O2 consumpation is a useful and accurate measure of the net chemical energy utilization for a single contraction.
Article
1. Methods are described whereby the soleus muscle of the rat may be used for the investigation of initial processes in the absence of oxidative recovery. 2. The anaerobic conditions employed had no effect on the concentration of phosphocreatine in resting muscle or the mechanical response during contraction. 3. Muscles were stimulated tetanically for 10 s at 17-18 degrees C. Measurements were made of the heat production and metabolic changes that occurred during a 13 s period following the first stimulus. 4. There was no detectable change in the concentration of ATP. Neither was there detectable activity of adenylate kinase or adenylate deaminase. The changes in the concentration of glycolytic intermediaries were undetectable or very small. 5. The change in the concentration of phosphocreatine was large and amounted to -127 +/- 11-4 mumol/mmol Ct (mean and S.E. of the mean, negative sign indicates break-down, Ct = free creatine + phosphocreatine) which is equivalent to about -2-13 mumol/g wet weight of muscle. The heat production was 6549 +/- 408 mJ/mmol Ct (mean and S.E. of mean) which is equivalent to about 110 mJ/g. 6. About 30% of the observed energy output is unaccounted for by measured metabolic changes. 7. The ratio of heat production (corrected for small amounts of glycolytic activity) to phosphocreatine hydrolysis was -49-7 +/- 5-6 kJ/mol (mean and S.E. of mean), in agreement with previous results using comparable contractions of frog muscle, but different from the enthalpy change associated with phosphocreatine hydrolysis under in vivo conditions (-34 kJ/mol). 8. The results support the notion that the discrepancy between energy output and metabolism is an indication of an unidentified process of substantial energetic significance that is common to a number of species.
Article
Full-text available
Recent data obtained from Rana temporaria sartorius muscles during an isometric tetanus indicate that the time-course of phosphocreatine (PC) splitting cannot account for the total energy (heat + work) liberation (Gilbert et al. 1971. J. Physiol. (Lond.) 218:)63). As this conclusion is important to an understanding of the chemical energetics of contraction, similar experments were performed on unpoisoned, oxygenated Rana pipiens sartorius muscles. The muscles were tetanized (isometrically) at 0 degrees C for 0.6, 1, or 5 s; metabolism was rapidly arrested by freezing the muscles with a specially designed hammer apparatus, and the frozen muscles were chemically analyzed. Comparable myothermal measurments were made on frogs from the same batch. Results of these experiments indicate: (a) The energy liberation parallels the PC and ATP breakdown with a proportionality constant of 10.7 kcal/mol; (b) comparably designed experiments with sartorius muscles of R. temporaria revealed that the ratio of energy liberation to PC splitting was significantly greater than that observed in R. pipiens sartorius muscles; (c) there is no systematic difference between experiments in which metabolism was arrested by the hammer apparatus and others using a conventional immersion technique.
Article
Full-text available
31P nuclear magnetic resonance spectra recorded from intact muophosphate, and the sugar phosphates. Quantitation of these metabolites by 31P nuclear magnetic resonance was in good agreement with values obtained by chemical analyses. The spectra obtained from various muscles showed considerable variation in their phosphorus profile. Thus, differences could be detected between (a) normal and diseased muscle; (b) vertebrates and invertebrates; (c) different species of the same animal. The time course of change in phosphate metabolites in frog muscle showed that ATP level remains unchanged until phosphocreatine is nearly depleted. Comparative studies revealed that under anaerobic conditions the Northern frog maintains its ATP content for 7 hours, while other types of amphibian, bird, and mammalian muscles begin to show an appreciable decay in ATP after 2 hours. Several lines of evidence indicated that ATP forms a complex with magnesium in the muscle water: (a) the phosphate resonances of ATP in the muscle were shifted downfield as compared to those in the alkaline earth metal-free perchloric acid extract of the muscle; (b) the coupling constants of ATP measured in various live muscles closely corresponded to those for MgATP in a solution resembling the composition of the muscle water; (c) in the muscle the gamma-phosphate group of ATP exhibited no shift change over a period of 10 hours under conditions where resonances of other phosphate compounds could be titrated. This behavior is similar to that of MgATP in model solutions in the physiological pH range, and it is different from that of CaATP. The chemical shifts of the phosphate metabolites were determined in several relevant solutions as a function of pH. Under all conditions only inorganic orthophosphate showed an invariant titration curve. From the chemical shift of inorganic phosphate observed during aging of intact muscle the intracellular pH of frog muscle was estimated to be 7.2.
Article
This chapter discusses the thermodynamic, physiological, and biochemical aspects of muscle contraction as well as the details of energy balance. Experiments relating to energy balance are essentially quantitative analyses and descriptions of the necessary equivalency between the energy inputs and outputs during and following muscular contractions. Energy balance is the study of muscle contraction and energy metabolism by means of quantitative balance measurements as a tool. The rapid freezing techniques do not measure the total net high energy phosphate splitting associated with the contraction–relaxation cycle. The myothermic energy balance studies relate main aspects of cellular energy metabolism such as the degree of coupling of oxidative phosphorylation, the control of respiration, and the possibility of futile metabolic cycling
Chapter
It was not too long ago, back in the early sixties, perhaps, when the use of nuclear magnetic resonance in the study of intact tissues at the clinical level was thought by most in the field of NMR spectroscopy to be beyond the realm of possibility. After all, it was argued, how much information could be gleaned from the proton resonance of tissue water, the only magnetic resonance signal from intact cellular masses which was then practically accessible. There were a few who did try to obtain NMR spectra from intact cellular masses, but the results were not very encouraging. Proton spectra could, in fact, be obtained, but they contained so many overlapping signal that all fine-structure was lost so that only the water signal could be assigned with certainty. Fluorine-19 spectroscopy was of little value, since living things contain little fluorine. Phosphorus-31, perhaps, has possibilities, since, in living systems, phosphorus has the status of a heteroatom, there are relatively few compounds that contain it, and these represent some of the more interesting and abundant biological chemicals. In the early sixties, however, 31P spectroscopy required, at a minimum, 0.1 M phosphate solutions, and these had to give rise to only one, or at most two, resonance lines, so phosphorus-31 was also of no practical value. Carbon-13 NMR was still in the hands of the physists as was all the other magnetic nuclides of the Periodic Table.
Article
D.R. Wilkie entered University College London (UCL), which was to be his lifelong academic home, in 1940 to study medicine on the shortened wartime course. He soon showed his great academic ability and won the Rockefeller Scholarship that took him to Yale University, New Haven, Connecticut, for the last year of his medical education, where he obtained his MD. He returned to University College Hospital as house physician in 1944 and, quite exceptionally, obtained his MRCP in that same academic year. The Physiology Department of UCL appointed him to an assistant lectureship in 1945 when he was 23 years old and, apart from a period of military service at the Institute of Aviation Medicine in Farnborough, from 1948 to 1950, he worked there until his retirement in 1988. During the period 1951–54 he held a Locke Fellowship of The Royal Society. In 1945 A.V. Hill, F.R.S., then nearly 60, had returned to his laboratories at UCL to resume the muscle research interrupted by the war. Wilkie evidently soon fell under his spell and he took up some of Hill's lifelong interests: the mechanics of muscle, its relation to human performance and the application of thermodynamics to muscle contraction. In addition, he adopted something of Hill's style of research, characterized by the application of basic principles and measurements from physics, mathematics and chemistry to the understanding of the behaviour of human or muscle, together with ingenuity in the invention of methods. Wilkie's research work started with the application of muscle mechanics to human movement. He critically tested the current theories of muscle mechanics and then took up the question of the supply of chemical energy for muscle contraction. Through initiating collaborations he brought together the experimental study of the chemical changes in muscle with that of the output of energy as heat and as work. These experiments, along with his 1960 review (12)*, put this subject of ‘chemical energetics of muscle contraction’ back on the thermodynamic rails from which it had strayed and allowed the subject to make further progress, exposing again the limitations of the current theories. In 1969 A.F. (later Sir Andrew) Huxley, F.R.S. (P.R.S. 1980–85), head of UCL's Physiology Department, stepped aside to take a Royal Society Chair and it was natural that Wilkie, by then holder of a personal chair and a major force in medical education, should be asked to lead the department. He filled that role conscientiously for 10 years. Although his personal involvement in scientific experimentation had consequently to be reduced during this period, his interest in muscle energy supply led to a new enthusiasm: the application of magnetic resonance spectroscopy, first to the study of isolated muscles, in collaboration with G.K. Radda (F.R.S. 1980) and D.G. Gadian in Oxford, and then, with his UCL colleagues R.H.T. Edwards (Medicine), D.T. Delpy (F.R.S. 1999) (Medical Physics) and E.O.R. Reynolds (F.R.S. 1993) (Paediatrics), to the study of the brains of newborn babies. Wilkie was elected to Fellowship of The Royal Society in 1971 and to Fellowship of UCL in 1972.
Article
In contracting muscle a chemical reaction takes place in such a way that part of the free energy of the reactants can be converted into mechanical work. Recently Caplan (1965, 1966) has made a theoretical study of certain types of energy converters, using the methods of irreversible thermodynamics. He has extended this study to muscle and he makes several predictions of practical importance, notably about the efficiency of muscular contraction. In this paper a simplified summary of Caplan's theory is given, with the equations rearranged, as far as possible, to provide relations between experimentally observable quantities. Four tests of the theory are then possible, of which the results are: 1. The theory correctly predicts the shape of the force-velocity curve. 2. The theory predicts a relation between the maximum efficiency of energy conversion and the degree of curvature of the force-velocity curve which is probably similar to what is found experimentally. 3. The theory predicts a relation between the force on the muscle and the rate of the driving chemical reaction which is markedly different from that which can be inferred from measurements of heat and work production. 4. The theory requires that there should be large variations in the free energy change of the driving reaction when the force on the muscle changes. It is difficult to see how these could occur in practice. No simple modification of the theory seems to avoid these difficulties. It is concluded that muscle is not an energy converter of the type described by the theory.
Article
A B S T R A C T The spectral characteristics of the pH-sensitive dyes neutral red (NR) and bromcresol purple (BCP) were utilized for studies of the changing intracellular pH (pH0 of sartorius muscles from Rana pipiens, both during the Course of an isometric twitch and during recovery metabolism subsequent to a train of twitches. The information from the two dissimilar dyes correlated to confirm the methodol- ogy. Neither the fast realkalinization observed during a twitch nor the slow alkaliz- ing phase of recovery metabolism was affected in an obvious manner when phos- phocreatine (PC) hydrolysis was blocked by l-fluoro-2,4-dinitrobenzene (FDNB). Iodoacetic acid (IAA) did inhibit the slow acidic phase of recovery metabolism. The conclusion is made that alkalizing reactions other than PC breakdown must be considered as operative at these levels of activity. Hypertonic solutions altered twitch tension and time course without altering the pHi shifts observed until approximately 75% of the twitch amplitude was abolished. Multiple effects of hypertonic solutions as the muscles approach tonic equilibrium are proposed.
Article
Chemical and thermal analyses have been carried out in parallel experiments at 0C using identical mechanical conditions. The large number of muscle pairs investigated under both conditions allows a statistically meaningful analysis. Only muscles of comparable weight have been used which under steady state tetanic conditions were able to maintain similar isometric tensions. To limit the considerations of the energy balance to the steady state, both muscles of the pair were initially stimulated at a frequency of 15 Hz for 0.6 sec under isometric conditions. At this time, which reflects the beginning of the steady state, one of the muscles was frozen while the other was allowed to shorten froml 0+2 mm tol 0–6 mm in an isotonic or isovelocity tetanus. The total energy output over the range of loads and shortening velocities is always the sum of mechanical work, shortening heat and maintenance energy. Although a considerable fraction of shortening heat is liberated particularly at the smaller loads and higher velocities the total amounts of heat plus work are clearly balanced by the expenditure of chemical energy. The molar free enthalpy change of PC hydrolysis obtained in this way was 10.98 kcal/mole. As it could be shown with steady state isometric tetani that the maintenance energy depends on the sarcomere length (or the tension maintained) it is not possible to subtract chemical and heat changes occurring within an isometric muscle at rest length from those of a twin muscle shortening over a wide range of sarcomere lengths during the same time period.
Article
1. The physiological responses to many hormones and neurotransmitters decrement with time in the continued presence of the agonist. 2. This process, variously termed desensitization, tachyphylaxis and receptor inactivation, has been demonstrated to occur at many receptors but remains poorly understood. 3. The effect of the application of metabolic inhibitors on the rate of desensitization at the neuromuscular junction of the frog has been investigated to test the possible role of intracellular calcium (Ca i ) in the desensitization mechanism. 4. Desensitization curves recorded under voltage clamp in response to the cholinergic agonist carbachol have been found to display two time constants. 5. The application of metabolic inhibitors decreased the slow time constant but not the fast one. 6. Treatment of the muscles with metabolic inhibitors also decreased the degree of recovery from desensitization. 7. The data are consistent with a role of Ca i in the desensitization process.
Article
1. Phosphorus nuclear magnetic resonance ((31)P NMR) can be used to measure the concentrations of phosphorus-containing metabolites within living tissue. We have developed methods for maintaining muscles in physiological condition, stimulating them and recording tension while at the same time accumulating their (31)P NMR spectra. Experiments were performed on frog sartorii and frog and toad gastrocnemii at 4 degrees C.2. The NMR signals from (31)P (the naturally occurring phosphorus) is weak, and signal averaging is required. In order to follow the time course of reactions it is necessary to maintain the muscles in a steady state for many hours while they are undergoing repeated contractions. Signals were accumulated in separate computer bins according to time after initiation of contraction. By these means spectra were obtained which corresponded to the different intervals during the contraction and recovery cycle.3. In the absence of stimulation, the spectra of frog sartorius muscles and of their extracts indicated concentrations of adenosine triphosphate (ATP), phosphoryl creatine (PCr), inorganic orthophosphate (P(i)) and sugar phosphates (sugar P) which are in reasonable agreement with the values obtained by chemical analysis.4. We have confirmed that unidentified resonances representing unknown compounds appear in the spectra of both frog and toad muscle; one of these is much larger in spectra from toad than from frog. We have found an additional small, unidentified resonance which appears to be specific to toad muscle.5. Spectra accumulated during actual contractions (1 s tetani every 2 min) did not differ dramatically from those accumulated throughout the 2 min cycle of contraction and partial recovery.6. Following 25 s tetanii, approximately 20% of the PCr had been hydrolysed; it was then rebuilt exponentially with a half-time of about 10 min. The increase in [P(i)] immediately after contraction and the time course of its disappearance corresponded to the changes in [PCr]. During the later half of the recovery period the concentration of P(i) was reduced to below that in resting muscle. The [sugar P] remained very high ( approximately 4 mmol kg(-1)) throughout the 56 min interval between contractions.7. When frog sartorii were tetanized for 1 s every 2 min, the changes in [PCr] and [P(i)] between contractions could not be observed because too little signal was obtained from these small muscles. However, when toad gastrocnemii were similarly stimulated, the changes in these compounds could be readily detected and were even greater than expected.8. The position of the P(i) resonance can be used to monitor intracellular pH and changes in pH. Under the conditions of our experiments the average intracellular pH in unstimulated frog sartorius muscles was 7.5. After a 25 s tetanus this was observed to move in the acid direction by a few tenths of a pH unit and to return to its pre-stimulation value before the end of the recovery period. After a 1 s contraction of toad gastrocnemius the environment of P(i) became slightly more alkaline for the first few seconds.
Article
A means of measuring pH spectrophotometrically with two pH-sensitive indicators, neutral red (NR) and bromcresol purple (BCP), is presented. Theoretical calculations and experimental measurements of pH in solution correspond in a satisfactory manner for both dyes. Spectrophotometric determinations of pH were made of dye-equilibrated frog sartorius muscles. A more accurate indication of shifting intracellular pH (pHi) was obtained with NR than with BCP, since only the spectra of NR-equilibrated muscles were insensitive to changes in external pH over the range 7.0–6.0. Muscles were stimulated in oxygenated Ringer's and the latent phase of acidification correlated with lactic acid production by analysis of tissue frozen after spectral determination of ΔpHi. Buffering capacities were calculated to be 0.041 to 0.048 g equiv per liter of strong acid or base per resultant ΔpH for a pHi range covering 0.25 unit.
Article
When, on addition of a suitable substrate, a chemical potential is applied to an enzymic process such as glycolysis or respiration, whether in solution or membrane-bound, all components of the process pass into a nonequilibrium state, which might be steady or non-steady and which produces the following phenomena: (1) The reactants of each enzymic reaction are displaced from their equilibrium concentration, and energy is dissipated; (2) Part of each enzyme is transferred to a transition state of its catalytic function as well as isosteric and allosteric controlling functions, displaying local and gross conformation changes, and a rate-controlling state is generated; (3) In cyclic portions of a process futile events and chemical interconversion may occur; (4) In self- and cross-coupled portions of a process, oscillation with periodic changes of states and spatial propagation as well as instabilities may be observed; (5) At each step of a process, depending on the rate of flux and the specific enzymic function, a varying proportion of the free energy changes--which are concentration-dependent and derived from the overall potential of the system-is contributed to the control of flux rates. This will be exemplified for enzymes of bioenergetic pathways.
Article
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Nuclear magnetic resonance is a new method for assaying the content of phosphate metabolites in intact tissues. Its nondestructive nature allows simultaneous and repeated determinations of these compounds with a minimum perturbation of tissue. Changes in the concentrations of the phosphates as a function of time characterize the metabolic machinery of the tissue and reveal alterations in enzymic activity that result from drug treatment or disease. The entire phosphate profile shows differences between normal and diseased muscle which should be of diagnostic value. Further, by examining phosphate profiles we detected a family of chemical compounds that were not previously known to exist as major constituents in muscle. Of these, two have been isolated and one has been identified as glycerol 3-phosphorylcholine. Finally, shifts in the positions of resonances monitor the internal environment of the living system, its hydrogen ion concentration, the complexing of alkaline earth metals with ATP, and compartmentalization within the cell.
Article
1. In an attempt to study the intracellular location of ATP in skeletal muscle the distribution of substances which absorb light at 260 nm wave-length has been studied in isolated muscle fibres with the aid of a modified U.V.-microscope.2. U.V.-absorption in resting frog fibres was found to be higher in the I band than in the A band which confirms earlier findings. In stretched fibres (sarcomere length 2.9-3.6 mum) an absorbing substance could be seen to be concentrated in a pair of narrow lines, centred at the Z-line. The separation of the lines increased with increasing sarcomere length.3. Snake fibres, with sparse triads located at the A-I junction, displayed an absorption pattern very similar to that of frog fibres. It is concluded that it is unlikely that the absorbing substance is associated with the sarcotubular system.4. The absorption pattern of frog fibres remained unchanged during a tetanus. No clear changes could be detected after a period of stimulation, neither after single twitches nor after repeated tetani.5. In further attempts to cause exhaustion, metabolically poisoned fibres were stimulated repetitively until they went into rigor. The absorption pattern was essentially unchanged also when rigor tension started to develop.6. The characteristic absorption pattern was observed also in glycerol-extracted fibres. It was confirmed by spectrophotometry that glycerol-extraction led to the disappearance of a large amount of a substance with the spectral characteristics of ATP.7. The higher U.V.-absorption in the I band does not prove that the major part of the ATP in the fibre is concentrated here; the absorption could either be due to a minor fraction of the ATP or to RNA.
Article
1. Measurements were made of the energy produced as heat and work (h + w) and the chemical changes which occurred between the beginning and end of each of two periods of stimulation. The muscles contracted tetanically under isometric conditions. Each period of stimulation (tetanus) lasted 5 sec and there was an interval of 3 sec between them. The tension developed in the second tetanus was 91% of that in the first.2. The observed (h + w) was greater in each tetanus than the quantity expected from the measured chemical changes. This excess was 230 +/- 43 mJ/g dry wt. (mean +/- S.E. of mean) in the first tetanus, but only 89 +/- 46 mJ/g in the second tetanus (mean +/- S.E. of mean). The result for the first tetanus agrees with previous findings.3. The observed (h + w) was divided into two parts, labile and stable, which were defined by Aubert (1956). This division was made on the basis of the time course of the (h + w) production, without reference to the chemical changes. The labile part of the (h + w) has an exponentially declining rate, and the stable part has a constant rate.4. The observed (h + w) was less in the second tetanus than in the first. This was due largely to the decrease in the labile part in the second tetanus; it was only ca. 0.35 of that in the first tetanus. However, the stable part remained relatively constant, 0.83 of that in the first tetanus. Aubert & Maréchal (1963) and Aubert (1968) have reported similar results.5. Having divided the (h + w) into the stable and labile parts, we found that the stable part could be accounted for by the chemical change, but the labile part could not. Thus, for both tetani, the stable part of the energy is about equal to the explained energy, and the labile part is about equal to the unexplained energy. The possible interpretations of these equalities are discussed.
Article
In addition to a strophanthidin-sensitive (SS) sodium efflux, a large component of the sodium efflux in freshly isolated frog skeletal muscle is sodium-activated and strophanthidin-insensitive (SASI). The amount of metabolic energy associated with sodium movement by each of these components was measured and the coupling between sodium movement and adenosine 5'-triphosphate (ATP) hydrolysis in muscle was calculated. Energy production was blocked by iodoacetate and cyanide. Energy turnover was estimated from the change in creatine phosphate (CrP) and ATP contents and expressed as potential energy (PE = CrP + 2ATP). After metabolic poisoning a linear fall of PE occurred (6.3 mumol/g.h). Metabolic poisoning had no effect on the magnitude of the SS or SASI components of sodium efflux. In 2 h the sodium moved, and PE change due to the SS component was 4.35 and 1.66 mumol/g.h, respectively, which gave a coupling factor of 2.6. The amount of sodium moved by the SASI component was similar to that moved by the SS component in 2 h whereas no energy change was observed. It was, therefore, concluded that sodium movement by the SASI component requires no energy input.
Article
Fatigue and recovery from fatigue were related to metabolism in single fibers of the frog semitendinosus muscle. The fibers were held at a sarcomere length of 2.3 microm in oxygenated Ringer solution at 15 degrees C and were stimulated for up to 150 s by a schedule of 10-s, 20-Hz tetanic trains that were interrupted by 1-s rest periods, after which they were rapidly frozen for biochemical analysis. Two kinds of fatigue were produced in relation to stimulus duration. A rapidly reversed fatigue occurred with stimulation for under 40 s and was evidenced by a decline in tetanic tension that could be overcome by 1 s of rest. A prolonged fatigue was caused by stimulation for 100-150 s. It was evidenced during stimulation by a fall in tetanic tension that could not be overcome by 1 s of rest, and after stimulation by a reduction, lasting for up to 82 min, in the peak tension of a 200-ms test tetanus. Fiber phosphocreatine (PCr) fell logarithmically in relation to stimulus duration, from a mean of 121 +/- 8 nmol/mg protein (SEM, n = 12) to 10% of this value after 150 s of stimulation. PCr returned to normal levels after 90-120 min of rest. Stimulation for 150 s did not significantly affect fiber glycogen and reduced fiber ATP by at most 15%. It is suggested that the prolonged fatigue caused by 100-150 s of tetanic stimulation was caused by long-lasting failure of excitation-contraction coupling, as it was not accompanied by depletion of energy stores in the form of ATP. One possibility is that H+ accumulated in fatigued fibers so as to interfere with the action of Ca2+ in the coupling process.
Article
A previous paper (Mahler, M. 1978 J. Gen. Physiol. 71:559--580) describes the time-course of the suprabasal rate of oxygen consumption (delta QO2) in the sartorius muscle of R. pipiens after isometric tetani of 0.1--1.0 s at 20 degrees C. To test whether these were the responses to impulse changes in the rate of ATP hydrolysis, we compared the total suprabasal oxygen consumption during recovery (delta[O2]) with the amount of ATP hydrolyzed during a contraction, measured indirectly as the decrease in creatine phosphate (delta[CP]O). If suprabasal ATP hydrolysis during recovery is negligible in comparison with that during contraction, delta[CP]0/delta[O2] should approximate the P:O2 ratio for oxidative metabolism, which has an expected value of 6.1--6.5. We found: formula; see text. We conclude that in this muscle at 20 degrees C: (a) after a tetanus of 0.2--1.0 s, delta QO2(t) can be considered the response to an impulse increase in the rate of ATP hydrolysis; (b) the reversal during recovery of unidentified exothermic reactions occurring during the contraction (Woledge, R. C. 1971. Prog. Biophys. Mol. Biol. 22:39--74) can be coupled to an ATP hydrolysis that is at most a small fraction of delta[CP]0; (c) the pooled mean for delta[CP]0/delta[O2], 6.58 +/- 0.55, sets an experimental lower bound for the P:O2 ratio in vivo.
Article
SHORTENING of muscle during single isotonic twitches and following a tetanic stimulus is accompanied by the development of extra heat1-3. The heat of shortening varies with the distance shortened and depends also on the load lifted by the muscle4. Although it is generally agreed that the heat developed during work done by the muscle can be accounted for by the energy of ATP splitting5,6, there appears to be no evidence of changes in the high-energy phosphate level accompanying the actual shortening7,8.
Article
The dependence of PC1 and ATP1 dephosphorylation on the number of isometric twitches in the iodoacetate-nitrogen-poisoned muscle has been examined. There is no net dephosphorylation of adenosinetriphosphate. PC dephosphorylation varies linearly with the number of twitches and produces equivalent amounts of C1 and P1i.1 Iodoacetate concentrations which block the enzyme, creatine phosphokinase, render the muscle non-contractile. A value of 0.286 µmole/gm. for the amount of PC split per twitch is obtained which gives a value of -9.62 kcal./mole for the "physiological" heat of hydrolysis of PC in agreement with expectations based on thermochemical data. In a single maximal isometric twitch it is estimated that 2 to 3 PC molecules are dephosphorylated per myosin molecule, or 1 per actin molecule. The results support the view that under the conditions of these experiments PC dephosphorylation is the net energy yielding reaction. The in vivo stoichiometry of the mechano-chemistry of contraction revealed by these studies on the one hand, and the known stoichiometry of actin polymerization and its coupling to the creatine phosphokinase system on the other are strikingly similar and strongly suggest that the reversible polymerization of actin is involved in a major way in the contraction-relaxation-recovery cycle of muscle.
Article
Lactic acid is converted quantitatively into acetaldehyde on being heated with concentrated sulfuric acid. The acetaldehyde is determined by measurement of the purple color formed with p-hydroxydiphenyl. Procedure of preparation involves removal of protein; treatment with copper and calcium; formation of acetaldehyde; development of color; color measurement; and calculation of results. For calculation of results duplicate colorimeter readings obtained is used to calculate the lactate content of the aliquot by reference to a calibration curve relating colorimeter readings and known concentrations of lactate. It is emphasized that standards should be included in every series of determinations and these compared to previously established values. Any considerable deviation should be investigated, because analytical conditions must be rigorously controlled if earlier calibration figures are to be valid. Analytical precautions should be taken while the procedure as contamination by lactic acid itself is the principal source of error. It is recommended that conditions be held as constant as possible for maximum reproducibility.
Isolated frog muscles were exposed to Ringer’s solution of widely varying pH. In the presence of oxygen they remained in good condition for a long time and continued to contract well at hydrogen-ion concentrations many times greater (up to x 200) than that of their normal environment in the body. If the Donnan equilibrium, which is believed to govern the K and Cl ion ratios across the fibre membrane, applied also to H and HCO 3 ions, the internal pH in these circumstances would be 3.8 or less. It is difficult to believe that the contractile mechanism would function so well under such conditions, but the question could be examined experimentally as follows. Muscles in oxygen-free Ringer at pH 7.4 to 3.3 were stimulated in a regular series of twitches to complete exhaustion, and the total tension developed was used to measure the energy liberated. If the energy was less than 0.4 cal/g muscle it could have been derived solely from the splitting of creatine phosphate and other phosphorus compounds; if it was greater than 0.4 cal/g muscle it must have been obtained in part from lactic acid production. The formation of lactic acid in response to stimulation ceases when the internal pH falls below about 6.3; but experiments show that at external pH 6.0 adjusted by phosphate buffers, lactic acid can be produced in practically normal amount, while some lactic acid can be formed even when the external pH is as low as 4.5. When muscles are stimulated in a medium saturated with 100% CO 2 and buffered with bicarbonate, there is seldom evidence of lactic acid formation at any external pH (from 6.8 downwards). The CO 2 itself appears to reduce the internal pH to about the critical level below which lactic acid production is inhibited. At lower CO 2 percentages (50% or less, in nitrogen) some lactic acid can be produced at all external pH’s, from 5.1 upwards. If the hydrogen-ion ratio across the fibre membrane were governed by the Donnan equilibrium, the internal pH of a normally excitable muscle would have to be at least 1.2 less than that of the outside fluid. At external pH 6.0 the internal pH would then be 4.8 or less, yet the nearly normal production of lactic acid shows that it must have been well above 6.3, while at external pH 4.5 the internal pH would not be greater than 3.3, yet some lactic acid was formed, so it cannot in fact have been less than 6.0 (allowing 0.3 for the increased alkalinity due to phosphagen splitting). When phosphate buffers are used, the internal pH certainly falls to some extent when the external pH is lowered, but far less than prescribed by the Donnan equilibrium. With CO 2 -bicarbonate buffers, there is no sign that the internal pH depends on anything but the partial pressure of CO 2 . Other evidence is considered, particularly that obtained by using an intracellular glass electrode (Caldwell, with crab fibres). The conclusion is that in normally excitable muscle the Donnan equilibrium does not control, and does not greatly influence, the distribution of hydrogen ions across the fibre membrane. In resisting diffusion and potential gradients the muscle fibre probably maintains its own internal pH, at least to a large extent, by active metabolic effort, If so, since CO 2 penetrates freely, the internal HCO 3 -ion concentration also must be actively maintained. When the controlling mechanism fails, the contractile function of the muscle deteriorates. The observed variability of muscles exposed to abnormal external conditions may depend on differences in their capacity to maintain their internal state.
Article
1.1. Experiments have been devised to test the suggestion of a number of investigators that the interconversion of adenine and hypoxanthine nucleotides may provid the energy for muscular contraction.2.2. Several methods for the assay of very small amounts of these nucleotides in muscle extracts have been examined. These involve spectrophotometry, radioactivity, electrophoresis and chromatography. They have been applied to a study of the production of the hypoxanthine nucleotides during single contractions of the frog rectus-abdominis muscles at 0°.3.3. Small amounts of the hypoxanthine di- and trinucleotides were detected in resting muscles but there were no significant net changes in either the adenine or hypoxanthine nucleotides during single contractions.
Article
The question was raised, what causes the sudden increase in the rate of glycolysis when muscle contracts and the equally rapid decrease in rate when the muscle relaxes. For the purpose of this study, isolated frog sartorii were stimulated electrically while immersed in anaerobic Ringer's solution without glucose. The rate was varied between 3 and 48 shocks per min and the duration was 30 min. Under these conditions, the conversion of glycogen to lactate is the main energy producing reaction. The results indicated that two reactions are of prime importance in the regulation of glycolysis in the working muscle: the formation of glucose-1-P from glycogen and inorganic P through the action of the phosphorylase system and the removal of fructose-6-P through the phosphofructokinase reaction. From the relation of the concentration of hexosemonophosphates to the flow rate over the glycolytic system it was concluded that during stimulation these two enzyme systems increase their activity synchronously and proportionately. This is in contrast to the action of epinephrine which causes a much greater increase in phosphorylase activity than in phosphofructokinase activity and thereby leads to a large accumulation of hexosemonophosphates. From measurements of tissue concentration and rate of efflux of lactate from muscle immediately after cessation of stimulation it was concluded that lactate formation returned to the resting rate within 5 min.On the basis of measurements of the tissue concentrations of ATP, ADP, AMP, and Pi it was concluded that changes in the concentrations of these compounds in stimulated muscle were too small to permit the increase in enzymatic rates actually observed in vivo. This applies particularly to the phosphorylase reaction for which in vitro data are available which permit predictions of rates at different concentrations of the above reactants. The concentrations of glucose-6-P, fructose-6-P and fructose-1,6-di-P were likewise not well correlated to the rate increase. The only two compounds that increased proportionately with the frequency of stimulation were end products of anaerobic glycolysis in muscle, namely lactate and α-l-glycero-P.A detailed investigation of the phosphorylase b [rlhar2] a interconversion in stimulated muscle suggested that the phosphorylase b kinase underwent an activation-inactivation cycle closely connected with the contraction and relaxation phases of muscle. An analysis of results obtained with a strain of mice incapable of forming phosphorylase a during stimulation indicated that this interconversion, while not essential for lactate formation per se, was of importance for the time of onset and the final speed of lactate formation.A hypothesis based on localization and structural organization of the glycolytic chain in muscle is proposed which could explain the results summarized above. A mechanism is suggested which is similar in nature to the activation and inactivation of actomyosin (and perhaps of phosphorylase b kinase) by Ca++ ions. It contains the idea of access of reactants to the catalyst during contraction and separation from reactants during relaxation.
Tension and heat production of frog muscle tetanized after intoxication with 1-fluoro-2,4-dinitrobenzene (FDNB)
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The wafer thermopile: a new device for measuring the heat production of muscles
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Zur Bestimmung des Orthophosphates neben saure-molybdat-labilen Phosphorsaureverbindungen
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WAHLER, B. E. & WOLLENBERGER, A. (1958). Zur Bestimmung des Orthophosphates neben saure-molybdat-labilen Phosphorsaureverbindungen. Biochem. Z. 329, 508-520.
Assay of ATP AMP aminohydrolase in the muscles of some vertebrates and invertebrates
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STREHLER, B. L. & MCELROY, W. D. (1957). Assay of ATP. In Methods in Enzymology, ed. COLOWICK, S. P. & KAPLAN, N. D., vol. III, p. 871. New York: Academic Press, Inc. UMIASTOWSKI, J. (1964). AMP aminohydrolase in the muscles of some vertebrates and invertebrates. Acta biochim. pol. 11, 459-464.
Breakdown of ATP during a single contraction of working muscle
  • D F Davies
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