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Effects of hydrostatic pressure on (Na+ + K+)ATPase and Mg2+ATPase in gills of marine teleost fish
Abstract
The effects of hydrostatic pressure on branchial NaK-ATPase and Mg-ATPase activities were studied in two species of marine teleost fish: the relatively shallow water Scorpaena guttata, collected from a depth of 20 m, and the deeper living Anoplopoma fimbria, collected from a depth of 310 m. Specific activities in gill microsomes, measured at 18oC, were similar in both species, with the NaK-ATPase usually comprising about 30–40% of the total ATPase activity. NaK-ATPase activity of S. guttata was activated by low hydrostatic pressure (68 atm) but activity was inhibited at pressures above 136 atm. The NaK-ATPase of A. fimbria showed a similar activation at low hydrostatic pressure but was less sensitive to a pressure of 340 atm than the enzymefrom S. guttata. At a pressure of 476 atm, the NaK-ATPase activity of both species was inhibited to the same extent. The Mg-ATPase of both species was activated by pressure (maximal activation occurred at 136 atm), but in contrast to the NaK-ATPase, remained activated at all pressures tested. Solubilization of the enzymes from gill microsomes of A. fimbria with Lubrol WX did not significantly alter the pressure responses. Inhibition of NaK-ATPase at high pressure was reversible upon release of pressure. Inhibition of branchial NaK-ATPase in both species occurred at pressures higher than the fish normally experience in their environment.
... La presión se contrapone a procesos moleculares que conllevan incremento de volumen. Este efecto se ha usado para el estudio de membranas, bombas [44] y canales iónicos, como los de sodio [45], potasio [46] y calcio [47], y aun de corrientes de compuerta (gating) de algunos de éstos [48]. La presión reduce las cinéticas de apertura y cierre de canales, y produce poco cambio en sus conductividades máximas [48]. ...
... La presión reduce las cinéticas de apertura y cierre de canales, y produce poco cambio en sus conductividades máximas [48]. Efectos similares se describieron para canales dependientes de ligandos, como receptores de acetilcolina [49], glicina [50], GABA [51] y glutamato [52], transportadores transmembranales, como bombas iónicas (ATPasas de Na-K) [44], o intercambiadores, como el de Na + / Ca 2+ [53]. Estos cambios llevarían a la reducción en la amplitud, al enlentecimiento de cinéticas de los potenciales de acción, y a reducción de la velocidad de conducción [54,55] de los axones (Fig. 2). ...
... Pressure opposes to molecular processes that involve volume expansion. This effect has been used for the study of membranes , pumps [44] and ionic channels like sodium [45], potassium [46], and calcium channels [47], and even for the study of gating currents in some of them [48]. Pressure reduced the kinetics of opening and close of the channels while inducing minimal effect on the channels' maximal conductance [48] . ...
... Pressure reduced the kinetics of opening and close of the channels while inducing minimal effect on the channels' maximal conductance [48] . Similar effects were described for channels gated by ligands, like receptors for acetyl- choline [49], glycine [50], GABA [51], and glutamate [52], transmembrane transporters such as the Na-K ATPase pump [44] or a Na + /Ca 2+ exchanger [53]. These changes may lead to the reduction in the amplitude and the slowing down of the kinetics of action potentials, and to the reduction of axonal conduction ve- locity [54,55] (Fig. 2a ). ...
Pressure is a thermodynamic variable that, like temperature, affects the states of matter. High pressure is an environmental characteristic of the deep sea. Immersion to depth brings about an increase in pressure of 0.1 MPa (1 atm) for each 10 m of seawater. Humans exposed to high pressure, mostly professional divers, suffer effects that are proportional to their exposure.
The nervous system is one of the most sensitive targets of high pressure. The high pressure neurological syndrome (HPNS) begins to show signs at about 1.3 MPa (120 m) and its effects intensify at greater depths. HPNS starts with tremor at the distal extremities, nausea, or moderate psychomotor and cognitive disturbances. More severe consequences are proximal tremor, vomit, hyperreflexia, sleepiness, and psychomotor or cognitive compromise. Fasciculations and myoclonia may occur during severe HPNS. Extreme cases may show psychosis bouts, and focalized or generalized convulsive seizures. Electrophysiological studies during HPNS display an EEG characterized by reduction of high frequency activity (alpha and beta waves) and increased slow activity, modification of evoked potentials of various modalities (auditory, visual, somatosensory), reduced nerve conduction velocity and changes in latency. Studies using experimental animals have shown that these signs and symptoms are progressive and directly dependent on the pressure. HPNS features at neuronal and network levels are depression of synaptic transmission and paradoxical hyperexcitability.
HPNS is associated with exposure to high pressure and its related technological means. Experimental findings suggest etiological hypotheses, prevention and therapeutic approaches for this syndrome.
Relatively little is known about the effects of high pressure on fish. Mammals have attracted much more attention, serving as models for human divers. It is a curious fact that few marine biologists in general, and fish biologists in particular, have responded to the extraordinary range of interesting hyperbaric phenomena which fish present.
The biological effects of hydrostatic pressure are extremely varied and depend not only upon the rate and magnitude of the pressure change, but also upon the organism and tissue being studied and the environmental (acclimatory) and evolutionary history of the animal.
This chapter discusses the effects of pressure on shallow-water fishes. The measurements of the concentrations of various substances in organs and/ or tissues are generally performed on tissue samples taken from fishes that are first exposed to high pressure and then decompressed. It is observed that when isolated muscle is pressurized, an increase in maximum twitch tension and in the time taken to reach peak tension is noted. It is found that when isolated gill preparations are incubated in artificial seawater and then submitted to hydrostatic pressure, various changes occur in tissue Na⁺, K⁺, and Cl⁻ contents, depending on the experimental pressure. Na⁺ content varies much more rapidly than CI⁻ and K⁺. In shallow-water fishes exposed to the pressures of about 50–100 ATA, changes in ion contents are principally because of changes in permeability and not to changes in active transport. The effects are mainly an increase in Na⁺ content and a decrease in K⁺ content. The information provided in the chapter shows that a shallow-water fish is able to acclimatize to high pressure and that during this acclimatization physiological changes occur that makes the fishes less sensitive to further compression. The acclimatization of trout and goldfish for at least 21 days at 101 ATA has also been successful, provided that care was taken to compress the fish slowly.
The effects of hydrostatic pressure on branchial NaK-ATPase and Mg-ATPase activities of Scorpaena guttata (shallow-water fish) and Anoplopoma fimbria (deep-water fish) were used to calculate volume changes that occur during catalysis. The apparent activation volumes (ΔVs) of the NaK-ATPase, determined at test pressures between 68 and 340 atm, were positive and not significantly different in the two species. ΔV for the Mg-ATPase of S. guttata was also positive but significantly lower than the ΔV value for the NaK-ATPase. Negative values for ΔV were obtained for the Mg-ATPase of A. fimbria. Solubilization of the enzymes from gill microsomes of A. fimbria with Lubrol WX had no effect on ΔVs. NaI treatment of gill microsomes resulted in significant changes in ΔV for both enzymes in A. fimbria and in the Mg-ATPase of S. guttata. The effect of hydrostatic pressure on Kmfor ATP was used to estimate apparent ATP binding volume (ΔVbind.). The Km values obtained at 1 atm for the Mg-ATPase and NaK-ATPase of A. fimbria (0.030 and 0.043 mM, respectively) were approximately an order of magnitude lower than those of S. guttata(0.24 and 0.28 mM, respectively). High hydrostatic pressure (340 atm) produced an increase in Km in the NaK-ATPase of both species, indicating that ΔVbind. is positive. The Km of the Mg-ATPase in S. guttata was relatively insensitive to pressure (small positive value for ΔVbind.) where it decreased at high pressure in A. fimbria, yielding a large negative value for ΔVbind..
1.1. Freshwater crayfish lose Na when subjected to hydrostatic pressures of 16–101 ata.2.2. Net Na loss is due to pressure inhibition of unidirectional Na influx.3.3. Na efflux is independent of pressure within the range tested.4.4. The inhibition of active Na influx by pressure is reversible, but requires 1–2 hr for recovery.
Cellular membrane processes are extremely sensitive to environmental perturbations. Thus, membrane function plays a critical role in determining the temperature and pressure tolerance limits of fishes and other animals. Adaptive and acclimatory changes in cellular membrane lipids and enzymes have received considerable attention from environmental physiologists. One of the most intensively studied membrane proteins is the sodium pump, sodium-potassium adenosine triphosphatase (Na+–K+ ATPase). The direct effects of temperature and pressure on Na+–K+ ATPase activity are relatively large compared to many other enzymes, making it a clear candidate for studies of biochemical adaptation. The aim of this chapter is to describe the biochemical mechanisms used by fishes to maintain Na+-K+ ATPase function in differing physical environments, particularly the role of changes in the membrane lipid environment (homeoviscous adaptation). The activity of Na+-K+ ATPase is strongly dependent upon the surrounding lipids, and numerous studies have used this enzyme to address the functional consequences of homeoviscous adaptation. Studies of temperature effects have primarily focused upon acclimatory responses, whereas pressure studies have been concerned more with interspecific comparisons.
Phosphorylated ATPases may be involved in the effective pH regulation seen in the hydrothermal vent tubeworm Riftia pachyptila. R. pachyptila appears not only to have a large concentration of ATPases, but the main function of these ATPases seems to have shifted from
other types of transport, such as Na+ and K+ movement, to the facilitation of H+ elimination. Plume and trophosome ATPase activity for R. pachyptila measured 646.2 ± 29.5 and 481.4 ± 32.0 μmol Pi (inorganic phosphate) g−1 wet wt h−1, respectively. Plume tissue ATPase activity (both mass-specific and protein-specific) in R. pachyptila was higher (between 7% and 55%) than the activity measured in any tissue for 7 other shallow- and deep-living species, in
this study. This supports the hypothesis that R. pachyptila regulates acid/base balance via high concentrations of H+-ATPases, including Na+/H+ and K+/H+ exchangers and possibly electrogenic H+-ATPases, as evidenced by a higher total ATPase concentration (646 μmol Pi g−1 wet wt h−1), lesser Na+/K+-ATPase activity (13% of the total, as compared to 20−40% found in other animals), and higher H+-ATPase activity (226–264 μmol Pi g−1wet wt h−1). Overall, R. pachyptila appears to demonstrate elevated ATPase activity, with a greater fraction of the enzymes devoted to proton elimination, in
order to effectively control its extracellular pH in the face of processes acting to acidify the internal environment.
1.1. The study was set up to investigate the effects of long-term exposure (30 days) at 101 ATA of hydrostatic pressure (HP), in normoxic conditions and at constant temperature, on enzyme activities involved in energy production and ionic transfers.2.2. Measurements of activities of LDH, PFK, IDH and CPK in liver, red and white muscle seem to show that HP induces an improvement of aerobic pathway in red muscle and anaerobic pathway in white muscle; in liver no significant change is observed.3.3. At the same time, there appears an increase of plasma Na+, Cl− and Mg2+ contents; in muscle and gill, only Na+ and Cl− increase under pressure. Concomitantly with the changes in ion contents, there is a decrease in maximum activity of gill (Na+ + K+)ATPase and Mg2+ ATPase activity.4.4. The results agree with a Na+ balance impairment occurring at the tissue level at the same time when a new state of energetic metabolism results from adjustments of intertissue coupling of anaerobic and aerobic metabolisms induced by a long-term exposure to hydrostatic pressure.
Temperature preference behaviour of gammarid crustaceans from depths between 600–2000 m in Lake Baikal was studied in a system which provided a stable temperature gradient at pressures ranging from 50–150 atm. At the pressure of their habitat, these animals show well-defined modal distributions of sojourn temperatures around mean values from 3.0–5.5°C, av. 3.9 ± 0.3°C; mean modal Tp is estimated at 3.5°C. Year-round habitat temperatures are 3.0–3.6°C. The effect of changing pressure upon sojourn temperatures was explored over the range 50–150 atm. The slope of the mean sojourn temperature/pressure curves was 2.1°C/100 atm, significantly greater than 0. Mean nodal temperature estimates indicate that the corresponding slope in the range of 50–100 atm is 3°C/100 atm, and in the range of 100–150 atm, is likely to exceed 5°C/100 atm.
It is clear from the literature reviewed that modifications in membrane lipid composition play a major role in the adaptation of diverse organisms to specific environments and physiological circumstances. Acyl chain and molecular species restructuring in phospholipids are the most ubiquitous adaptations to environmental insult, being implicated in membrane adjustments to temperature, pressure, water activity, pH and salinity. In contrast, other adaptations (e.g. modulation of anionic phospholipids (salinity adaptation), trehalose content (dehydration) and the PC/PE ratio (temperature acclimation] appear to be more context specific. Although the volume of correlative data relating membrane composition to environmental state is impressive, several questions must be explicitly addressed in future research if a mechanistic understanding of the role of lipids in fine tuning membrane function is to be achieved. These include: (1) Adaptation thresholds--How much environmental variation is required before an acclimatory response is initiated, and is the extent of membrane perturbation induced by such minimally effective stimuli similar for different stress vectors? Interspecific comparisons of the Na+/K(+)-ATPase of fish collected at different depths indicate that species must be separated in depth by a distance corresponding to a pressure difference of 20 MPa before pressure adaptation is evident. Assuming a dT/dP value of 0.23 (Table 1), a 20 MPa change in pressure corresponds to ca. a 5 degrees C change in temperature, which agrees well with the minimal temperature change required to elicit changes in the lipid composition of plasma membranes in kidney tissue of thermally-acclimating trout. A pressure of 20 MPa also corresponds approximately to the maximum depth from which deep sea animals survive being brought to the surface. Collectively, these observations suggest that the minimally effective stimuli for both temperature and pressure adaptation are similar. Comparable data are not available for other environmental variables. (2) Signal transduction--What signals are being sensed and how are they transduced into an adaptational response? In some cases, it is clear that the enzymes of lipid metabolism respond directly (either by a variation in catalytic rate or substrate preference) to variations in the physical environment in an apparently adaptive manner (e.g. refer Sections VI.A.1 and VI.B.2). It seems unlikely, however, that such direct effects can explain the totality of the adaptive capacity of organisms, especially given the evidence for the induction of desaturase synthesis in cold adaptation (refer to Section VI.A.2).(ABSTRACT TRUNCATED AT 400 WORDS)
The effects of pressure and temperature on an integral membrane protein, Na+/K+-adenosine triphosphatase (Na+/K+-ATPase), were studied in fish gill membrane preparations from shallow- and deep-living marine teleosts. The inhibition by pressure of maximal velocity of the enzyme is nonlinear, increasing at higher pressures. Na+/K+-ATPases from deep-sea fish were less inhibited by pressure than those of shallow-living species. Habitat temperature also affected the pressure response of the enzyme. As a function of physiological pressure and temperature, the order of increasing pressure-sensitivity was cold, deep-sea less than warm, deep-sea (hydrothermal vents) less than polar = shallow and mid-depth, cold less than shallow, warm. Activation volumes in all species were conserved at 30-60 ml mol-1 at physiological pressures, which may reflect a similar membrane physical state at the actual pressure the animal experiences. Arrhenius plots [In(Na+/K+-ATPase activity) vs 1/T] were steeper for warm-water and shallow-living species than for deep-sea species. The depth at which adaptation was first observed was about 2000 m (approximately equal to 200 atm: 1 atm = 101.3 kPa). The data are consistent with a model of increased membrane fluidity resulting in reduced pressure-sensitivity of Na+/K+-ATPase from deep-sea species.
The binding properties of A1 adenosine receptors in brain membranes were compared in two congeneric marine teleost fishes which differ in their depths of distribution. Adenosine receptors were labeled using the A1 selective radioligand [3H]cyclohexyladenosine ([3H]CHA). The A1 receptor agonist [3H]CHA bound saturably, reversibly and with high affinity to brain membranes prepared fromSebastolobus altivelis andS. alascanus; however, the meanK
d values differed significantly (Figs. 1–3, Table 1). Saturation data fit to a one site model indicated that the A1 receptor inS. alascanus exhibited a higher affinity (K
d=1.49 nM) for [3H]CHA whereas A1 receptors inS. altivelis exhibited a significantly lower affinity (K
d=3.1 nM). Moreover,S. altivelis, but notS. alascanus, parameter estimates for [3H]CHA binding to two sites of receptor were obtained (Fig. 3, Table 1). The mean dissociation constant values for the high and low affinity sites for [3H]CHA inS. altivelis were 0.43 nM and 16.3 nM, respectively. In equilibrium competition experiments the adenosine analogs R-phenylisopropyladenosine (R-PIA), N-ethylcarboxamidoadenosine (NECA) and S-phenylisopropyladenosine (S-PIA) all displayed higher affinities for A1 receptors inS. alascanus as compared toS. altivelis brain membranes (Table 2, Fig. 6). The specific binding of [3H]CHA was significantly increased by 0.1 and 1.0 mM MgCl2 in both fishes; however, the sensitivity (95–131% increase) ofS. altivelis to this effect was significantly greater than that ofS. alascanus (48–91% increase) (Fig. 5). The results of kinetic, equilibrium saturation and equilibrium competition experiments all suggest that A1 adenosine receptors ofS. altivelis andS. alascanus brain membranes differ with respect to their affinities for selected adenosine agonists.
This study describes the preliminary isolation of substances from beef brain cortex which are required to produce an oxygen-induced enhancing effect on Na, K-ATPase. Evidence is presented that at least 3 fractions--a heat stable, low molecular weight proteinaceous substance, a cholesterol rich, membranous component, and an as yet unidentified substance--are required to produce oxygen enhancement of Na, K-ATPase activity. These findings have specific ramifications in neurocellular physiology, especially as related to seizures.
Exposure to high pressures (HP) has been associated with the development of the high pressure neurological syndrome (HPNS) in deep-divers and experimental animals. In contrast, many diving mammals are naturally able to withstand very high pressures. Although at a certain pressure range humans are also able to perform to some extent, the severe signs of HPNS at higher pressures motivated the research on the pathophysiology underlying this syndrome rather than on possible adaptive mechanisms. Thermodynamically, high pressure resembles cooling. Both conditions usually involve reduction in the entropy and slowing down of kinetic rates. We have observed in rat corticohippocampal brain slices that high pressure slows and reduces excitatory synaptic activity. However, this was associated with increased gain of the system, allowing the depressed inputs to elicit regular firing in their target cells. This increased gain was partially mediated by elevated excitability of their dendrites and reduction in the background inhibition. This compensation is efficient at low-medium frequencies. However, it induces abnormal spike reverberation at the high frequency band (> 50 Hz). Synaptic depression that requires less vesicles/transmitter turn over may serve as an energy-saving mechanism when enzymes and membrane pumps activity are slowed down at pressure. It is even more efficient if a similar reduction is induced in inhibitory synaptic activity. Unfortunately, the frequency response characteristics at this mode of operation may make the system vulnerable to external signals (noise, auditory, visual, etc) at frequencies that elicit 'resonance' responses. Therefore, it is expected that humans exposed to pressures above 1.5 MPa display lethargy and fatigue, certain reduction in cognitive and memory functions when the system is working in an 'economic' mode. The more serious signs of HPNS such as nausea, vomiting, severe tremor, disturbance of motor coordination, and seizures, may be the consequence of an interaction between the 'economic' mode of operation and resonance-inducing environmental disturbances.
Membranes have been isolated from fresh rectal glands of the dogfish shark, Squalus acanthias, in which the specific activity of the (sodium + potassium)-activated adenosine triphosphatase (NaK ATPase) is as high as
400 µmoles of Pi per mg of protein per hour. Membranes isolated from frozen rectal glands have only one-fourth to one-third this specific
activity. Solubilization of membranes from frozen glands with the nonionic detergent Lubrol WX activates the NaK ATPase 2-fold,
but there is no activation of the enzyme with membranes from fresh glands. Purification of Lubrol extracts by zonal centrifugation
and a novel ammonium sulfate fractionation gives an enzyme preparation with a specific activity of 1,500 µmoles of Pi per mg of protein per hour. The same specific activity and gel pattern is obtained with enzyme purified from membranes from
either fresh or frozen glands. The loss of Lubrol activation of the enzyme from frozen glands occurs on zonal centrifugation,
where free Lubrol is removed. The over-all yield of enzyme from the membrane stage is 70%. A mince of 10 rectal glands weighing
about 10 to 15 g fresh weight yields about 20 to 30 mg of purified enzyme. The enzyme is completely stable at 0° for 10 days
in either the membranous form or in the most highly purified form. It is stable on freezing at -70°. Disc gel electrophoresis
shows a gradual elimination of proprotein bands on purification. At the final stage of purification, scanning of Coomassie
blue-stained gels gives 72% of the protein as the 97,000 molecular weight catalytic subunit, 19% as the 55,000 molecular weight
glycoprotein, and 8.5% as the protein running with the tracking dye. The catalytic subunit and the glycoprotein can be isolated
in milligram quantities by solubilization and chromatography on Sephadex G-150. About 90% of the protein applied to the column
can be recovered. Sephadex chromatography gives a protein composition of 66% for the catalytic subunit, 28% for the glycoprotein,
and 5% for the protein running with the tracking dye. The catalytic subunit and the glycoprotein enrich more or less in parallel
as purification proceeds. At the last stage of purification, both proteins are found in the form of vesicles, which bear a
striking resemblance on negative staining to purified cytochrome oxidase. Electron-translucent rods and rings which are approximately
80 A in diameter are also formed with projections of about 35 to 55 A at regular intervals. These rods and rings resemble
reconstituted oligomycin-sensitive mitochondrial ATPase. The projections appear to be more hydrophilic, and this, coupled
with their size, suggests that they are the glycoprotein. Incubation of the enzyme with [γ-32P]ATP, magnesium, and sodium gives 4,080 pmoles of acyl phosphate per mg of protein, which is 2 to 3 times higher than the
highest levels of phosphorylation previously reported, thus attesting to the high purity of the enzyme preparation (the level
of phosphorylation is generally accepted as being proportional to the number of NaK ATPase molecules in the preparation).
The turnover number of the preparation is 6,300 min-1, which is within the range found for most NaK ATPase preparations from different organs and species. Arguments are presented
for and against the view that the glycoprotein is a subunit of the NaK ATPase. If the catalytic subunit of molecular weight
of 97,000 and the glycoprotein of molecular weight of 55,000 are both integral components of the NaK ATPase, the enzyme is
90 to 95% pure. If the 97,000 molecular weight subunit is the only subunit in the enzyme, the present preparation is 66 to
72% pure.
The effects of a wide range of hydrostatic pressures (from 50 to 1000 kg/cm2) have been investigated on the spontaneous potential difference (PD), the short-circuit current (SCC) and the activity of the membrane ATPases of the isolated abdominal skin from the frog Rana temporaria L. Two types of variations in PD are induced by pressure changes: short and transient potential variations which appear to be related to the pressure change (compression and decompression) and lasting variations which persist as long as pressure is applied and whose nature appears to be related to the pressure magnitude.
Long-lasting potential changes have particularly been investigated. At pressures lower than 500 kg/cm2, the skin potential increases while a pressure over 500–600 kg/cm2 induces a depolarization. Both variations consecutively occur at 500 + 100 kg/cm2. These effects of pressure have been shown to be reversible up to about 800 kg/cm2.
The question of the origin of the potential changes is discussed and it is proposed that the lasting hyperpolarization results from an effect on the passive permeabilities to Na+, K+ and Cl- ions inducing in turm a secondary readjustment (stimulation) of the Na+ active transport while the depolarization at high pressures reflects a direct inhibition of the Na+ pump.
These interpretations are supported by experimental data on the effects of pressure on the short-circuit current and on the activity of the skin (Na+ + K+) ATPase.
The teleostean gill is a multi-purpose organ, specialized for
respiratory gas exchanges, clearance of waste products of nitrogenous
metabolism and maintenance of acid-base and mineral balances. Structural
studies reveal a complex epithelium. The 'chloride-cells are almost
certainly the site of ion exchange in relation to salt balance.
Functional studies show that the gill is responsible for the net
absorption of Na^+ and Cl^- occurring in fresh water and extrusion of
these ions in sea water. In fresh water, a coupling between endogenous
NH^+_4 or H^+ and HCO^-_3 excretion and Na^+ and Cl^- absorption is
observed. In sea water active Na^+ excretion is linked with K^+
absorption from the external medium. In parallel, active Cl^- excretion
occurs. The gill is also the site of Na^+/Na^+ and Cl^-/Cl^- exchanges
which involve 25 to 75% of the internal NaCl per hour. The relative
importance of simple diffusion and exchange-diffusion in these exchanges
is assessed. Biochemical studies reveal two enzymes playing important
roles in the ionic pumps: carbonic anhydrase and Na-K activated ATPase.
Studies involving transfer of euryhaline fishes from low to high
salinity, show that the switch from fresh-water to seawater types of
gill function is far from instantaneous. Synthesis or destruction of
functional sites and renewal of specialized cells are involved. The role
of external or internal NaCl concentration changes as stimuli for these
'inductive processes' and the endocrine control of these functional
changes are briefly discussed.
SYNOPSIS. Muscle pyruvate kinase from an abyssal Coryphaenoides species occurs as a single electrophoretic form with an isoelectric point of about pH 6.0. Maximum catalytic rates are dramatically reduced by pressure. For catalysis at 3°C, the volume change of activation, ΔV*, is about 44 cm3/mole (calculated between 14.7 and 8000 psi). The value ot ΔV* decreases at higher temperatures but is pH independent. The activation energy for rattail muscle pyruvate kinase at 14.7 psi is about 13 Kcal/mole and doubles at 12,000 psi. Mg2+ saturation kinetics involve positive site-site interactions. Hill plots yield n values of about 2.4 and Ka values of about 2 mM (at 3°C), and these constants are pressure independent. The Km values for ADP increase slightly with pressure. PEP saturation curves are complex: at high PEP concentrations, the n values are about 2-2.5, while at low PEP levels, values for the Hill constant are about 1.0. The Hill constant lor PEP is not affected by pressure, but the apparent Km increases somewhat with pressure. FDP dramatically activates rattail muscle pyruvate kinase (500% activation with 0.1 mM FDP) by (1) reducing the KmPEP, (2) increasing the maximum velocity, and (3) overriding negative ATP modulation of the enzyme. The latter control feature is strictly dependent upon pressure and is not observed at low pressure. In the presence of FDP, the Km for PEP decreases at high pressures, in this way counteracting the inhibitory effects of pressure. Under low concentrations of substrates, pyruvate kinase activity is probably determined by its kinetic properties and not by energy-volume relationships.
SYNOPSIS. A free vehicle is a timed and weighted device released from a ship in a free fall to the ocean bottom. Instruments carried on the free vehicle have been built to take biological samples, sediment samples, water samples, and photographs, and to measure currents, tides, and temperature. The instrument then returns to the surface where it is recovered by the ship. A free vehicle system for biological sampling in the deep sea is described in detail. It consists of a mast assembly, flotation, hookline and traps, and a magnesium release attached to weights. Different types of magnesium links used include a rod, a wire on pliers, and a series of diamond-shaped beads that drop through a hole after dissolving. A deck plan for launching the free vehicle and its retrieval at sea are described.
Changes in the pH of buffer solutions under pressure have been monitored using the optical indicators 2,5-dinitrophenol and p-nitrophenol. Data were obtained to 6500 kg/cm2 for acetate, cacodylate, phosphate, and Tris buffers. The results give the pressure dependences of the ionization constants for the weak acid components of these buffers. These can be described by the equation RT In (Kp/Ko) = -ΔVa°P + 0.5Δk°P2 where ΔVa° is the limiting volume change on ionization at atmospheric pressure and Δk° reflects the pressure dependence of ΔVa. Respective values of ΔVa° (cc/mol) and Δk°; (cc/mol/ (103kg/cm2)) determined using acetic acid (-11.2, -1.44) as a reference are 2,5-dinitrophenol, -11.3, -1.28;p-nitrophenol, -11.3, -1.41; cacodylic acid, -13.2, -1.94; H2PO4-, -24, -3;TrisH+, +1, ∼0.
Hydrostatic pressure applied to isolated eel gills induces changes in the tissue, Na+, K+ and Cl– contents. It also inhibits the activity of the (Na++K+) ATPase. Results are discussed in terms of an effect of pressure on the Na+ and Cl– pumps and on the passive permeability processes.
1.1. Microsomal ATPase activities from gills of fresh water- and salt water-adapted rainbow trout (Salmo gairdneri) were examined at temperatures of 13 °C and 37 °C.2.2. The alkali metal-stimulated activity measured at 13 °C was enhanced by preincubating the reaction mixture, minus ATP, for 30 min at 37 °C. This procedure nearly eliminated “baseline” Mg2+-ATPase activity. The salt water “baseline” showed a greater temperature sensitivity than that of fresh water.3.3. The enzyme from salt water fish required both Na+ (100 mM) and K+ (20 mM) for maximal activation at 13 °C. The for Na+ was 7 mM and for K+, 0.8 mM. 5·10−4 M ouabain completely inhibited for alkali metal stimulation (). Na+ alone was ineffective in stimulating activity at 13 and 37 °C.4.4. The enzyme from fresh water fish required only Na+ (200 mM) for maximal stimulation at 13 °C, although a small amount of K+ stimulation was sometimes seen. The for Na+ was 25 mM. This activity was unaffected by 5·10−4 M ouabain; 5·10−3 M ouabain inhibited only about 30%. At 37 °C, K+, in addition to Na+, was required for maximal activity. This K+ stimulation was inhibited by 5·10−4 M ouabain while the Na+ stimulation remained relatively insensitive to the inhibitor.5.5. The fresh water enzyme required 2.5 mM Mg2+ () for optimal activity at 13 °C and the salt water enzyme required .6.6. The fresh water enzyme showed maximal activity over wide ranges of pH (6.6–8.0) whereas the salt water enzyme showed a distinct optimum at pH 7.1.7.7. The alkali metal-activated ATPase activity was greater in the gills of salt water fish than those adapted to fresh water.
1.1. The activities of Na+K+ ATPase in the gills of stenohaline teleosts were much higher in marine species than in fresh-water species.2.2. In euryhaline species (eel, trout and goby) the enzyme activities of the gills were also higher in fish adapted to sea water than in fish adapted to fresh water.
1.1. A comparison of gill Na-K-ATPases isolated from the surface dwelling sea water adapted coho salmon, Oncorhynchus kisutch, and a benthic marine teleost, Antimora rostrata, was initiated to study the effects of hydrostatic pressure on gill ion flux.2.2. Specific activities of the two enzymes are similar, as are the effects of high hydrostatic pressure which results in large + ΔV‡ values (enzyme inhibition). However, at physiological pressures, activation is seen in the Antimora enzyme. This observation, plus ouabain sensitivity and enzyme stability, suggest these two enzymes have distinctive catalytic properties.3.3. Due to an inability to prepare a stable Na-K-ATPase preparation from Antimora gills, affinity parameters could not be determined. However, the significant alteration in these parameters with high pressure, especially the Km(Na+), seen for the coho enzyme suggest that substantial changes in Na+ efflux could occur which would be detrimental in an abyssal habitat.4.4. It has been postulated that Antimora rostrata gills lack chloride cells, and that an alternative extrarenal salt secretory mechanism may be utilized. This may account for the results reported in this paper.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
Membranes and Ion Transport
- S. L. Bonting
- Péqueux