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Intracellular protein breakdown in growing cells of Escherichia coli
Abstract
1. When Escherichia coli was grown exponentially in a defined medium at 35 degrees , the rate of protein breakdown was initially rapid, but decreased to 0.6%/hr. after about 30min. The latter rate was maintained for at least 3.5hr. 2. The initial rapid rate may have been due to the presence of a small protein fraction (about 1%) that was degraded with a half-life of 13min. 3. The rate of protein degradation was the same during balanced growth at low rates imposed in a bactogen. However, it increased during the period immediately after a decrease of the growth rate.
... Protein turnover has been studied extensively in a variety of organisms (for reviews, see Goldberg & St John, 1976). In bacteria, the degradation of total cell protein is very low in growing cells, 0.67; h-l (Willetts, 1967), but may increase to 4 to 576 h-l in nutrient-starved cells (Mandelstam, 1958) and to 8 to 107; h-l in sporulating cells (Mandelstam & Waites, 1968). The enhanced rate of protein breakdown apparently results from the activity of proteolytic enzymes synthesized during starvation (Doi, 1972)' A similar system seems to exist in cyanobacteria. ...
... The enhanced rate of protein breakdown apparently results from the activity of proteolytic enzymes synthesized during starvation (Doi, 1972)' A similar system seems to exist in cyanobacteria. Lau et al. (1977) have reported increased degradation of phycocyanin, which may function as a storage protein, in nitratestarved Anacystis nidulans, while proteases which hydrolyse phycocyanin have recently been described in species of Anabaena (Wood & Haselkorn, 1976;Foulds & Carr, 1977). ...
Nitrogen starvation and heterocyst development were induced in the cyanobacterium Anabaena 7120 by growth in nitrogen-free medium or by treatment with the amino acid analogue methionine sulphoximine. During the first 6 h of nitrogen deprivation, amino acid levels and rates of protein synthesis, as measured by the incorporation of [3H]leucine, decreased to 50 to 70% of those in ammonia-grown organisms; after this time there was no difference between the rates of protein synthesis in ammonia-grown and nitrogen-starved cultures. The period 4 to 12 h after the onset of starvation was marked by the release of [3H]leucine from previously labelled proteins at a rate 6 to 7·5 times that of ammonia-grown organisms. These results are consistent with the idea that nitrogen starvation in cyanobacteria causes a reduction in protein synthesis and leads to the rapid degradation of storage proteins. In rapidly growing Anabaena 7120, the doubling time for total cell protein was estimated to be 14·9 ± 1·0 h and the half-life was 139 ± 88 h.
... Although unrecognized as such, evidence in the lterature for the existence of a limited labile class of protein similar to rapidly degrading protein that represents 2 to 8% of the total cellular protein in various strains of E. colz is overwhelming (3,13,(17)(18)(19)(20)(21). Thus, after a IO-min incorporation of [i4C]leucine, 7% of the total radioactivity was released by growing cells of strain 113-3, with nearly half of this radioactivity appearing by 60 min (17). ...
... Thus, when strains K-12 and ML were grown in radioactive leucine or valine (amino acids of the same family) and then degradation was measured in the presence of carrier amino acid it was observed that about 3 to 4% of the cellular protein radioactivity was released within 2 to 4 hours and no more thereafter (13,21). This value should correspond to a value of 8% if correction for inhibition of amino acid exchange is applied. ...
Based on leucine exchange measurements of cells labeled with [14C]leucine, we had previously reported (J. Biol. Chem., 245, 2889 (1970)) that only a limited class of protein of Escherichia coli B is subject to a first order process of rapid degradation. The rate of degradation (half-life of 60 min or faster) and the
total amount of protein undergoing degradation (2 to 7%) was the same during growth and during various kinds of starvation.
We extend this finding and report here that the release of this "rapidly degrading protein" is unaltered not only during growth
and starvation but also during growth in an enriched medium (step-up), in a poor medium (step-down), and during the diauxic
phase of induced β-galactosidase synthesis.
The extent of this observed degradation (less than 10%) is lower than reported by other workers (20 to 35%). This discrepancy
is not a consequence of experimental manipulation or cellular damage but reflects differences in bacterial strains. Although
strains ML and K-12 under conditions of growth release radioactivity similar in amount and half-life to the rapid protein
degradation process observed in strain B, during several conditions of starvation, degradation of an additional class of cellular
protein can be measured by leucine exchange in these two strains but not in B. The starvation-induced protein degradation
occurs at a rate of 2.5 to 6% per hour, and the amount of the cellular protein that degrades as a result of starvation amounts
to 20 to 40% of the total bacterial protein. Simultaneous with this starvation-induced protein degradation is the excretion
of nucleic acid degradation products into the medium amounting to 40 to 60% of the ordinarily stable nucleic acid of growing
cells. Starvation-induced nucleic acid degradation occurs in all strains equally. With the findings of these strain differences,
much of the conflict in the literature can be explained satisfactorily.
We conclude that a continuous process of protein degradation occurs in bacterial cells at all times, and an additional process
of protein degradation concomitant with nucleic acid degradation is initiated following starvation of nutrients. Some of the
properties of the degradation processes under starvation conditions are as follows. While almost all of the protein degradation
products released in the presence of carrier leucine are acid soluble, a large portion of nucleic acid degradation products
released from the cells are precipitable by cold trichloroacetic acid. Both protein and nucleic acid degradation occur under
starvation of either glucose, nitrogen, or phosphate. Inhibition of protein synthesis by chloramphenicol at 100 µg per ml
inhibits the starvation-induced protein degradation, but does not affect the degradation of the rapidly degrading protein.
... However, there are conflicting claims in the literature that the proteins in growing cells do turn over appreciably (6)(7)(8)(9). ...
... We have previously derived the correction factor for this case (Reference 25, Equation 31 or 33). It can be rewritten in the present notation as: (6) Where X is the bacterial growth rate constant, a' is the apparent turnover rate constant, and t; is the time for the uptake of isotope. ...
The rate of degradation of intracellular radioactive protein of bacterial cells supported by a membrane filter has been measured
directly from the appearance of radioactivity in the carrier-containing perfusate. For this purpose a perfusion apparatus
has been constructed from a microsyringe filter holder. The conditions inside the apparatus are physiological and permit exponential
growth up to 1 x 108 cells. Exchange of extracellular carrier leucine-12C with the intracellular leucine-14C pool is so rapid that at least 87% of the amino acid arising from protein breakdown appears in the perfusate and is not
recycled into protein even in growing cultures.
Only a limited portion of the cellular protein is subject to rapid degradation. It decays with a half-life of approximately
1 hour and constitutes 2 to 7% of the total cellular protein of cells growing in glucose minimal medium under various nutritional
conditions (including growth and starvation). The proportion of the total protein synthesis which is directed to the synthesis
of this rapidly degrading protein component increases with decreasing growth rate. At very slow growth rates 10 to 40% of
the radioactivity that is incorporated into protein is incorporated into the rapidly degrading protein. Treatment which removes
nonproteinaceous material and Pronase digestion of cells before and after the decay of the rapid component substantiates the
conclusion that the rapidly decaying component is derived from bacterial protein.
There is another radioactive component in the perfusate which is released at the very slow rate of 0.2 to 0.6% per hour for
as long as the experiments are carried out (48 hours). This does not represent degradation of intracellular protein to amino
acids since this process is independent of leucine exchange, and 30 to 50% of the radioactivity in the perfusate is acid-insoluble.
... However, there are conflicting claims in the literature that the proteins in growing cells do turn over appreciably (6)(7)(8)(9). ...
... We have previously derived the correction factor for this case (Reference 25, Equation 31 or 33). It can be rewritten in the present notation as: (6) Where X is the bacterial growth rate constant, a' is the apparent turnover rate constant, and t; is the time for the uptake of isotope. ...
... Although unrecognized as such, evidence in the lterature for the existence of a limited labile class of protein similar to rapidly degrading protein that represents 2 to 8% of the total cellular protein in various strains of E. colz is overwhelming (3,13,(17)(18)(19)(20)(21). Thus, after a IO-min incorporation of [i4C]leucine, 7% of the total radioactivity was released by growing cells of strain 113-3, with nearly half of this radioactivity appearing by 60 min (17). ...
... Thus, when strains K-12 and ML were grown in radioactive leucine or valine (amino acids of the same family) and then degradation was measured in the presence of carrier amino acid it was observed that about 3 to 4% of the cellular protein radioactivity was released within 2 to 4 hours and no more thereafter (13,21). This value should correspond to a value of 8% if correction for inhibition of amino acid exchange is applied. ...
... Low level metabolites can be mainly associated with acetylated amino acids and proteolytic breakdown products. Degradation of proteins can be interpreted as a means to increase the availability of amino acids required for the synthesis of new proteins (Willetts, 1967). This observation can be linked with reduced growth of animals related to cluster B and goes in line with observed AFDW loss in I-to-C animals in the second part of the experiment. ...
The current knowledge on bioaccumulation of emerging contaminants (ECs) in aquatic invertebrates exposed to the realistic environmental concentrations is limited. Even less is known about the effects of chemical pollution exposure on the metabolome of aquatic invertebrates. We conducted an in situ translocation experiment with passive filter-feeding caddisfly larvae (Hydropsyche sp.) in an effluent-influenced river in order to i) unravel the bioaccumulation (and recovery) dynamics of ECs in aquatic invertebrates, and ii) test whether exposure to environmentally realistic concentrations of ECs will translate into metabolic profile changes in the insects. The experiment was carried out at two sites, upstream and downstream of the discharge of an urban wastewater treatment plant effluent. The translocated animals were collected at 2-week intervals for 46 days. Both pharmaceuticals and endocrine disrupting compounds (EDCs) were detected in water (62 and 7 compounds, respectively), whereas in Hydropsyche tissues 5 EDCs accumulated. Overall, specimens from the upstream site translocated to the impacted site reached higher ECs concentrations in their tissues, as a reflection of the contaminants' water concentrations. However, bioaccumulation was a temporary process susceptible to change under lower contaminant concentrations. Non-targeted metabolite profiling detected fine metabolic changes in translocated Hydropsyche larvae. Both translocations equally induced stress, but it was higher in animals translocated to the impacted site.
... Until recently, intracellular protein degradation in micro-organisms has been mainly studied in 'standard' bacteria such as Escherichia coli or Bacillus megaterium. Under optimum growth conditions, the overall rates of protein breakdown within these cells are low (Calandruccio & Larrabee, 1981;Chaloupka & Strnadova, 1982;Willets, 1967), and most of the proteins synthesized during exponential growth are stable (Goldberg & St John, 1976;Larrabee et al., 1980;Pine, 1972). However, changes in growth conditions can cause a rapid and selective degradation of certain enzymes (Rivett et a/., 1985). ...
Proteolysis of endogenous and exogenous substrates in cell-free extracts of the psychrotrophic bacterium Arthrobacter sp. S155 has been compared. Endogenous proteins were degraded only after treatment with cyanogen bromide. The hydrolysis of exogenous proteins of high Mr, (i.e. casein) was optimum at alkaline pH and was stimulated by Ca2+, Mg2+, Mn2+ and ATP. The serine protease inhibitor phenylmethylsulphonyl fluoride had no effect on ATP stimulation. Small peptides (i.e. insulin) were degraded at very high rates. This activity was optimum at slightly acidic pH and was stimulated by Ca2+, strongly inhibited by Mn2+, but not affected by ATP. Degradation of cyanogen bromide-treated cellular proteins displayed two pH optima which corresponded to the optimum pH for the degradation of insulin and casein. The characteristics of these acidic and alkaline activities were identical to those active against insulin and casein respectively. The proteases which degraded casein were much more heat resistant than those which degraded insulin.
... The hypothesis that these proteins, synthesized at the onset of starvation, play a role in longterm survival is supported by the observation that the addition of chloramphenicol or amino acid analogs at zero time for starvation had a delayed effect on culture viability. Willetts (20) demonstrated the presence of a pool of labile proteins in logarithmically growing E. coli, and it is possible that some of the protein synthesis occurring at the onset of starvation serves to replenish these rapidly degraded proteins. However, this resynthesis of previously existing proteins would not explain the relatively greater importance to survival of protein synthesis during the initial hours of starvation; resynthesis of labile proteins would be expected to occur at the same rate throughout starvation. ...
tocarbon-starved cultures toinduce synthesis ofabnormal proteins hadaneffect on viability similar tothat observed when50,ugofchloramphenicol permlwasaddedatzerotimeforstarvation. Bothchloramphenicol andtheaminoacidanalogs haddelayed effects onviability, compared withtheir effects onsynthesis ofnormal proteins. Theneedforprotein synthesis didnotarise fromcryptic growth, since no cryptic growth ofthestarving cells wasobserved undertheconditions used. Fromthese andprevious results obtained fromworkwithpeptidase-deficient mutants ofE.coli K-12andSalmonella typhimurium LT2(Reeve etal., J.Bacteriol. 157:758-763, 1984), we concluded thata numberofsurvival-related proteins are synthesized byE.coli K-12cells asaresponse tocarbon starvation. Theseproteins arelargely synthesized during theearly hours ofstarvation, buttheir continued activity isrequired forlong-term survival. Starving bacteria areofinterest inbothanecological and anapplied context, andwehavebeeninterested incharac- terizing thephysiological changes that occurduring starva- tion (12, 16,22). Wehavepreviously presented evidence (16) thatprotein degradation playsa roleinthesurvival of carbon-starved bacteria. Mutants ofEscherichia coliand Salmonella typhimurium that weredeficient inprotein deg- radation werefoundtopossess agreatly decreased stability underconditions ofcarbon starvation. Although these mu- tantshadnoinnate deficiency intheir protein-synthetic machinery, theywereunable tosynthesize protein atthe samerateastheir corresponding wild-type strains during carbon starvation. Thissuggested that aminoacids derived fromprotein degradation wereutilized bythese cells fornew protein synthesis andthattheir increased susceptibility to carbon starvation originated fromtheir inability tosynthe- size these proteins. InE.coli cells subjected tocarbon starvation, therate of protein synthesis drops toabout 20%oftheinitial rate during thefirst hourofstarvation (16)andthenremains roughly constant foratleast thenext47h(unpublished data). We present evidence herethat this protein synthesis isimportant forthesurvival ofcarbon-starved E.coli K-12; inhibition of normal protein synthesis during starvation greatly compro- misedsurvival.
... The size of the labile fraction increases in slowly growing cells [13,21] although the bulk of the proteins synthesised during growth are not subject to degradation even after prolonged starvation [22]. The rate of protein turnover is increased by amino acid [23], glucose [24] or inorganic nutrient starvation [25] or on entering stationary phase [26]. The magic spot nucleotide ppGpp has been implicated in the regulation of protein degradation [14], and the rate of intracellular proteolysis has been correlated with the cellutar level of ppGpp [27]. ...
Vibrio strain 14 supports phage alpha 3a growth in standing stationary phase cells but not in shaking (aerated) stationary phase cells. In exponential cells, protein was turned over at 1.8% h-1, and the rate was increased by starvation or inhibition of protein synthesis. In shaking stationary phase cells the rate of protein turnover was low (1.0% h-1) for proteins synthesised during growth but high (20% h-1) for recently synthesised proteins. In contrast recently synthesised proteins in standing stationary phase cells were stable over 60 min and proteins synthesised during growth were turned over at 2.9% h-1. ppGpp and pppGpp were detected in exponential cells, but were not detected in stationary phase cells.
Towards the end of the 19th century, plant physiologists considered the possibility that proteins undergo continuous breakdown and resynthesis. Despite the objections of Pfeffer, the idea of protein turnover was revived in the 1920’s and a great deal of work on the nitrogen balance of detached leaves was interpreted in terms of the temporal separation of degradation and synthesis. Mothes (1933) however, proposed that in leaves there was simultaneous synthesis and degradation of protein involving separate systems for synthesis and degradation. Paech (1935), on the other hand, argued that control of the nitrogen balance was exercized through mass action. Gregory and Sen (1931) noted that in leaves of potassium-starved barley there was an accumulation of amino acids, which, according to the mass action proposals of Paech, should have led to enhanced protein synthesis. However, the reverse happened and the continued accumulation of amino nitrogen suggested to them that the routes of protein synthesis and degradation were separable and that the rates of these processes might vary independently. They proposed a “PROTEIN CYCLE” according to which proteins undergo a cycle of synthesis and degradation (i.e., protein turnover), but also included the proposition that protein degradation is linked to the production of CO2. The extent to which protein turnover contributes to respiration is discussed in Section 3, but it is not central to the concept of turnover itself. The first direct evidence for protein turnover in plants was provided independently by Hevesey et al. (1940) and Vickery et al. (1940), who reported the assimilation of [15N]H3 into leaf protein, despite a net loss of protein from leaves.
This chapter discusses the roles of synthesis and degradation in regulation of enzyme levels in mammalian tissues. Information concerning the regulation of enzyme concentrations or levels has been obtained largely from studies with microbial systems, most specifically with Escherichia coli. The simplified organization uniform cell population and ready availability of mutants have allowed for a rapid advancement of knowledge of mechanisms involved in such regulation. An increasing wealth of evidence indicates that in mammals, marked changes in enzyme levels occur in response to a variety of physiologic, hormonal, nutritional, and pharmacologie stimuli. The nature of mechanisms responsible for the regulation of enzyme levels in mammalian tissues are discussed in the chapter.
Measurements of the metabolic stability of ribosomal proteins of exponentially growing Escherichia coli B/r showed that their rate of degradation to free amino acids is zero to within limits of the experimental accuracy of 0.7% per hour or 0.5% per doubling period.
We found that cycling of cystine-14C through solutions of soil samples at steady-state was influenced by two kinetically-defined biontic components. We report work aimed at further characterizing these two components. Surface horizons from a cultivated Gray Luvisol and a Dark Brown Chemozemic soil were sampled. They were incubated with cystine at 60 ng g−1 to maintain steady-state conditions and with glucose at 1000 μg g−1 to perturb the system. Soil solution 14C and 14CO2 were monitored. The data were analyzed for their fit to several alternate models. The kinetic model which described 14C cycling through the solution of perturbed soil samples is consistent with C allocation into a pool of small intermediate molecules of biosynthesis and into macromolecules such as proteins and RNA. The protein component did not degrade measurably during the period of study. A second kinetic model described non-perturbed soil conditions in which 14C-cystine was partitioned between microbial cytoplasmic and protein components. Both models are consistent with pure culture studies. Neither model can be considered to distinguish between microbial taxa. We have inferred that taxonomic controls do not determine short-term 14C kinetics and that a physiologic basis for the kinetic distinctions may be most reasonable. This appears to hold both under conditions which favor growth (i.e. excess glucose) and which preserve steady-state (small cystine additions) in soils incubated under laboratory conditions.
The modes of action of insulin and of inhibitors of protein synthesis on the degradation of labeled cellular proteins have been studied in cultured hepatoma (HTC) cells. Protein breakdown is accelerated upon the deprivation of serum (normally present in the culture medium), and this enhancement is inhibited by either insulin or cycloheximide. An exception is a limited class of rapidly turning over cellular proteins, the degradation of which is not influenced by insulin or cycloheximide. Alternative hypotheses to explain the relationship of protein synthesis to the regulation of protein breakdown, viz., control by the levels of precursors of protein synthesis, regulation by the state of the ribosome cycle, or requirement for a product of protein synthesis, have been examined. Protein breakdown was not influenced by amino acid deprivation, and measurements of valyl-tRNA levels in HTC cells subjected to various experimental conditions showed no correlation between the levels of charged tRNAVal and the rates of protein degradation. Three different inhibitors of protein synthesis (puromycin, pactamycin, and cycloheximide) suppressed enhanced protein breakdown in a similar fashion. A direct relationship was found between the respective potencies of these drugs to inhibit protein synthesis and to block enhanced protein breakdown. When cycloheximide and insulin were added following a prior incubation of HTC cells in a serum-free medium, protein breakdown was maximally suppressed within 15-30 min. Actinomycin D inhibited protein breakdown only after a time lag of about 90 min. It is suggested that the regulation of protein breakdown in hepatoma cells requires the continuous formation of a product of protein synthesis, in a manner analogous to the mode of the control of this process in bacteria.
Nitrogenous metabolites, cyclic adenosine 3':5'-monophosphate (cAMP), and the stage of culture growth all influence the levels of glutaminase A in Escherichia coli, but no variables in culture conditions alter the levels of glutaminase B. Growth of E. coli on culture media containing glucose and excess ammonia results in a rise in the level of glutaminase A as the cultures enter stationary phase; this rise is abolished by ammonia limitation. cAMP or glycerol reduce the level of glutaminase A. In mutants deficient in cAMP receptor protein, glutaminase A levels are unchanged by cAMP, but they are still susceptible to regulation by ammonia. We consider glutaminase B to be a constitutive enzyme, since its levels appear independent of nutritional conditions.
We have previously demonstrated the existence of two types of endopeptidase in Escherichia coli. A purification procedure is described for one of these, designated protease II. It has been purified about 13,500-fold with a recovery of 24%. The isolated enzyme appears homogeneous by electrophoresis and gel filtration. Its molecular weight is estimated by three different methods to be about 58,000. Its optimal pH is around 8. Protease II activity is unaffected by chelating agents and sulfhydryl reagents. Amidase and proteolytic activities are stimulated by calcium ion, which decreases the enzyme stability. Like pancreatic trypsin, this endopeptidase catalyses the hydrolysis of alpha-amino-substituted lysine and arginine esters. It appears distinct from the previously isolated protease I, which is a chymotrypsin-like enzyme. The apparent Michaelis constant for hydrolysis of N-benzoyl-L-arginine ethyl ester is 4.7 X 10(-4) M. The esterase activity is inhibited by diisopryopylphosphorofluoridate (Ki(app) equals 2.7 X 10(-3) M) and tosyl lysine chloromethyl ketone (Ki(app) equals 1.8 X 10(-5) M), indicating that serine and histidine residues may be present in the active site. However, protease II is insensitive to phenylmethanesulfonyl fluoride and several natural trypsin inhibitors. Its amidase and esterase activities are competitively inhibited by free arginine and aromatic amidines. The proteolytic activity measured on axocasein is very low. In contrast to trypsin, protease II is without effect on native beta-galactosidase. It easily degrades aspartokinase I and III. Nevertheless both enzymes are resistant to proteolysis in the presence of their respective allosteric effectors. These results provide further evidence that such differences in protease susceptibility can be related to the conformational state of the substrate. The possible implication of structural changes in the mechanism of preferential proteolysis in vivo, is discussed.
Growth of Streptococcus faecalis in complex media with various fuel sources appeared to be limited by the rate of supply of adenosine-5' -triphosphate (ATP) at 1 atm and also under 408 atm of hydrostatic pressure. Growth under pressure was energetically inefficient, as indicated by an average cell yield for exponentially growing cultures of only 10.7 g (dry weight) per mol of ATP produced compared with a 1-atm value of 15.6. Use of ATP for pressure-volume work or for turnover of protein, peptidoglycan, or stable ribonucleic acid (RNA) did not appear to be significant causes of growth inefficiency under pressure. In addition, there did not seem to be an increased ATP requirement for ion uptake because cells growing at 408 atm had significantly lower internal K(+) levels than did those growing at 1 atm. Pressure did stimulate the membrane adenosine triphosphatase (ATPase) or S. faecalis at ATP concentrations greater than 0.5 mM. Intracellular ATP levels were found to vary during the culture cycle from about 2.5 mumol/ml of cytoplasmic water for lag-phase or stationary-phase cells to maxima for exponentially growing cells of about 7.5 mumol/ml at 1 atm and 5.5 mumol/ml at 408 atm. N,N'-dicyclohexylcarbodiimide at a 10 muM concentration improved growth efficiency under pressure, as did Mg(2+) or Ca(2+) ions at 50 mM concentration. These agents also enhanced ATP pooling, and it seemed that at least part of the growth inefficiency under pressure was due to increased ATPase activity. In all, it appeared that S. faecalis growing under pressure has somewhat reduced ATP supply but significantly increased demand and that the inhibitory effects of pressure can be interpreted largely in terms of ATP supply and demand.
This chapter focuses on the current assessment of peptide utilization by microorganisms from the standpoints of cleavage and transport. It also lays emphasis on the effects of peptides on other aspects of microbial physiology. It is clear that prior to their nutritional utilization peptides must be hydrolyzed to yield free amino acids, and that this cleavage may occur before or after absorption. Variations in these two essential features of cleavage and transport produce circumstances in which particular peptides may give a growth response that is greater than, equal to, or less than an equivalent mixture. Simple peptides offer a vast reservoir of biologically active compounds and techniques exist for the synthesis of any desired sequence. It is hoped that studies on the structural requirements of microbial peptide permeases and peptidases, and of the effects of peptides on microbial growth, may be useful not only in their immediate context, but they may also act as a model system to provide information relevant to peptide interactions in more complex biological systems of free amino acids.
The synthesis and degradation of the soluble and sodium dodecyl sulfate-(SDS)-solubilized protein fractions of Escherichia coli were studied in both growing and nongrowing cultures. When separated according to molecular weight on SDS-polyacrylamide gels, the proteins of both fractions of growing cells undergo no measureable differential synthesis or degradation during logarithmic growth. However, when a leucine auxotroph is suspended in medium containing 5.3 muM leucine (a level that will not sustain growth), the SDS-solubilized protein of such a nongrowing culture shows a rapid synthesis of two protein components (32,000 and 12,000 daltons) found only in the out membrane.
One of the main functions of intracellular proteinases lies in their participation in protein turnover by the hydrolytic degradation of proteins. The rates of synthesis and degradation differ for the various enzymes and groups of enzymes. Moreover, they are influenced by many variables, such as nutritional conditions, growth phase, processes of differentiation. Protein synthesis is regulated at the level of transcription and translation by positive and negative control. The degradation of proteins is generally assumed to occur through the combined action of proteinases and peptidases, yielding amino acids after complete hydrolysis. On the other hand, it is possible that “limited proteolysis” leads to an accumulation of distinct macromolecular products that are either inactive or different in their catalytic properties from their uncleaved precursors. Microbial proteinases are commonly divided into intracellular and extracellular enzymes. Extracellular enzymes usually occur in the active state in the growth medium and are quite stable. Many of them are even available in large quantities allowing their application to medicine and industry.
Methods are described for measuring soluble pool magnitudes in steady-state or exponentially growing cultures, and for distinguishing between anabolic, catabolic and total metabolic pools within cells. These methods were applied to the measurement of pool magnitudes for several amino acids and other precursors in Escherichia coli THU. Our results support the independence of the magnitudes of total metabolic pools and growth rate in steady-state cultures. Our results also show that the total metabolic pool size is much larger than previously published estimates, which failed to include the contribution of catabolic pools. The average value of the total soluble material in exponential-phase cells is estimated to be 8 to 9% of the cell dry mass; pool values could be almost twice as large during midcycle because of the known increase in the magnitudes of protein and RNA precursor pool during the cell cycle.
Immunological cross-reaction was employed for identification of proteolytic fragments of E. coli RNA polymerase genered both in vitro and in vivo. Several species of partially denatured but assembled RNA polymerase were isolated, which were composed of fragments of the two large subunits, β and β′, and the two small and intact subunits, α and σ. Comparison of the rate and pathway of proteolytic cleavage in vitro of unassembled subunits, subassemblies, and intact enzymes indicated that the susceptibility of RNA polymerase subunits to proteolytic degradation was dependent on the assembly state.
Using this method, degradation in vivo was found for some, but not all, of the amber fragments of β subunit in merodiploid cells carrying both wild-type and mutant rpoB genes. Although the RNA polymerase is a metabolically stable component in exponentially growing cells of E. coli, degradation of the full-sized subunits was found in two cases, i.e., several temperature-sensitive E. coli mutants with a defect in the assembly of RNA polymerase and the stationary-phase cells of a wild-type E. coli. The in vivo degradation of RNA polymerase was indicated to be initiated by alteration of the enzyme structure.
1. The rate of protein breakdown was determined on growing and non-growing cultures of thermophilic and mesophilic fungi. 2. In growing cells protein breakdown was negligible. 3. In non-growing cells the breakdown rate of total protein varied between 5.2%/h and 6.7%/h. These values were found to be dependent on both the temperature of the protein breakdown assay and the temperature of growth of the organism. 4. The rate of breakdown of soluble protein in thermophilic fungi was 9-15%/h whereas the rate in mesophilic fungi for the soluble protein fraction was only 4%/h.
This article is in the nature of a review with theoretical comments on protein turnover in cells, concentrating on possible modes of control of synthesis and degradation rates. While our knowledge of protein synthesis is well-developed, that of protein degradation is rudimentary. Thus, the review covers data on protein degradation with regards the influence of substrate, of the environment in which the protein finds itself, of the size and amino acid composition of protein, and on the proteolytic enzymes involved. It is stressed that a linkage must exist between synthesis and degradation, to explain the steady-state concentration of specific proteins in cells, and it explores ideas as to the nature of this linkage. Possible explanations are given of the apparent necessity of having proteins being constantly degraded; these include the need for a basic mechanism which can be easily modulated in order that concentrations of specific proteins may change, the need for the correction of possible errors arising from the transcription and translation processes, and the apparent need to make the most efficient usage, in terms of energy input, of the information machinery of the cell. Finally, ideas are suggested of the possible relevance of protein turnover to the informational molecules and complexes in the cell.
In E. Coli ML 304 G, 90 p. cent of the proteolytic activity determined by hydrolysis of (125I) casein is located as endocellular enzymes. Most of these endocellular proteases are soluble and are separated into several molecular species on a DEAE-cellulose column. A small fraction of the endocellular proteolytic enzyme is associated with ribosomes, though only 50 p. cent remains bound to ribosomes after washing with 1,5 M NH4Cl. The proteases eluted with high salt are mainly basic. The exocellular proteases, which comprise 10 p. cent of the cellular proteases and are released into the medium under osmotic shock in the presence of Mg++, are basic proteins and can be separated into two peaks of activity by DEAE-cellulose chromatography. Under various conditions, e.g. osmotic shock in the presence or in the absence of Mg++, spheroplast formation, or disintegration of whole cells by sonication or by alumina grinding, about 15 p. cent of the total proteolytic activity is found to be associated with membrane fragments. When cells are ground with alumina, the extract contains much less proteolytic activity than that found in a sonic extract. The difference is due neiher to higher release of proteases by sonication nor to absorption of the enzymes onto alumina. The grinding procedure is probably a suitable method for the preparation of enzymes and ribosomes with low proteolytic activity.
Individual nitrogenous metabolites have been examined as regulating agents for the breakdown of intracellular proteins in Escherichia coli. Generally, NH(4) (+) is the most effective regulator. Its depletion progressively increases the basal proteolytic rate to maximum in most strains when the doubling time is increased to 2 h. In E. coli 9723, the rate is further increased at longer doubling times. Amino acids have individual effects on intracellular proteolysis. The basal rate in amino acid-requiring auxotrophs of E. coli 9723 is stimulated weakly by starvation for histidine, tryptophan, or tyrosine, moderately by four other amino acid depletions, and more strongly by eight others. The degree of stimulation roughly correlates with the frequency of the amino acid in the cell proteins. Amino acid analogues that incorporate extensively into protein generally slightly inhibit intracellular proteolysis, except for selenomethionine, which is slightly stimulatory. Metabolic inhibitors were studied at graded concentrations. Chloramphenicol inhibits the basal level of intracellular proteolysis when protein synthesis is slightly or moderately inhibited, and stimulates proteolysis slightly at higher levels. Graded inhibition of ribonucleic acid synthesis with rifampin progressively stimulates intracellular proteolysis. Uracil depletion is also stimulatory. Inhibition of deoxyribonucleic acid synthesis with mitomycin C or by thymine starvation slightly inhibits intracellular proteolysis. Intracellular proteolysis is postulated to be regulated primarily by active ribosomal function. At 43 to 45 C, intracellular proteolysis becomes maximally induced and unresponsive to normal regulatory control by metabolites. Most regulation is directed towards the breakdown of the more stable cell proteins. Total proteolysis in all cell proteins is no more than doubled by the most effective conditions of starvation.
Regulation of intracellular proteolysis has been compared during amino acid deficiencies in seven double auxotrophs of Escherichia coli 9723f with a common phenylalanine requirement. Individual deficiencies were either more effective than, less effective, or equal to phenylalanine deficiency in stimulating intracellular proteolysis. For each amino acid, the same relationship prevailed in inhibiting uracil incorporation into nucleic acids, a reaction series regulated by the rel gene for stringent control. The three amino acids least abundant in the cellular protein were the least effective regulators. These findings are interpreted as supportive evidence for stringent control of intracellular proteolysis by the rel gene.
Protein turnover was found to take place in cells of the asporogenic strain ofBacillus mega, terium KM during the stationary phase brought about by exhaustion of a nitrogen source. Its rate measured by degradation of prelabelled
proteins varied around 4%/h. however, the synthesis of proteins at the beginning of the stationary phase was slightly higher
(7–8%/h). Protein turnover started already during growth in the medium with a limiting nitrogen concentration. Addition of
low doses of ammonium chloride (2 μg NH4Cl/ml and higher) to the nongrowing population at thirty min intervals stimulated protein synthesis. This resulted both in
the increased incorporation of14C-leucine into proteins and in the increased synthesis of exocellular protease. On the other hand, the intracellular degradation
of proteins decreased only slightly. The number of “colony forming units” in the starving population as well as in the population
which was given 2 μg NH4Cl/ml/30 min did not change during 4 h. The number of cells not exhibiting protein synthesis was negligible in both cases.
Protein breakdown of 5 to 7% per hr was found in nitrogen-starved cells of an unclassified prototrophic thermophilic bacillus; a similar protein-breakdown rate (6.5% per hr) was found in resting cells of Escherichia coli. In the thermophile, the rate of protein breakdown was markedly influenced by the temperature; it was maximal between 45 and 55 C, and it decreased considerably at 35 and 75 C, temperatures which are only slightly below or above the minimal and maximal growth temperatures. Growing cultures of the thermophile showed little, if any, protein breakdown, a finding similar to that of others with E. coli.
The increased protein turnover that accompanies amino acid starvation was examined in isogenic auxotrophs of Escherichia coli K-12 which differ only in the genetic locus responsible for control of RNA synthesis under conditions of amino acid starvation. The multiple auxotrophs investigated were CP78 (stringent RNA control) and CP79 (relaxed RNA control). The protein degradation which occurs following amino acid starvation in the stringent CP78 consists of two processes. (a) A chloramphenicol-insensitive breakdown which plays a large role early in the starvation period due to the presence of a pool of labile proteins, and (b) a chloramphenicol-sensitive proteolysis which causes the bulk of the observed protein degradation. Under normal conditions of amino acid starvation, there is considerably less protein degradation in the relaxed CP79 relative to the stringent CP78. This decreased level of degradation in the relaxed CP79 appears to result from a deficiency in the chloramphenicol-sensitive proteolytic process. However, under conditions of simultaneous starvation for both glucose and amino acids, proteolysis occurs to the same extent in CP78 and CP79. Whereas the withdrawal of glucose from the starvation medium has no effect on proteolysis in the stringent CP78, it increases degradation in CP79 to the level occurring in CP78. These results are discussed in terms of the pleiotropic nature of the control exercised by the RNA control locus.
Turnover of cellular protein has been estimated in Escherichia coli during continuous exponential growth and in the absence of extensive experimental manipulation. Estimation is based upon the cumulative release into carrier pools of free leucine-1-(14)C over a number of time intervals after its pulsed incorporation into protein. Breakdown rates obtained with other labeled amino acids are similar to those obtained with leucine. Two kinetically separate processes have been shown. First, a very rapid turnover of 5% of the amino acid label occurs within 45 sec after its incorporation, most likely indicating maturative cleavages within the proteins after their assembly. A slower heterogeneous rate of true protein turnover follows, falling by 39% in the remaining proteins for each doubling of turnover time. At 36 C, the total breakdown rate of cellular protein is 2.5 and 3.0% per hr over a threefold range of growth rate in glucose and acetate medium, respectively. This relatively constant breakdown rate is maintained during slower growth by more extensive protein replacement, one fifth of the protein synthesized at any time in the acetate medium being replaced after 4.6 doubling times. Intracellular proteolysis thus appears to be a normal and integral reaction of the growing cell. The total rate equals minimal estimates obtained by others for arrested or decelerated growth but is kinetically more heterogeneous. Quantitatively proteolysis is not directly affected by growth arrestment per se as caused by alpha-methylhistidine, chloramphenicol, or uncouplers of oxidative phosphorylation, but qualitatively it can gradually become more homogeneous kinetically as a secondary event of starvation. Under more extreme conditions as with extensive washing, prolonged phosphorylative uncoupling, or acidification of the growth medium, the proteolytic rate can increase severalfold.
The mechanism by which the levulinate locus (Lv) regulates the tissue activity of δ-aminolevulinate dehydratase in mice has been examined. The assayable hepatic enzyme activity in homozygous Lvb mouse strains represented by C57B1/6 is between one-third and one-half that of homozygous Lva strains. This difference in enzyme activity is due to a difference in amount of enzyme protein as demonstrated by immunochemical techniques with an antibody specific for δ-aminolevulinate dehydratase. Combined immunochemical and isotopic techniques show that the levulinate locus regulates the concentration of hepatic δ-aminolevulinate dehydratase by acting at the level of enzyme synthesis. The rate of degradation of hepatic enzyme is the same in both low and high activity strains; when expressed as a half-life, this is equal to 5 to 6 days.
The pattern of δ-aminolevulinate dehydratase development with age is similar in livers of both low and high activity strains. The specific activity of the enzyme is high in fetal liver, decreases during the several days prior to birth, and increases to the adult level during the first 3 weeks of postnatal life. The activity of δ-aminolevulinate dehydratase in fetal liver is also regulated by the levulinate locus. The enzyme in fetal liver is approximately twice as active catalytically as the enzyme in adult liver relative to its function as an antigen. The fetal enzyme also appears to be different in stability to heat and proteolytic inactivation. However, fetal and adult enzymes are similar by other physiochemical criteria, including electrophoretic mobility, sedimentation coefficient, and Km for the substrate.
1. When Escherichia coli leu(-) was incubated at 35 degrees in a medium based on minimal medium, but with the omission of phosphate ions, or glucose, or NH(4) (+) ions and leucine, intracellular protein was degraded at a rate of about 5%/hr. in each case. If Mg(2+) ions were omitted, however, the rate of degradation was 2.9%/hr. 2. Under certain conditions of incubation, protein degradation was inhibited. The inhibitor was neither NH(4) (+) ions nor amino acids, and its properties were not those of a protein, but it might be an unstable species of RNA. 3. Although a large part of the cell protein was degraded at about 5%/hr. during starvation of NH(4) (+) ions and leucine, some proteins were lost at more rapid rates, whereas others were lost at lower rates or not at all. 4. In particular, beta-galactosidase activity was lost at about 8%/hr. during starvation of NH(4) (+) ions and leucine, whereas d-serine-deaminase and alkaline-phosphatase activities were stable. During starvation of Mg(2+) ions, all three enzyme activities were stable.
A labile class of proteins in the range of Mr = 30 000–60 000 which turn over rapidly have been demonstrated in Leishmania tropica promastigotes. The rate of protein degradation is increased by exhaustion of nutrients or inhibition of energy metabolism. Proteolysis is reduced when the parasites are provided with a readily utilizable carbon and energy source such as glucose, glutamate or proline. The breakdown of proteins in L. tropica is dependent on continuous protein synthesis probably for protease synthesis. It is suggested that the relatively high rate of intracellular protein degradation is an auxiliary means for generating carbon and energy sources during nutritional stress.
In a typical Escherichia coli K-12 culture starved for glucose, 50% of the cells lose viability in ca. 6 days (Reeve et al., J. Bacteriol. 157:758-763, 1984). Inhibition of protein synthesis by chloramphenicol resulted in a more rapid loss of viability in glucose-starved E. coli K-12 cultures. The more chloramphenicol added (i.e., the more protein synthesis was inhibited) and the earlier during starvation it was added, the greater was its effect on culture viability. Chloramphenicol was found to have the same effect on a relA strain as on an isogenic relA+ strain of E. coli. Addition of the amino acid analogs S-2-aminoethylcysteine, 7-azatryptophan, and p-fluorophenylalanine to carbon-starved cultures to induce synthesis of abnormal proteins had an effect on viability similar to that observed when 50 micrograms of chloramphenicol per ml was added at zero time for starvation. Both chloramphenicol and the amino acid analogs had delayed effects on viability, compared with their effects on synthesis of normal proteins. The need for protein synthesis did not arise from cryptic growth, since no cryptic growth of the starving cells was observed under the conditions used. From these and previous results obtained from work with peptidase-deficient mutants of E. coli K-12 and Salmonella typhimurium LT2 (Reeve et al., J. Bacteriol. 157:758-763, 1984), we concluded that a number of survival-related proteins are synthesized by E. coli K-12 cells as a response to carbon starvation. These proteins are largely synthesized during the early hours of starvation, but their continued activity is required for long-term survival.
Most bacteria have evolved a number of regulatory mechanisms which allow them to maintain a balanced and rather constant cellular composition in response to nutritional variations. In particular, when the availability of any aminoacyl-tRNA species becomes limiting (namely through amino acid starvation or inactivation of an aminoacyl-tRNA synthetase), several biochemically distinct physiological processes are significantly modified. This coordinate adjustment of cellular activity is termed the "stringent response". Under such conditions of aminoacyl-tRNA limitation, protein synthesis still proceeds, but various quantitative as well as qualitative changes in polypeptide metabolism can be observed. In this review, after a brief recall of the main characteristics of the stringent response, several aspects concerning protein synthesis in deprived bacteria have been presented. First, the rates of residual protein formation, peptide chain growth and protein degradation, and the molecular weight distribution of proteins newly synthesized have been analyzed. Then, the data relative to the biosynthetic regulation of non-ribosomal and ribosomal proteins have been summarized and compared to the results obtained from in vitro experiments using transcription-translation coupled systems. Finally, the problem of translational fidelity during deprivation has been discussed in connection with the metabolic behavior of polysomal structures which are still maintained in cells. The stringent dependence of cellular activity on aminoacyl-tRNA supply is known to be abolished by single-site mutations which confer to bacteria a phenotype referred to as "relaxed". These mutant strains provide an useful analytical tool in the scope of understanding the stringency phenomenon. Therefore, their proteosynthetic activity under aminoacyl-tRNA deprivation has also been studied here, in comparison to that of normal wild-type strains.
An investigation has been made to determine the extent of turnover synthesis in the subcellular components of resting cells of Escherichia coli, and the extent to which this synthesis differs from that in normal growth.Estimates of turnover vary with the strain of organism examined, the condition of starvation and the amino acid used as an indicator of turnover. Estimates further depend on whether breakdown of growth-labeled protein or resynthesis of new protein is examined. Turnover of protein was found to occur to varying extents in all subcellular fractions examined including the cell walls.Most of the protein synthesized in turnover differs from the protein of growth in being for the most part metabolically unstable and associated with the cell membranes. It is maximally revealed by pulsed labeling, the most unstable elements disappearing within a minute thereafter. The cumulative rate of cellular replacement can thus amount to 20% per h. The proteins synthesized in turnover are postulated to comprise a generally stable class derepressed by nutritional deficiency, and a more labile class which recycle non-specifically to contend with environmental alteration.In the soluble subcellular component, turnover synthesis is favored among the low molecular weight proteins.Cell-free preparations retain a limited capacity for preferential breakdown of pulse-labeled protein.
1. When Escherichia coli leu(-) was incubated at 35 degrees in a medium based on minimal medium, but with the omission of phosphate ions, or glucose, or NH(4) (+) ions and leucine, intracellular protein was degraded at a rate of about 5%/hr. in each case. If Mg(2+) ions were omitted, however, the rate of degradation was 2.9%/hr. 2. Under certain conditions of incubation, protein degradation was inhibited. The inhibitor was neither NH(4) (+) ions nor amino acids, and its properties were not those of a protein, but it might be an unstable species of RNA. 3. Although a large part of the cell protein was degraded at about 5%/hr. during starvation of NH(4) (+) ions and leucine, some proteins were lost at more rapid rates, whereas others were lost at lower rates or not at all. 4. In particular, beta-galactosidase activity was lost at about 8%/hr. during starvation of NH(4) (+) ions and leucine, whereas d-serine-deaminase and alkaline-phosphatase activities were stable. During starvation of Mg(2+) ions, all three enzyme activities were stable.
When Staphylococcus aureus strain 524/SC/55 is inoculated from an overnight culture into fresh broth only small amounts of hyaluronidase are formed at first, but while growth proceeds at a constant exponential rate an increasing proportion of the cell protein is secreted into the medium as hyaluronidase. This increase in proportion continues for eleven generations. Thereafter, it remains constant even when the organisms are transferred to fresh medium. The organisms of the inoculum taken from overnight cultures are deficient in thiamine and a partial deficiency of this and other essential growth factors suppresses the proportion of hyaluronidase formed. The accumulation of α-aminobutyric acid in organisms from overnight cultures was demonstrated; the addition of this substance to cultures decreases the proportion of cell protein which is turned into hyaluronidase.
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