Protein synthesis and degradation in a leucine auxotroph of Escherichia coli.

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.     

Protein turnover and cellular autophagy in growing and growth-inhibited 3T3 cells

The relationship between growth, protein degradation, and cellular autophagy was tested in growing and in growth-inhibited 3T3 cell monolayers. For the biochemical evaluation of DNA and protein metabolism, growth-inhibited 3T3 cell monolayers with high cell density and growing 3T3 cell monolayers with low cell density were labeled simultaneously with [14C]thymidine and [3H]leucine. The evaluation of the DNA turnover and additional [3H]thymidine autoradiography showed that 24 to 5% of 3T3 cells continue to replicate even in the growth-inhibited state, where no accumulation of protein and DNA can be observed. Cell loss, therefore, has to be assumed to compensate for the ongoing cell proliferation. When the data of protein turnover were corrected for cell loss, it was found that the rate constant of protein synthesis in nongrowing monolayers was reduced to half the value found in growing monolayers. Simultaneously, the rate constant of protein degradation in nongrowing monolayers was increased to about 1.5-fold the value of growing monolayers. In parallel to the increased rate constant of protein degradation, the cytoplasmic volume fraction of early autophagic vacuoles (AVs) as determined by electron microscopic morphometry was found to be increased twofold in nongrowing 3T3 cell monolayers when compared with the volume fraction of early AVs in growing 3T3 cell monolayers. These data are in agreement with the assumption that cellular autophagy represents a major pathway of regulating protein degradation in 3T3 cells and that the regulation of autophagic protein degradation is of relevance for the transition from a growing to a nongrowing state.     

Turnover of cell wall polysaccharides of a Vinca rosea suspension culture

Turnover of cell wall components was examined in two growth phases of a batch suspension culture of Vinca rosea L. Three-day-cultured cells (cell division phase) and 5-day-cultured cells (cell expansion phase) were incubated with d -[U-¹⁴C]glucose. After various periods of incubation, extra-cellular polysaccharides (ECP) and cell walls were isolated, and then the cell walls were fractionated to pectic substance, hemicellulose, and cellulose fractions. The results of the measurement of radioactivities and amounts of total carbohydrate in the ECP and cell wall fractions indicated that synthesis of pectic substance was more active in the cell division phase than in the cell expansion phase. From the results of the pulse-chase experiments, in which cells prelabelled by incubation with d -[U-¹⁴C]glucose for 3 h were incubated in a medium containing unlabelled glucose for various periods, the gross degradation, net synthesis, and gross synthesis of cell wall components were estimated. Active degradation and synthesis were observed in the hemicellulose fraction, indicating that active turnover occurred in the hemicellulose fraction, while little degradation was found in the pectic substance and cellulose fractions.     

Turnover of the plasma membrane proteins of hepatoma tissue culture cells.

The turnover of the plasma membrane proteins of hepatoma tissue culture cells was examined by three different methods--loss of polypeptides labeled in situ by lactoperoxidase-catalyzed iodination, loss of membrane polypeptides labeled with amino acid precursors, and loss from the membrane of fucose-labeled polypeptides. In both logarithmically growing and density-inhibited cells the proteins of the membrane are degraded with a half-life of about 100 hours. This is longer than the half-life of total cell protein, 50 to 60 hours, and longer than the doubling time of the cells, about 30 hours. Similar values for the rate of degradation of the membrane proteins were obtained by each of the three techniques. The same fucose-labeled polypeptides are present in the microsomal and the plasma membrane fractions of hepatoma tissue culture cells as analyzed by electrophoresis in dodecyl sulfate-acrylamide gels. But the fucose-labeled polypeptides were lost from the microsomal fraction at a faster rate than from the plasma membrane. Autoradiographic and double labeling techniques using 125I and 131I, or [3H]leucine and [14C]leucine were used to measure the relative rates of degradation of the proteins in the plasma membrane. All of the leucine-labeled polypeptides and the iodinated polypeptides had similar rates of degradation. These results support a model for the biogenesis of the plasma membrane in which the proteins are incorporated and removed in large structural units.     

Turnover of Phospholipids in an Unsaturated Fatty Acid Auxotroph of Escherichia coli

The membrane phospholipids of an unsaturated fatty acid auxotroph of Escherichia coli were found to undergo turnover. These phospholipids were excreted into the culture medium, and were replaced in the cell with newly synthesized phospholipids. Phospholipids of growing cells supplemented with elaidic acid underwent rapid turnover, while those of cells supplemented with oleate, or cis-vaccenate plus palmitoleate, underwent slow turnover. Starvation for required amino acids stimulated this turnover in the latter two cases. Protein was also lost from growing cells. However, after amino acid starvation this loss ceased while phospholipid turnover continued. Electron micrographs of growing cells indicated that large pieces of membrane-like material were separating from the cell surface.     

Parallel changes in protein synthesis and messenger RNA content in growing and resting epithelial cells of Xenopus laevis

The regulation of cell growth can be achieved at many levels but ultimately the regulatory factors must alter protein synthesis since growing cells always exhibit an increased rate of protein synthesis compared to resting cells. Some studies using growing and nongrowing mammalian cells have shown that the rate of protein synthesis compared to resting cells. Some studies using growing and nongrowing mammalian cells have shown that the rate of protein synthesis is directly dependent on mRNA content. Other studies have shown that growing and resting cells have similar amounts of mRNA and that protein synthesis is regulated by the proportion of mRNA in polysomes. We have analyzed mRNA content in growing and resting epithelial cells of Xenopus laevis. Quantitation of poly(A)⁺ mRNA by uniform labeling with ³H-uridine and by ³H-poly(U)hybridization demonstrated a direct relationship between mRNA content and the relative rate of protein synthesis in growing and resting cells. Likewise, after serum stimulation of resting cells the increase in mRNA content closely paralleled the increase in protein synthesis. Our results suggest that control of protein synthesis in growing and nongrowing cells is exerted before the translational level.     

Synthesis and accumulation of mannose-containing glycopeptides in human fibroblast cells possible separate pathways for neutral and acidic glycopeptides

The normal human fibroblast, WI-38, was labelled with radioactive mannose and its incorporation, as well as the accumulation of acidic and neutral glycopeptides on the cell surface, was followed as a function of time. The transit time of newly made Pronase-released cell surface glycopeptides from their intracellular site of synthesis to the cell surface was slower in nongrowing cells than in a rapidly growing culture. When the surface glycopeptides were separated by high-voltage paper electrophoresis into neutral and acidic species, it was observed that the cell surface material was initially enriched with neutral glycopeptides. However, with time the relative proportion of acidic species increased so that by 3 h the ratio between the acidica and neutral species approached a constant value. Our data are consistent with the hypothesis that multiple pathways for asparagine-linked glycoprotein biosynthesis are possible.     

Characterization and Regulation of Protease Synthesis and Activity in Bacillus licheniformis

Extracts of growing and sporulating cells contain a protease activity that has a broad pH optimum and an unusually broad specificity. The activity, which resides in at least two protein fractions, hydrolyzes all peptide bonds and can reduce a mixture of proteins into a mixture of free amino acids with a high efficiency. No inhibitors of the activity were found, but the protease showed a definite preference for denatured protein as substrate. The synthesis of the intracellular protease activity is under catabolite repression control, as is the extracellular activity. However, the synthesis of the two activities is not coordinate, making the relationship between the two unclear. Due to (i) the specificity of the intracellular activity, (ii) the fact that it is synthesized most rapidly under slow or nongrowing conditions, and (iii) our inability to measure in vivo protein turnover in cells containing high levels of enzyme, a scavenger role is postulated for the enzyme. The rate of protein turnover is not a function of the protease content of the cells.     

Degradation of intracellular endogenous proteins following serum deprivation in mammalian cell cultures: theoretical considerations of the role of very-fast turnover proteins in growth regulation.

This paper discusses the way in which serum deprivation affects the turnover of nascent or newly synthesised proteins in mammalian cells. A theoretical treatment of their turnover relative to changes in rate of protein synthesis and the turnover of existing or "resident" proteins is presented. Previous experimental work has not seriously addressed this question because the pulses of radiolabelling of proteins have been too long to identify the very-fast turnover population (Wheatley et al., 1980; Bohley, 1987). Logically one would expect cell growth rate to be regulated by the rate at which new proteins become incorporated into the cell within the first 30 min of their existence. This requires their successful integration at what we will refer to as the "growing point", recognizing that at any time there may be thousands of such sites. Growth is a simple term betraying the complexity of the processes involved--synthesis, processing, sorting, targeting, and stabilization of macromolecules, and their successful integration into functional assemblies at appropriate locations. Turnover of the truly short-lived, very-fast turnover proteins at the "growing point" is affected by serum adjustment, but it is not the only change since synthetic rate quickly responds, as also does the turnover rate of long-lived proteins. Our theoretical discussion will relate to recent findings in 3T3 and HeLa cell cultures after serum modulation, lines with quite different dependencies on serum growth factors.     

The measurement of the relative turnover rates of proteins of the surface membranes and other fractions of Schistosoma mansoni in culture.

The double isotope labelling method of Arias, Doyle & Schimke (1969) and Dehlinger & Schimke (1971) was used to determine the relative rates of turnover of proteins in various tissue fractions and in the culture medium of adult Schistosoma mansoni. The majority of proteins in the surface membrane turned over at about the same rate. This is consistent with a model for membrane assembly and degradation involving multi-protein units. The proteins in the other fractions examined, except frozen-thawed supernatant fluid, also turned over at about the same rate. In the frozen and thawed supernatant fluid, the higher molecular weight (greater than 40 000 daltons) proteins turned over at a much greater rate than the lower molecular weight proteins (less than 40 000 daltons). The antigens in the culture medium, both particulate and soluble, had a higher turnover rate than the worm tissue proteins. It is concluded that the culture medium antigens are released as the result of two distinct processes: (a) surface membrane turnover and (b) a rapid secretory process. It is possible that the high molecular weight proteins found in the forzen-thawed supernatant fluid are involved in the rapid secretory process. The culture media of 6-day and 16-day schistosomula were also examined by the double isotope method. The culture medium of the adult worm and 16-day schistosomula contains more material presumed to be secreted than does that from the 6-day schistosomula. This may explain the poor immunogenicity of young schistosomula.     

Turnover and spreading of old wall during surface growth of Bacillus subtilis.

The steady-state concentration of cell wall turnover products in the medium of Bacillus subtilis 168 growing exponentially on a casein hydrolysate-supplemented medium is equivalent to an overall rate of turnover of less than 10% per generation. After transfer of a steady-labeled culture to nonradioactive medium, the rate of release of labeled turnover products increased exponentially for up to two generations. The rate of turnover finally attained by this culture reached an apparently first-order rate of about 50% per generation. The addition of soluble autolytic activity to growing cultures of a mutant possessing a reduced rate of wall turnover resulted in a marked stimulation in the rate of solubilization of the cell wall fraction. The increased rate of solubilization produced was proportional to the concentration of added enzyme and remained constant until less than 20% of the wall originally present was left. Autolytic activity added under these conditions was bound entirely to wall at least one generation old. The results are interpreted in terms of a model for cell wall growth in which wall two or more generations old covers a total surface area at least four times larger than that occupied at the time of synthesis, forming a shallow outer layer (overlying newer wall) from which all turnover takes place. The model is discussed in relation to previous attempts to determine the pattern of surface expansion in bacilli.     

Products of phospholipid metabolism in Bacillus stearothermophilus.

The products of phospholipid turnover in Bacillus stearothermophilus were determined in cultures labeled to equilibrium and with short pulses of [32P]phosphate and [2-3H]glycerol. Label lost from the cellular lipid pool was recovered in three fractions: low-molecular-weight extracellular products, extracellular lipid, and lipoteichoic acid (LTA). The low-molecular-weight turnover products were released from the cells during the first 10 to 20 min of a 60-min chase period and appeared to be derived primarily from phosphatidylglycerol turnover. Phosphatidylethanolamine, which appeared to be synthesized in part from the phosphatidyl group of phosphatidylglycerol, was released from the cell but was not degraded. The major product of phospholipid turnover was LTA. Essentially all of the label lost from the lipid pool during the final 40 min of the chase period was recovered as extracellular LTA. The LTA appeared to be derived primarily from the turnover of cardiolipin and the phosphatidyl group of phosphatidylglycerol. Three types of LTA were isolated; an extracellular LTA was recovered from the culture medium, and two types of LTA were extracted from membrane preparations or whole-cell lysates by the hot phenol-water procedure. Cells contained 1.5 to 2.5 mg of cellular LTA per g of cells (dry weight), over 50% of which remained associated with the membrane when cells were fractionated. Over 75% of the 3H label incorporated into the cellular LTA pool during a 90-min labeling period was released from the cells during the first cell doubling after the chase. Label lost from the lipid pool was incorporated into cellular LTA which was then modified and released into the culture medium.     

Flow and shuttle of plasma membrane during endocytosis

A striking feature of endocytosis is the large amount of surface membrane that is brought into the cells through the formation of endocytic vesicles. Little is known about the fate of this membrane material. It is implausible that it would be destroyed in lysosomes, as the rate of turnover of the constituents of plasma membrane is much too low with respect to the rate of endocytosis in all cells studied so far. Conversely, plasma membrane fragments, internalized by endocytosis cannot merely be incorporated in lysosomes, as these organelles have been shown to maintain their size, despite continuous and active endocytosis. We present evidence that plasma membrane antigens, detected by means of specific antibodies, are internalized during endocytosis and reach lysosomes. They are thereafter returned back to cell surface. These results indicate the existence of a shuttle of membrane elements between the cell surface and lysosomes.     

Dual pathways for ribonucleic acid turnover in WI-38 but not in I-cell human diploid fibroblasts.

The turnover rates of 3H-labeled 18S ribosomal ribonucleic acid (RNA), 28S ribosomal RNA, transfer RNA, and total cytoplasmic RNA were very similar in growing WI-38 diploid fibroblasts. The rate of turnover was at least twofold greater when cell growth stopped due to cell confluence, 3H irradiation, or treatment with 20 mM NaN3 or 2 mM NaF. In contrast, the rate of total 3H-protein turnover was the same in growing and nongrowing cells. Both RNA and protein turnovers were accelerated at least twofold in WI-38 cells deprived of serum, and this increase in turnover was inhibited by NH4Cl. These results are consistent with two pathways for RNA turnover, one of them being nonlysosomal and the other being lysosome mediated (NH4Cl sensitive), as has been suggested for protein turnover. Also consistent with the notion of two pathways for RNA turnover were findings with I-cells, which are deficient for many lysosomal enzymes, and in which all RNA turnover was nonlysosomal (NH4Cl resistant).     

Induction of autolysis in nongrowing Escherichia coli

Unless relaxation of the stringent response is achieved, all nongrowing bacteria rapidly develop resistance to autolysis induced by a variety of agents, including all classes of cell wall synthesis inhibitors. We now describe inhibitors of cell wall synthesis which were unusual in that they could continue to effectively induce autolysis in relA+ Escherichia coli even after prolonged amino acid starvation. The process of cell wall degradation seems to be catalyzed by similar hydrolytic enzymes in nongrowing and growing cells, yet the activity of these new agents capable of inducing autolysis in the nongrowing relA+ cells did not involve relaxation of RNA or peptidoglycan synthesis. We propose that the suppression of autolysis characteristic of nongrowing cells can be bypassed by a novel mechanism of autolytic triggering which is independent of the relA locus.     

Biopolymer particulate turnover in biological waste treatment systems: a review

Microorganisms — the major component in most biological waste treatment processes and a number of industrial fermentations — are not able to directly assimilate biopolymeric particulate material. Such organic particulates must first be solubilized into soluble polymers or monomers before they can diffuse through the capsular slime layer surrounding most bacteria, then transported across the cell membrane, to be used as either a carbon, energy or other essential nutrient source. Throughout these events, new cells are synthesized, which are themselves biopolymer particulates.The turnover of biopolymer particulates in biological treatment systems has not been examined with respect to its impact on system performance and culture physiology. The aim of this paper is to review the observations of particulate turnover in various biological treatment systems and to identify those fundamental mechanisms which govern microbial conversion of biopolymer particulates.Current address: Department of Chemical Engineering, California, Institute of Technology, Pasadena, Ca 91125 USA     

Metabolic activities of histones in rat liver and spleen.

Synthesis and turnover of histone I and II in normal rat liver and spleen were studied by Amberlite CG 50 column chromatography. Histone I was separated into three or four subfractions, each of which showed a different rate of incorporation of [3H]lysine. This was verified by a more shallow gradient chromatography developed by Kinkade and Cole [3] for very lysine-rich histone (F1), which showed tissue specific differences between liver and spleen in both the elution pattern and synthetic rates. These subfractions were distinguished from each other by dodecylsulphate electrophoresis. The turnover, or disassociation of histone I and II in chromatin was measured by double-labelling of normal rat liver with [3H] and [14C]lysine. A good correspondence was found between the synthesis and turnover patterns of individual histone I fractions, while the histone II synthesized was conserved for over a month. From consideration of the turnover in relation to the cell population of normal liver tissue, which consists of a very small fraction of growing cells and a very large fraction of resting ones, it was concluded that turnover of histone I must occur even in resting cells. When DNA synthesis in the spleen was completely inhibited by hydroxyurea, the synthesis of histone II was inhibited but that of histone I was only partially inhibited. The remaining synthesis seemed to occur in cells in the resting state. It was concluded tentatively, the continuous replacement of very lysine-rich histones of chromatin must occur even in resting cells in which DNA synthesis has ceased. The biological significance of disassociation of histones from chromatin was discussed.     

Protein turnover and proliferation. Turnover kinetics associated with the elevation of 3T3-cell acid-proteinase activity and cessation of net protein gain.

1. At least 95% of the total protein of A31-3T3 cell cultures undergoes turnover. 2. First-order exponential kinetics were used to provide a crude approximation of averaged protein synthesis, Ks, degradation, Kd, and net accumulation, Ka, as cells ceased growth at near-confluent density in unchanged Dulbecco's medium containing 10% serum. The values of the relationship Ka = Ks - Kd were : 5%/h = 6%/h - 1%/h in growing cells, and 0%/h = 3%/h - 3%/h in steady-state resting cells. 3. As determined by comparison of the progress of protein synthesis and net protein accumulation, the time course of increase in protein degradation coincided with the onset of an increase in lysosomal proteinase activity and decrease in thymidine incorporation after approx. 2 days of exponential growth. 4. After acute serum deprivation, rapid increases in protein degradation of less than 1%/h could be superimposed on the prevailing degradation rate in either growing or resting cells. The results indicate that two proteolytic mechanisms can be distinguished on the basis of the kinetics of their alterations. A slow mechanism changes in relation to proliferative status and lysosomal enzyme elevation. A prompt mechanism, previously described by others, changes before changes in cell-cycle distribution or lysosomal proteinase activity. 5. When the serum concentration of growing cultures was decreased to 1% or 0.25%, then cessation of growth was accompanied by a lower steady-state protein turnover rate of 2.0%/h or 1.5%/h respectively. When growth ceased under conditions of overcrowded cultures, or severe nutrient insufficiency, protein turnover did not attain a final steady state, but declined continually into the death of the culture.     

Vectorial transport of proteins by membrane-bound ribosomes of nongrowing and growing Jerusalem artichoke tuber cells

Both nongrowing (water-incubated) and growing (hormonally stimulated) Jerusalem artichoke tuber cells contain membrane-bound (mb) ribosomes. Using a rapid flotation procedure, a membrane fraction was prepared from both types of cells. This fraction was enriched in mb ribosomes, contained NADH cytochrome c reductase activity, had RNA:phospholipid and RNA:protein ratios similar to those reported for rough microsomes from animal tissues, and supported synthesis of preinitiated proteins in vitro. Using puromycin and detergent release, vectorial transport of labelled polypeptides was measured in the in vitro system. Of proteins made by mb ribosomes from nongrowing cells, on 12% remained associated with microsome membranes following chain termination. The comparable figure for proteins from mb ribosomes of growing tissue was 42%. The membrane-associated proteins were preferentially protected from protease digestion. Some possible reasons are suggested for the correlation between cell growth and the association of newly synthesized proteins with microsomes. The role of proteins synthesized by mb ribosomes but not vectorially transported, in both growing and nongrowing cells, is unknown.     

Biosynthesis of the glycoproteins present in plasma membrane, lysosomes and secretory materials, as visualized by radioautography

Synopsis The three major types of glycoproteins present in animal cells, that is, the secretory, lysosomal and plasma membrane glycoproteins, were examined with regard to the sites of synthesis of their carbohydrate side chains and to their subsequent migration within cells.The site at which a monosaccharide is added to a growing glycoprotein depends on the position of that monosaccharide in the carbohydrate side-chain. Thus, radiauutography of thyroid cells within minutes of the intravenous injection of labelled mannose, a sugar located near the base of the larger side-chains, reveals that it is incorporated in rough endoplasmic reticulum, whereas the more distally located galactose and fucose are incorporated in the Golgi apparatus. Recently [³H]N-acetylmannosamine, a specific precursor for the terminally located sialic acid residues, was shown to be also added in the Golgi apparatus. Presumably synthesis of glycoproteins is completed in this organelle.Radioautographs of animals sacrificed a few hours after injection of [³H]N-acetylmannosamine show that, in many secretory cells, labelled glycoproteins pass into secretory products. In these cells, as well as in non-secretory cells, the label may also appear within lysosomes and at the cell surface. In the latter site, it is presumably included within the plasma membrane glycoproteins whose carbohydrate side-chains form the cell coat. The continual migration of glycoproteins from Golgi apparatus to cell surface implies turnover of plasma membrane glycoproteins. Radioautographic quantitation of [³H]fucose label at the surface of proximal tubule cells in the kidney of singly-injected adult mice have shown that, after an initial peak, cell surface labelling decreases at a rate indicating a half-life of plasma membrane glycoproteins of about three days.