Synthesis of cytoskeletal and contractile proteins by cultured IMR-90 fibroblasts

Models of the assembly of cytoskeletal and contractile proteins of eukaryotic cells require quantitative information about the rates of synthesis of individual component proteins. We applied the dual isotope technique of Clark and Zak (1981, J. Biol. Chem., 256:4863-4870) to measure the synthesis rates of cytoskeletal and contractile proteins in stationary and growing cultures of IMR-90 fibroblasts. Fibroblast proteins were labeled to equilibrium with [14C]leucine over several days, at the end of which there was a 4-h pulse with [3H]leucine. Fractional synthesis rates (percent per hour) were calculated from the 3H/14C ratio of cell protein extracts or protein purified by one- or two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of medium-free leucine. The average fractional synthesis rate for total, SDS- or urea-soluble; Triton-soluble; and cytoskeletal protein extracts in stationary cells each was approximately 4.0%/h. The range of values for the synthesis of individual proteins from total cell extracts or cytoskeletal extracts sliced from one-dimensional gels was similar, though this range was greater than that for major proteins of Triton-soluble protein extracts. Three specific cytoskeletal proteins--actin, vimentin, and tubulin--were synthesized at similar rates that were significantly slower than the average fractional synthesis rate for total protein. Myosin, on the other hand, was synthesized faster than average. Synthesis rates were the same for beta-and gamma-actin and polymerized (cytoskeletal extract) vs. Triton-soluble actin. The same was true for alpha- and beta-tubulin and two different forms of vimentin. Synthesis rates were uniformly higher in growing cells, though the same pattern of differential rates was observed as for stationary cells. Synthesis rates in growing cells were higher than the rate necessary to maintain the growth rate, even for those cytoskeletal proteins being synthesized slowly. Therefore, there appears to be some turnover of these cytoskeletal elements even during growth. We conclude that proteins in cytoskeletal extracts may have nonuniform rates of synthesis, but at least one important subclass of cytoskeletal proteins that comprise filament subunits have the same synthesis rates.     

Effects of lactation on L-leucine metabolism in the rat. Studies in vivo and in vitro.

1. The turnover rate of L-[1-14C]leucine was increased by 35% in lactating rats compared with virgin rats. Starvation or removal of pups (24 h) returned the value to that of the virgin rat. 2. Incorporation of L-[U-14C]leucine into lipid and protein of mammary glands of lactating rats in vivo increased 7-fold and 6-fold respectively compared with glands of virgin rats. Lactation caused no change in the incorporation of L-[U-14C]leucine into hepatic lipid and protein. 3. The production of 14CO2 from L[l-14C]leucine (in the presence of glucose) was similar in isolated acini from glands of fed (chow) and starved lactating rats. Feeding with a 'cafeteria' diet caused a slight decrease, and removal of pups a large decrease, in the oxidative decarboxylation of leucine. 4. Oxidation of L-[2-14C]leucine to 14CO2 was increased about 3-fold in acini from starved lactating rats or lactating rats fed on a 'cafeteria' diet compared with rats fed on a chow diet. Insulin decreased the formation of 14CO2 in all three situations. 5. Incorporation of L-[U-14C]- and [2-14C]-leucine into lipid was decreased in acini from starved lactating rats and lactating rats fed on a 'cafeteria' diet. Insulin tended to increase the conversion of [2-14C]leucine into lipid, but this was significant only in the case of the acini from 'cafeteria'-fed rats. 6. Experiments with (-)-hydroxycitrate indicate that the major route for conversion of leucine carbon into lipid in acini is via citrate translocation from the mitochondria. 7. The physiological implications of these findings are discussed.     

Effects of hormones on protein and amino acid metabolism in mammary-gland explants of mice.

The effects of insulin, cortisol and prolactin on amino acid uptake and protein biosynthesis were determined in mammary-gland explants from mid-pregnant mice. Insulin stimulated [3H]leucine incorporation into protein within 15 min of adding insulin to the incubation medium. Insulin also had a rapid stimulatory effect on the rate of aminoiso[14C]butyric acid uptake, but it had no effect on the intracellular accumulation of [3H]leucine. Cortisol inhibited the rate of [3H]leucine incorporation into protein during the initial 4h of incubation, but it had no effect at subsequent times. [3H]Leucine uptake was unaffected by cortisol, but amino[14C]isobutyric acid uptake was inhibited after a 4h exposure period to this hormone. Prolactin stimulated the rate of [3H]leucine incorporation into protein when tissues were exposed to this hormone for 4h or more; up to 4h, however, no effect of prolactin was detected. At all times tested, prolactin had no effect on the uptake of either amino[14C]isobutyric acid or [3H]leucine. Incubation with actinomycin D abolished the prolactin stimulation of protein biosynthesis, but this antibiotic did not affect the insulin response. A distinct difference in the mechanism of action of these hormones on protein biosynthesis in the mammary gland is thus apparent.     

Non-parallel secretion of newly synthesized rat pancreatic proteins after feeding a diet containing raw soybean flour

The relative rate of secretion of rat pancreatic proteins was studied in vivo using a double label method. Rats were injected with [3H]leucine and after different time intervals with [14C]leucine. At a fixed time after administration of the second precursor the animals were killed, and the pancreatic proteins were separated by polyacrylamide gel electrophoresis. The dpm of tritium to dpm of 14C ratio of several identified enzymes was assessed. The percentage secretion of a newly synthesized secretory protein was derived from the difference between the actual 3H/14C ratio and the 3H/14C ratio that was found for non-secretory proteins. In pancreata of rats fed with a standard diet several identified proteins, viz. three trypsinogens, chymotrypsinogen and three amylases were secreted in "parallel". When a diet containing raw soybean flour was fed, the secretory pattern for the amylases differed from that of the other proteins.     

PURIFICATION OF RAT BRAIN CHOLINE ACETYLTRANSFERASE AND AN ESTIMATION OF ITS HALF-LIFE

—Acetyl-CoA:choline-O-acetyltransferase (ChAc, EC 2.3.1.6) was purified from rat cerebral cortex and its half-life determined. The molecular weight of the enzyme under non-denaturing conditions was estimated by gel filtration to be in the range of 60,000–65,000. On SDS acrylamide gels, the purified enzyme migrated as a single band with a molecular weight estimated as 62,000. The turnover rate of ChAc in the mature rat was determined by the double label method, employing l -[1-¹⁴C]leucine and l -[4,5-³H]leucine. Its half-life under steady-state conditions was estimated to be 5.2 days. As a control, tubulin was isolated from the same preparation and its half-life measured. Under these conditions tubulin exhibited a half-life of 3.8 days.     

Modulation of proliferation in epidermal keratinocyte cultures by lowered oxygen tension

The modulation of proliferation and differentiation in primary epidermal keratinocyte cultures by lowered gas phase oxygen tensions was studied. Neonatal mouse epidermal keratinocyte cultures were grown in an Heraeus type B 5060 EK/O2 incubator in oxygen tensions between 5% and 15% (within the physiologic range); the oxygen tension of ambient air being 21%. Cell morphology was studied using histochemical stains and electron microscopy. Differentiation was assessed using autoradiography of SDS PAGE gels of six serially extracted cell protein fractions with [3H]leucine as a marker. Autoradiographs using [14C]glucosamine and 32Pi as markers were also assessed as a measure of other cell functions. Proliferation was studied using autoradiography of [3H]thymidine ([3H]TdR) pulse-labeled cultures and [3H]TdR incorporation into isolated DNA fractions. The results of these studies showed that lowering the oxygen tension in the gas phase reversibly inhibited cell proliferation. There was a direct arithmetic relationship between the proliferative rate of the cultures and the oxygen tension. No change in differentiation as defined by [3H]leucine indexing of protein synthesis was seen. Other markers of cell function, such as [14C]glucosamine glycosylation and [32P] phosphorylation of proteins were also unchanged. These results suggest that oxygen tension regulates only proliferation in epidermal keratinocytes. This epidermal response is well adapted to its role in the healing wound, and is an example of a tissue-specific modification of a regulatory function.     

Degradation of proteins in steady-state cultures of Escherichia coli.

The degradation of proteins in Escherichia coli was investigated in cells grown under steady-state conditions in a glucose-limited chemostat. During the first 24 h, approximately 25% of pulse-labeled proteins were degraded and after 72 h up to 58% of the proteins were broken down. To examine the stability of subcellular components steady-state cultures were labeled with an initial pulse of [14C]leucine, 24 h were allowed for turnover of these proteins, and the cells were then labeled with a short pulse of [3H]leucine. By this double-label protocol, the labile proteins were preferentially labeled with [H]leucine and had high 3H/14C ratios, while the more stable proteins had lower 3//14C ratios. The 3/-labeled proteins were degraded approximately five times as rapidly as the 14C-labeled proteins in exponentially growing cells. The relative stability of subcellular fractions was determined by comparing their 3H/14C ratios to the ratio of the cells at harvest. The soluble fraction contained the most labile proteins, while the ribosomal and membrane fractions were at least as stable as the average cell protein.     

HE TURNOVER RATE OF TUBULIN IN RAT BRAIN

The turnover rate of tubulin in rat brain was determined from the decay in specific radioactivity of the protein after pulse-labeling. When precursors were administered by a parenteral route, the shortest half-life, 9.8 days, was obtained with [¹⁴C]NaHCO₃; the longer half-lives obtained with [U-¹⁴C]glucose or [4,5-³H]leucine suggest significant reutilization of label. Furthermore, with leucine as precursor maximal specific radioactivity of tubulin was not obtained until eight days after administration of label. Labeling and decay kinetics obtained with [4,5-³H]leucine were markedly different when the isotope was administered directly into the lateral ventricle. The difference between the turnover rates of the -α and β subunits of tubulin purified by means of high resolution polyacrylamide gel electrophoresis was not statistically significant. A half-life for tubulin of 6.2 days was measured by continuous intravenous infusion of [U-¹⁴C]tyrosine.     

Studies on the biosynthesis of pancreatic glucagon in the pigeon (Columba livia).

The biosynthesis of glucagon was studied in microdissected pigeon pancreatic, islets. [3H]-Tryotophan and [3H]leucine were incorporated into big and little glucagon. No precursor-product relationship was evident between big and little glucagon after radioactive pulsechase and immunoreactive chase incubations. Radioactive and immunoreactive little glucagon and immunoreactive big glucagon were actively secreted and the synthesis of both glucagons was inhibited by high concentrations of glucose. [3H]Tryptophan and [3H]leucine were incorporated into an islet protein of about 20000mol.wt. Gel filtration of extracts of turkey pancreas revealed the presence of an immunoreactive peak of mol.wt. approx. 20000. This glucagon-immunoreactive component was also present in dog and ox pancreas and was stable to chaotropic agents and elution at various pH values. A similar-sized glucagon-immunoreactive species was present in the dog circulation. These results are discussed in the light of the presently accepted mechanisms of glucagon biosynthesis.     

DNA binding of a nonstructural reovirus protein

The specific early inhibition of DNA synthesis in reovirus-infected cells suggests that the cell nucleus is a target for virus-induced damage. We have now examined the affinity of reovirus proteins for DNA, postulating that such affinity could provide a mechanism for the inhibition. Cytoplasmic and nuclear extracts of cells labeled with [35S] methionine from 6 to 8.5 h after infection at high multiplicity was subjected to chromatography on denatured DNA - cellulose columns. Fractions from both cytoplasm and nucleus eluted with 0.6 N NaCl contained a protein with the same electrophoretic mobility of polyacrylamide slab gels as the nonstructural (NS) reovirus protein of the sigma size class. The protein also exhibited affinity for native DNA - cellulose and denatured DNA - agarose. Electrophoretic analysis is tube gels of cell extracts labeled for 48 h before infection with [14C] leucine and from 6 to 8.5 h after infection with [3H] leucine showed increased 3H label in this protein indicating it is reovirus specific. Small amounts of mu proteins also had DNA affinity. Purified virus did not bind strongly to DNA, suggesting that the binding protein is not a structural protein of the sigma size class on the outer surface of the virus. Our results provide evidence that the sigma NS protein binds to DNA. This affinity could interfere with chromosome function in the infected cell.     

Interconversion of valine and leucine by Clostridium sporogenes.

Clostridium sporogenes has been found to require L-leucine and L-valine for growth in a minimal medium, although valine can be replaced by isobutyrate and leucine by isovalerate. Cells grown in minimal media incorporated significant 14C from [14C]valine into leucine and from [14C]leucine into valine. Growth with [4,5-3H]leucine also resulted in the incorporation of 3H into valine. These results indicate that these bacteria can interconvert leucine and valine.     

Measurement of protein turnover in rat brain

Abstract— Degredation rates of rat brain proteins were measured by following the decay in specific radioactivity of carboxyl labelled aspartate and glutamate over a 17-day period. Initial labelling of these amino acids was achieved by a single intraperitoneal injection 0f NaH¹⁴CO₃. The non-linear decay curve for total brain proteins could be approximated by assuming that the mixture contained two classes of proteins with half-lives of 3.3 and 8.7 days, respectively. Half-lives of 2.5 and 7.7 days were estimated for such protein classes in the microsomal fraction. The half-lives of soluble proteins, synaptic membranes, cell body and synaptic mitochondria were 3.1, 5.8, 5.6 and 8.4 days, respectively. Identical results were obtained if the change in specific activity of intact protein labeled by NaH¹⁴CO₃ was followed. Two-fold slower decay rates were obtained when brain proteins were labeled with a pulse of [4,5-³H]leucine or [l-¹⁴C]leucine. Half-lives calculated for the two classes of proteins in whole brain were 8.4 and 16.5 days, respectively with [4,5-³H]leucine and 8.9 and 14.2 days, respectively with [1-¹⁴C]leucine. These results indicate the very significant reutilization of this amino acid in brain. Sodium [¹⁴C]bicarbonate is a more satisfactory isotopic precursor for accurate assessment of rates of protein turnover in brain.     

Incorporation of leucine into phospholipids of Bacteroides thetaiotaomicron.

L-[4,5-3H]- or L-[U-14C]leucine was incorporated by Bacteroides thetaiotaomicron into acid-precipitable material even when the bacteria were treated with concentrations of tetracycline high enough to prevent growth. Similar results were obtained when L-[2,3,4-3H]valine or L-[4,5-3H]isoleucine was used instead of leucine. In bacteria which had been treated with tetracycline, the acid-precipitable label was not solubilized by treatment with protease, lysozyme, or deoxyribonuclease. However, virtually all of the label was extractable with chloroform-methanol, indicating that the label had been incorporated into membrane lipids. Since L-[1-14C]leucine was not incorporated into lipids, leucine was probably decarboxylated before incorporation. When a chloroform extract from bacteria which had been labeled with both [32P]phosphate and [3H]leucine was resolved into component phospholipids by two-dimensional thin-layer chromatography, 3H was incorporated into all of the phospholipids. When these phospholipids were deacylated, the 3H from leucine was associated with released fatty acids rather than with the head groups. Thus, it appears that B. thetaiotaomicron can utilize leucine and similar amino acids not only by incorporating them into protein but also by incorporating portions of these amino acids into membrane phospholipids.     

Chemical measurement of steady-state levels of ten aminoacyl-transfer ribonucleic acid synthetases in Escherichia coli.

Polypeptide chains of 10 aminoacyl-transfer ribonucleic acid synthetases (those for arginine, glutamine, glutamic acid, glycine, isoleucine, leucine, lysine, phenylalanine, threonine, and valine) have been identified in lysates of Escherichia coli resolved by the O'Farrell two-dimensional gel system. By labeling cells uniformly with [14C]glucose and by measuring the total amounts of these polypeptides by their radioactivity, estimations of the steady-state, molecular amounts of these enzymes were made and compared to the number of ribosomes and elongation factors in these cells. Portions of a reference culture grown on glucose and labeled with [14C]leucine or [35S]sulfate were mixed with four cultures grown in widely different media containing [3H]leucine or [3H]leucine plus [3H]isoleucine. From the isotope ratios of the total protein and of the spots containing the synthetase chains, the chemical amount of each synthetase relative to that of the reference culture was determined. The results, where comparable, show reasonable agreement with enzyme activity measurements. In general, these synthetases each exhibit a positive correlation with growth rate in unrestricted media, indicating a strong tendency for the levels of transfer ribonucleic acid, synthetases, elongation factors, and ribosomes to remain approximately, though not exactly, in balance at different growth rates.     

Haem a, cytochrome c and total protein turnover in mitochondria from rat heart and liver

Haem a and cytochrome c were isotopically labelled in mitochondria from rat heart and liver after injection of delta-amino[2,3-(3)H(2)]laevulate, a specific haem precursor. [guanido-(14)C]Arginine or l-[4,5-(3)H(2)]leucine were used to label mitochondrial proteins. Half-lives were measured from biological decay in vivo and were similar (5.5-6.2 days) for haem a, cytochrome c and [(14)C]arginine-labelled proteins. Labelling of hepatic mitochondrial proteins with [(3)H(2)]leucine resulted in a prolonged apparent half-life.     

Stimulation of the biosynthesis of membrane glycoproteins from Zajdela ascites hepatoma cells by Robinia lectin.

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [14C]glucosamine and [3H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated cells by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [14C]glucosamine and [3H]leucine and having different molecular weights, one over 200000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [3H]-glucosamine and from lectin stimulated cells labeled with [14C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.     

L-Leucine activates branched chain alpha-keto acid dehydrogenase in rat adipose tissue

The activity of branched chain alpha-keto acid dehydrogenase in extracts of adipose tissue was elevated after homogenization of tissue segments which had been incubated in buffer containing 0.3 mM leucine. A maximum increase (4-fold) was observed in extracts of tissues incubated in buffer containing 2.5 mM leucine, alpha-Ketoisocaproate and leucine caused maximum increases which were of similar magnitude and which required the same length of incubation of the tissue segments (5 to 15 min). The effect of leucine on branched chain alpha-keto acid dehydrogenase activity was observed both in the presence and absence of insulin, which also increased the activity of the enzyme in tissue extracts. Intact adipose tissue segments oxidized [I-14C]leucine at a maximum rate approximately 4 times that of [1-(14)C]valine. The rate of valine oxidation by intact tissue segments was doubled by addition of 0.2 to 0.5 mM unlabeled leucine, but not isoleucine, to medium containing 2 mM [1-(14)C]valine. Leucine, but not valine, also stimulated the rate of oxidation of 2 mM [U-14C]isoleucine by intact tissue segments. These results suggest that branched chain alpha-keto acid dehydrogenase activity, which is thought to limit the rate of branched chain amino acid oxidation in adipose tissue, may be sensitive to changes in the concentration of leucine in rat blood.     

Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems

Leucine incorporation was examined as a method for estimating rates of protein synthesis by bacterial assemblages in natural aquatic systems. The proportion of the total bacterial population that took up leucine in three marine environments was high (greater than 50%). Most of the leucine (greater than 90%) taken up was incorporated into protein, and little (less than 20%) was degraded to other amino acids, except in two oligotrophic marine environments. In samples from these two environments, ca. 50% of the leucine incorporated had been degraded to other amino acids, which were subsequently incorporated into protein. The degree of leucine degradation appears to depend on the organic carbon supply, as the proportion of 3H-radioactivity incorporated into protein that was recovered as [3H]leucine after acid hydrolysis increased with the addition of pyruvate to oligotrophic water samples. The addition of extracellular leucine inhibited total incorporation of [14C]pyruvate (a precursor for leucine biosynthesis) into protein. Furthermore, the proportion of [14C]pyruvate incorporation into protein that was recovered as [14C]leucine decreased with the addition of extracellular leucine. These results show that the addition of extracellular leucine inhibits leucine biosynthesis by marine bacterial assemblages. The molar fraction of leucine in a wide variety of proteins is constant, indicating that changes in leucine incorporation rates reflect changes in rates of protein synthesis rather than changes in the leucine content of proteins. The results demonstrate that the incorporation rate of [3H]leucine into a hot trichloroacetic acid-insoluble cell fraction can serve as an index of protein synthesis by bacterial assemblages in aquatic systems.     

Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems.

Leucine incorporation was examined as a method for estimating rates of protein synthesis by bacterial assemblages in natural aquatic systems. The proportion of the total bacterial population that took up leucine in three marine environments was high (greater than 50%). Most of the leucine (greater than 90%) taken up was incorporated into protein, and little (less than 20%) was degraded to other amino acids, except in two oligotrophic marine environments. In samples from these two environments, ca. 50% of the leucine incorporated had been degraded to other amino acids, which were subsequently incorporated into protein. The degree of leucine degradation appears to depend on the organic carbon supply, as the proportion of 3H-radioactivity incorporated into protein that was recovered as [3H]leucine after acid hydrolysis increased with the addition of pyruvate to oligotrophic water samples. The addition of extracellular leucine inhibited total incorporation of [14C]pyruvate (a precursor for leucine biosynthesis) into protein. Furthermore, the proportion of [14C]pyruvate incorporation into protein that was recovered as [14C]leucine decreased with the addition of extracellular leucine. These results show that the addition of extracellular leucine inhibits leucine biosynthesis by marine bacterial assemblages. The molar fraction of leucine in a wide variety of proteins is constant, indicating that changes in leucine incorporation rates reflect changes in rates of protein synthesis rather than changes in the leucine content of proteins. The results demonstrate that the incorporation rate of [3H]leucine into a hot trichloroacetic acid-insoluble cell fraction can serve as an index of protein synthesis by bacterial assemblages in aquatic systems.     

Soluble polypeptides from meristematic and mature cells of Allium cepa L. roots

By using electrophoresis on SDS polyacrylamide gels we have studied the soluble protein fraction from a section of the meristem, where most cells are in the division last cycle, and from the mature region of Allium cepa L. roots. In order to estimate the apparent rate of synthesis of these polypeptides we labeled a series of roots with [¹⁴C]leucine and another with [³H]leucine. Coelectrophoresis was carried out by using polypeptides from both regions, their mol.wt. being between 20,000 and 100,000 daltons. The results show that most of the polypeptides in the soluble fraction are constantly present in a cell throughout its development. These constant polypeptides are synthesized at a high rate in the meristematic region. In the mature cells these polypeptides show only a low labeling rate, while a small number of specific polypeptides appear to have a very high rate of metabolism.