Inhibition Of Amino Acid Incorporation Research Articles

Fatty acid inhibition of somatomedin (serum sulfation factor)-stimulated protein and RNA synthesis in embryonic chicken cartilage

The effects of fatty acids and serum containing somatomedin on in vitro synthesis of collagen, chondromucoprotein, general proteins and RNA were studied in embryonic chicken cartilage. Somatomedin (5 % rat serum added) stimulated 35SO 2− 4, [ 3H]leucine and [ 3H]tryptophan incorporation into chondromucoprotein-rich and collagen-rich cartilage protein fractions; and [ 3H]proline incorporation and [ 3H]hydroxyproline synthesis in the collagen-rich fraction. Somatomedin also stimulated [ 3H]uridine incorporation into RNA. In the non-stimulated state (no serum added) butyrate and octanoate had little or no effect on amino acid incorporation into protein or uridine incorporation into RNA. In the serum stimulated state, butyrate and octanoate inhibited amino acid incorporation into protein and uridine incorporation into RNA. Such inhibition was observed only with serum containing growth hormone-dependent sulfation factor (somatomedin). Agarose column chromatography of the chondromucoprotein-rich fraction revealed that while somatomedin stimulates synthesis of all proteins of this fraction equally, butyrate inhibits stimulated chondromucoprotein synthesis to a greater extent than certain other cartilage proteins (which are inhibited little, if at all). The data indicate that somatomedin stimulates embryonic chicken cartilage RNA, collagen, chondromucoprotein and general protein synthesis. Fatty acids have little or no effect on unstimulated cartilage macromolecular synthesis. The somatomedin stimulation of RNA, collagen and chondromucoprotein synthesis, and some, but not all of protein synthesis other than collagen or chondromucoprotein, is inhibited by fatty acids.

Relationship between dimorphology and respiration in Mucor genevensis studied with chloramphenicol.

Growth of Mucor genevensis, a facultatively anaerobic dimorphic mold, in high concentrations of chloramphenicol (4 mg/ml) leads to increased numbers of yeast-like cells and small club-like mycelial forms. This change in morphology is accompanied by a threefold increase in the mass doubling time, the loss of cyanide-sensitive respiration, and the development of cyanide-insensitive respiration. Associated with these changes is the absence of cytochromes aa(3) and b and the inability of the organism to utilize ethanol; in addition, mitochondria appear more numerous and have less internal membrane. A further inhibitory action of the antibiotic, other than eliminating functional mitochondria, appears likely since microaerobic cultures which lack respiratory ability have twice the mass doubling time in the presence of the drug. Although a small inhibition of amino acid incorporation by cytoplasmic ribosomes is found with a high chloramphenicol concentration, it is insufficient to account for the effect on growth of the microaerobic culture. The nature of this additional effect of chloramphenicol remains to be determined, but it has been shown that increasing the glucose concentration can partially reverse this action of the antibiotic. The effect of the drug on the morphology of the organism is not as dramatic as that of phenethyl alcohol in producing yeast-like forms. However, in view of the action of chloramphenicol in eliminating functional mitochondria in M. genevensis the suggestion that phenethyl alcohol exerts its effect in promoting yeast-like morphology by uncoupling oxidative phosphorylation should be re-examined.

Inhibition of amino acid incorporation into protein of human neutrophils by phagocytosis.

The incorporation of uniformly labeled (14)C-amino acids into total protein of human polymorphonuclear leukocytes is inhibited when the cells are challenged with heat-killed Escherichia coli or latex particles. Parallel studies of amino acid incorporation and glucose oxidation indicate that the inhibition is dependent upon ingestion of the particles. It is suggested that the process of phagocytosis might inhibit the transport of amino acids into the cell either by competing for available energy or by internalizing specific transport sites.

The dependence of brain ATP content on cerebral electroshock current

A question of continuing interest is the nature of the biochemical lesion responsible for the amnesia caused by cerebral electroshock. Very many studies have been concerned with this problem, but apparently the effect of electroshock is so gross that almost all brain chemicals that have been studied are affected (see ref. 6). It is likely that many of the changes result from the convulsions induced by the electroshock. Since the occurrence of bodily convulsions is not necessary for the amnesia3,7,9,11,12, examination of the biochemical effects of subconvulsive electroshock may simplify the problem. The convulsions associated with electroshock may be prevented either by the use of anesthetics or by decreasing the electroshock current. The effect of subconvulsive electroshock on the incorporation of radioactive amino acids into brain protein has been examined using both these techniques. Cotman et aL ~ showed that diethyl ether, which inhibited the convulsions but not the brain seizure, did not prevent the inhibition of amino acid incorporation caused by the electroshock. Significant inhibitions of the incorporation were only observed when brain seizures occurred. Dunn 4 showed that the inhibition of amino acid incorporation was linearly correlated with the current used in the electroshock; the presence or absence of convulsions did not especially disturb this correlation. A similar relationship between electroshock current and retrograde amnesia has also been observed 8,7, 9,12. Thus a convenient way of studying the interrelationships of the effects of electroshock is to relate the behavioral, electrophysiological and biochemical data to the electroshock current. The depression of amino acid incorporation into brain protein may be caused by a depression of cerebral ATP levels or the cation imbalance caused by the electroshock4, s. Severe disturbances of brain ATP are induced by the electroconvulsive shock s, but, since these may be prevented by administration of oxygen 1, the disturbances may be provoked by the anoxia associated with the convulsions, rather than the electroshock itself. Nevertheless, anesthetization of mice with secobarbitone prevented the convulsions but not the depression of ATP associated with electroconvul-

Hypolipemic action of glucagon in experimental endogenous lipemia in the rat

The effect of glucagon on serum lipids and very low density lipoproteins (VLDL) has been examined in the eulipemic and the hyperlipemic rat. An inhibition of amino acid incorporation into hepatic lipoprotein apoprotein was observed, with an associated decrease in circulating VLDL apoprotein, decreased serum triglyceride concentration, and a loss of the pre-beta band as judged by serum lipoprotein electrophoresis. The data suggest that an important action of this hormone is to decrease the synthesis of the protein moiety of the VLDL; this may contribute to the hypolipemic action of glucagon by introducing a limitation in hepatic lipoprotein production.

Inhibition of peptide bond formation by cytidyl derivatives of puromycin

Preparation of cytidine-2′(3′)-P-5′- puromycin mixed isomers as well as cytidine 3′-P-5′- puromycin and cytidine-2′-P-5′- puromycin are described. The ability of these substances to inhibit amino acid incorporation in a cell-free protein-synthesizing system from rabbit reticulocytes has been determined over a wide range of concentration. On the basis of the concentration required to produce 50 % inhibition of amino acid incorporation, cytidine-3′-P-5′- puromycin was approximately 7 times more potent than puromycin itself. Cytidine-2′-P-5′- puromycin , on the other hand, was 22 times less effective than puromycin. The implications of these results regarding the substrate specificity of peptidyl transferase and the ribosomal acceptor site are discussed.

Morphogenetic disturbances from timed inhibitions of protein synthesis in Fundulus

ABSTRACT The reversible inhibition of protein synthesis at the 75 –95% level in the early zygote of Fundulus results in a specific series of developmental failures dependent upon the times of inhibitor pulse initiation. The severity of the morphogenetic failure is inversely related to the time of initiation and directly to the length of the pulse. The defects reflect the time dependent serial order of events in morphogenesis. The defects range from failure of cleavage through disorders of blastulation, failure of axiation, anencephaly to microcephaly and are entirely predictable. With the exception of cleavage failure the pattern is identical with that found using pulses of actinomycin D in a similar manner. The agent used was pactamycin, an antibiotic which reversibly inhibits amino acid incorporation into protein by disturbing the assembly of the functional ribosomal complex. The significance of time dependent protein synthesis as an active expression in morphogenesis of similarly time dependent information flow via RNA synthesis is discussed.

Cerebral anoxia: protein metabolism during recovery in in vitro model.

Amino acid incorporation into proteins of rabbit brain slices was studied during the recovery stage after various anoxic periods in an in vitro experimental model. There was a distinct difference between anoxic periods up to five minutes and those beyond ten minutes. In the former, inhibition of amino acid incorporation was of lesser degree and was reversible. On the other hand, more than 50% inhibition was observed following anoxic periods of 30 minutes and this inhibition was irreversible. Amino acid incorporation into proteins was more sensitive to anoxia than was amino acid transport into brain slices.

Inhibition of the assembly of cytochrome oxidase in Neurospora crassa by chloramphenicol.

Cytochrome oxidase was prepared from Neurospora crassa by chromatography on oleyl poly‐methacrylic acid resin and separated into seven polypeptides by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Incorporation of labelled amino acids into the single polypeptides was investigated after a pulse labelling in the absence and presence of chloramphenicol, and after washing out the inhibitor.Chloramphenicol (4 mg/ml) inhibited amino acid incorporation into all polypeptides 90–95% while labelling of the whole membrane protein was inhibited only 30%. After washing out the inhibitor and further growth of the cells, the four smaller polypeptides were highly labelled, whereas the other polypeptides showed only a small increase in radioactivity. It is concluded that the four small‐sized polypeptides of cytochrome oxidase are synthesized but not integrated into the functional enzyme under the action of chloramphenicol.

Amino acid incorporating activity of cell-free preparations from chicken liver and the factors that control the process

The amino acid incorporating activity of chicken liver cell-free preparation was only one third of that noted with a similar preparation from rat liver. Similar preparations from rat and chicken kidneys also showed very low amino acid incorporating activities. The cell-free preparation obtained by homogenizing chicken liver in rat liver cell sap was significantly more active than the chicken liver preparation whereas a similar preparation obtained by homogenizing rat liver in chicken liver cell sap was considerably less active than the rat liver preparation. The incorporation of amino acid was also higher when chicken liver was homogenized in the presence of bentonite and dextran but not in the presence of RNA. Maximum incorporation occurred at 40 °. A comparison of 14C — leucine incorporation into proteins by homologous and heterologous liver preparations from rat and chicken livers indicated higher activity of rat liver ribosomes than the chicken liver ribosomes measured in cell-free amino acid incorporating systems in the presence of cell sap or pH 5.0 fraction. Higher activity was also found to be associated with the ribosomes obtained by homogenizing chicken liver in rat liver cell sap compared to that of the ribosomes obtained by homogenizing rat liver in chicken liver cell sap. Co 2+ and Ni 2+ inhibited amino acid incorporation. Ni 2+ was more effective than Co 2+. Pre-incubation of cell-free preparations (or microsome — pH 5.0 fraction) from rat and chicken livers with pancreatic ribonuclease and venom phosphodiesterase rendered the preparations inactive. The degree of inhibition was dependent upon the amount of enzyme added. Chicken liver preparation was more sensitive than rat liver preparation to ribonuclease, but they both were equally affected by venom phosphodiesterase. The results presented in this paper indicated an inverse relationship between amino acid incorporating activity and ribonuclease activity. The study of polysome profiles of deoxycholate treated post-mitochondrial supernates from rat and chicken livers showed fewer polysomes in chicken liver preparation compared to the number of polysomes found in rat liver preparation. The low amino acid incorporating activity of chicken liver cell-free preparation is due to the presence of fewer polysomes in the preparation because of high ribonuclease activity in chicken liver.

Effects of Protease Inhibitors on Protein Breakdown in Escherichia coli

Abstract In an attempt to learn more about the proteolytic enzymes responsible for protein turnover in cells, the effects of various protease inhibitors on protein catabolism in Escherichia coli were examined. Diisopropyl fluorophosphate, phenylmethane sulfonyl fluoride, and toluenesulfonyl fluoride, all of which inhibit serine proteases, were found to decrease protein breakdown in E. coli, starved for a carbon source. These compounds partially inhibited protein breakdown at doses which did not inhibit growth on minimal medium. The effects of the sulfonyl fluorides were reversed by washing the cells. Protein breakdown in starving cells could also be inhibited reversibly by the aromatic diamidines, dibromopropamidine and pentamidine, which are potent trypsin inhibitors. The effects of pentamidine and p-toluenesulfonyl fluoride did not require concomitant protein synthesis. Tosyl lysine chloromethyl ketone, an inhibitor of trypsin, also inhibited protein breakdown in starving cells. A number of other protease inhibitors were tested and found either to have no effect on intact cells or to prevent normal cell growth. These findings suggest that at least one of the enzymes responsible for the increased protein breakdown in starving cells is a serine protease, possibly similar to trypsin. In cells deprived of a carbon or nitrogen source, synthesis of new enzymes should be dependent upon the breakdown of previously existent cell proteins. Treatment with the sulfonyl fluorides or aromatic diamidines inhibited amino acid incorporation into proteins under such conditions, but did not do so in growing cells. Similarly these compounds reversibly inhibited the induction of β-galactosidase and tryptophanase in cells deprived of a carbon or nitrogen source, but did not do so in cells growing on glycerol. The sulfonyl fluorides, the aromatic diamidines, and tosyl lysine chloromethyl ketone did not reduce the relatively low amount of protein breakdown found in growing cells. Growing cells degrade selectively or aberrant proteins, and the rate of protein breakdown in such cells increased markedly when they were grown on amino acid analogs, canavanine or threo-α-amino-β-chlorobutyric acid. This accelerated degradation also was not inhibited by the sulfonyl fluorides or the diamidines. These results thus suggest that there may exist at least two proteolytic systems in E. coli: one operating in all cells, which can degrade abnormal proteins, and one found in starving cells which is sensitive to sulfonyl fluorides and aromatic diamidines.

Studies on the inhibition of amino acid incorporation into protein by isolated rat liver ribosomes by protein kinase

Studies on amino acid incorporation into protein by isolated ribosome-pH 5 enzyme system show that incorporation was inhibited by the addition of protein kinase and cyclic AMP. The inhibition observed was 30, 50 and 70% with 14, 28 and 56 γ of protein kinase, respectively. Added protein kinase also inhibited poly U stimulated amino acid incorporation into protein. This inhibition observed was not due to the decrease in the availability of ATP. Protein kinase had no effect on either the activation of amino acid or the transfer of activated amino acid to soluble RNA. These studies indicate the phosphorylation of the ribosome fraction bringing about the inhibition of amino acid incorporation into proteins.

Protein synthesis in human leucocytes. IV. Mutual inhibition of amino acid incorporation by amino acids in cell suspensions and cell-free systems.

Article Protein Synthesis in Human Leucocytes, IV. Mutual Inhibition of Amino Acid Incorporation by Amino Acids in Cell Suspensions and Cell-free Systems was published on January 1, 1972 in the journal Biological Chemistry (volume 353, issue 1).

Effect of method of cell isolation on the metabolic activity of isolated rat liver cells.

ABSTRACT Rat liver cells isolated with a tissue press, by tetraphenylboron (TPB) chelation, or by hyaluronidase and collagenase digestion were compared as to morphology, cell yield, and biosynthetic activity. The cells were intact by light microscopy; ultrastructural changes were present on electron-microscopic examination of all cell types except those prepared by a modified enzyme incubation method. TPB chelation gave the largest and enzyme techniques the smallest yield of cells. All cell types incorporated labelled amino acids into cellular protein; however, amino acid incorporation was greatest in cells isolated by the revised enzyme technique. Only enzyme and mechanical cells incorporated acetate into cellular lipid. Cofactor supplementation was not required in the modified enzyme cells. Acetate incorporation was more sensitive to preincubation than was amino acid incorporation. Calcium, which was required to prevent aggregation of enzyme cells, inhibited amino acid incorporation moderately and acetate incorporation completely in mechanical cells.

On the requirement for calcium during the steroidogenic effect of ACTH.

The requirement for calcium during the steroidogenic effect of both ACTH and adenosine-3’,5’-cyclophosphate (cyclic-AMP) has been studied. Upon incubating rat adrenal sections, deletion of calcium from the incubation media decreased the steroidogenic effectiveness of both ACTH and cyclic-AMP by approximately 40-50%. Leucine incorporation into protein was diminished to a comparable degree, and there was a direct correlation between inhibition of steroidogenic responsiveness and inhibition of amino acid incorporation into protein. Whereas protein synthesis was inhibited by deleting calcium, incorporation of orotic acid into RNA was increased, and it would appear that the inhibitory effects of deleting calcium are not simply due to a generalized inhibition of cellular functions which require energy (ATP) production. The present results suggest that a major reason for the calcium requirement during ACTH and cyclic-AMP action is the maintenance of optimal adrenal protein synthesis, which in turn is thought to ...

Concerning the sites of synthesis of proteins of chloroplast ribosomes and of fraction I protein (ribulose-1,5-diphosphate carboxylase)

Summary Chloramphenicol and spectinomycin selectively inhibit amino acid incorporation into Fraction I protein but have no effect on incorporation into Fraction II protein. Neither antibiotic depresses specific radioactivities of either 70 or 80 S ribosomes. Cycloheximide inhibits incorporation of amino acid into Fraction I and Fraction II proteins equally. The specific radioactivities of 70 S and 80 S ribosomes are depressed equally by cycloheximide. These results suggest that most of the proteins of 70 S chloroplast ribosomes are made outside the chloroplast. They also confirm previous findings that accumulation of ribulose-1,5-diphosphate carboxylase is inhibited by both chloramphenicol and cycloheximide.

Effects of 3′deoxyadenosine (cordycepin) and 2′deoxyadenosine on nucleoside transport, macromolecular synthesis, and replication of cultured Novikoff hepatoma cells

3′Deoxyadenosine (cordycepin) inhibits the incorporation of uridine and adenosine into RNA and, to a lesser extent, DNA by Novikoff rat hepatoma cells growing in suspension culture without affecting the intracellular phosphorylation of the nucleosides to the triphosphate level. Although 3′deoxyadenosine competitively inhibits adenosine and uridine transport into the cell, this effect plays a minor role in its inhibition of nucleoside incorporation into nucleic acids. In contrast, 2′deoxyadenosine inhibits uridine and adenosine incorporation into nucleic acids in direct proportion to its effect on nucleoside transport and it is three to four times more effective as competitive inhibitor of nucleoside transport than is 3′deoxyadenosine. The latter inhibits preferentially the synthesis of ribosomal precursor RNA and transfer RNA, whereas the synthesis of heterogeneous nucleoplasmic RNA is little affected. It also inhibits to a much greater extent uridine incorporation into CMP than UMP of RNA. In addition, 3′deoxyadenosine inhibits amino acid incorporation into protein to about the same extent as DNA synthesis and, at 0.4 m m, stops very rapidly and completely cell division. These and other results suggest that most of the multiple effects of 3′deoxyadenosine might be related to an interference by 3′deoxyadenosine nucleotides with various normal functions of ATP and 2′deATP.

Studies on the origin of proteins in the nucleoli of mouse ascites tumour cells: A test for protein transfer to the nucleoli

Since it has been reported from this laboratory that the nucleoli of mouse ascites tumour cells are incapable of synthesizing proteins via a typical ribosomal system, the possibility of transfer of newly synthesized protein molecules to the nucleoli from other cellular components was tested. 1. 1. Pulse-chase experiments of amino acids were carried out on ascites tumour cells cultivated temporarily in a defined medium, RPMI 1640 supplemented with 5 to 10 % calf serum. Virtually complete inhibition of amino acid incorporation into subcellular components was attained by administration of both cycloheximide (100–200 μg/ml) and an excess of unlabeled amino acids (7500 times). 2. 2. After a 1- to 2-min pulse, radioactivity in the nucleoli was increased by 70 to 100 % during a 20-min chase. Accumulation of the labeled protein during the chase was also observed, but to a smaller extent, in an extranucleolar nuclear fraction. These increments found in the subnuclear fractions were always accompanied by a significant decline of the radioactivity in the cytoplasmic 25 000 × g supernatant fraction. 3. 3. Polyacrylamide gel electrophoresis demonstrated that the pattern of basic proteins labeled by a 1-min pulse and a subsequent 20-min chase was exactly the same as that obtained by a 1-min pulse only. 4. 4. The decline of radioactivity in cytoplasmic 25 000 × g supernatant fraction during the chase was shown to be the result of a remarkable decrease in the 105 000 × g sediment, microsomes and a concomitant but smaller increase in the 105 000 × g supernatant fraction. 5. 5. Protein transfer to the nucleoli was affected by temperature and by both actinomycin D and 2,4-dinitrophenol. These effects suggest that the protein transfer may not be a simple diffusion process, but an active one mediated by RNA content and by ATP level. 6. 6. These results strongly suggest that active incorporation of amino acids into the nucleoli in vivo is largely, if not entirely, based on the uptake of protein molecules synthesized on the outside of this organelle, probably in the cytoplasmic polysomes.

Effects of 1-( o-chlorophenyl)-1-( p-chlorophenyl) -2, 2-dichloroethane and puromycin on adrenocorticotropic hormone-induced steroidogenesis and on amino acid incorporation in slices of dog adrenal cortex

In adrenal slices from control dogs, ACTH in vitro produced a 3-fold increase in steroid production but had no effect on incorporation of 14C-labeled amino acids into adrenal cortical protein. Puromycin in vitro partially inhibited the steroidogenic response to ACTH and almost completely inhibited amino acid incorporation. Administration in vivo of 1-( o-chlorophenyl)-1-( p-chlorophenyl)-2, 2-dichloroethane ( o, p'-DDD), 2–48 hr prior to adrenalectomy, completely blocked the steroidogenic response to ACTH and had the paradoxical effect of producing a marked increase in incorporation of labeled amino acids into protein. Possible implications of these data are presented.

Relationships between the influence of cortisol on tissue amino acid accumulation and amino acid incorporation in protein, and the cortisol inhibition of substrate metabolism.

The in vitro effect of cortisol on the cellular accumulation of free amino acid or a-aminoisobutyric acid and the incorporation of amino acid into protein was studied using the mouse ear strip preparation. It was determined that cortisol strongly inhibited amino acid incorporation into protein in glucose medium, weakly inhibited this incorporation in pyruvate medium and did not inhibit it at all in succinate medium. The cortisol inhibition of the metabolism of these substrates to CO2 followed the same sequence, i.e., glucose, pyruvate and no inhibition of succinate. Cortisol usually shows at least a 3-hr latent period in glucose medium before an inhibition of amino acid incorporation into protein can be demonstrated. This latent period is decreased to less than 30 min by preincubation of the tissue with cortisol in succinate medium. These data are interpreted to mean that this cortisol action on protein synthesis is manifest through inhibition of specific substrate metabolism and subsequent energy generat...