Protein synthesis during the cell cycle of L5178Y cells

There are two peaks of ³H-leucine incorporation in the cell cycle of L5178Y cells. The first, during S stage, corresponds to a peak of ³H-leucine incorporation into the nuclear fraction. The second, during S or early G2, corresponds to a peak of ³H-leucine incorporation into the mitochondrial fraction. The rate of protein synthesis is unique for the proteins from each of the four fractions, nuclear, mitochondrial, microsomal, and soluble.The SDS polyacrylamide-gel electrophoretic patterns of ³H-leucine incorporation were different among three subcellular fractions: nuclear, mitochondrial, and microsomal + soluble. However, the incorporation pattern for each fraction remains qualitatively the same throughout the cell cycle.     

Circadian rhythm of tRNA aminoacylation in hepatocyte culture

In the monolayer of rat hepatocytes in vitro, the circadian rhythms were revealed of 3H-leucine incorporation in proteins and in aminoacyl-tRNA(Leu) fraction. The oscillations were mainly synphasic, though coordination between aminoacylation and protein synthesis was not stable in the coarse of time. In cell free system with the excess of ATP and aa-tRNA, the rhythm of 3H-leucine incorporation was also clear. This means, that oscillatory kinetics of the protein synthesis rate is not caused by oscillation of ATP which has been revealed earlier in the hepatocyte monolayer.     

Long-term trophic effect of ACTH on rat adrenocortical cells

Summary The changes occurring in rat adrenocortical cells (zona fasciculata) during an 8 day period of treatment with ACTH, were investigated by morphometric and autoradiographic methods.The most important ultrastructural change consists in a conspicuous increase in the smooth endoplasmic reticulum, that accounts for about 50% of the total increase of cellular volume. Also the mitochondrial fraction shows a significant increase, which is found to be due both to the increment in the number of mitochondria per cell and to the increase in the mean volume of organelles themselves.The quantitative autoradiographic data, indicating an increment in the incorporation of 3H-orotate and 3H-leucine into adrenocortical cells of the treated animals, allow us to conclude that the ACTH-induced ultrastructural changes are the morphological expression of a stimulation of the cellular protein synthesis.Since mitochondria are largely autonomous in the synthesis of their enzymes and structural proteins, it is possible to hypothesize that ACTH also intervenes in the regulation of the mitochondrial protein synthesis.The authors wish to express their sincere appreciation to Mr. G. Gottardo for his excellent technical assistance.     

Changes in RNA and protein synthesis in the neural retina during lens regeneration in newts

Since neural retina stimulates regeneration of a lens from the dorsal iris in newts, RNA and protein synthesis in the neural retina was investigated during this process. Incorporation of 3H-uridine and 3H-leucine using liquid scintillation counting was employed to compare RNA and protein synthesis in the neural retina from sham-operated control eyes with that in eyes during lens regeneration. An initial increase in 3H-uridine uptake was seen one to three days after lentectomy. This was followed by greater incorporation of 3H-leucine, indicating increased protein synthesis between 5 to 15 days after lens removal. A decrease in 3H-uridine uptake was also seen at 5 to 12 days after lentectomy. After 20 days both the RNA and protein synthesis returned to the normal level. Since the increase in protein synthesis is preceded by an increase in RNA synthesis, the two processes might be related. The results indicate significant changes in the synthesis of macromolecules by the neural retina following lentectomy. These may be indirectly related to the production of the neural retinal factor with stimulates lens differentiation.     

The inhibition by high concentrations of (2-chloroethyl)-trimethylammonium chloride (CCC) of chlorophyll and protein synthesis in excised barley leaf sections

Summary CCC at concentrations of 10^-3 M and higher inhibits chlorophyll synthesis and ³H-leucine incorporation into a protein fraction by barley leaf sections. Neither of these effects is reversed by exogenous GA₃. No effect of CCC was observed on oxygen uptake by the leaf sections. The results indicate that high concentrations of CCC may act through an inhibition of protein synthesis, rather than through a direct effect on endogenous gibberellin production.     

Effect of polymyxin-B on T-lymphocyte protein synthesis

The role of protein kinase-C (PK-C) protein phosphorylation on the mitogen triggered responses of T-lymphocytes was examined by observing the effect of polymyxin-B (an inhibitor of PK-C) on mitogen induced protein and DNA synthesis. Polymyxin-B inhibited 3H-thymidine incorporation by PHA activated T-lymphocytes over a range of PHA concentrations. 3H-leucine incorporation by PHA activated T-lymphocytes was inhibited by polymyxin-B in a dose dependent manner. A partially purified PK-C fraction from polymyxin-B treated PHA activated T-lymphocytes demonstrated less than 25% of the phosphorylating activity of untreated lymphocytes. These results suggest that protein synthesis during the T-lymphocyte activation process is dependent on PK-C activity.     

TRANSCRIPTIONAL CONTROL OF PROTEIN SYNTHISIS IN THE DEVELOPING OVARIES OF BOMBYX MORI*

Amino acid incorporation was studied with cell-free extracts and ribosomes prepared from pupal ovaries at different ages of Bombyx mori. Poly(U)-directed ³H-phenylalanine incorporation attained a maximum rate at a certain stage of development, but soon dropped to a low level and was replaced by ³H-leucine incorporation, which was due to endogenous mRNA. The latter incorporation occurred at the stage when actual protein synthesis takes place in the ovaries. “Run-off” of the ribosomes which had a high endogenous activity resulted in an enhancement of the poly(U)-dependent activity. The results indicate that the protein synthesis in the ovary is mainly controlled at the level of mRNA. This was further supported by the fact that the relative amount of an ovarian poly(A)-containing “mRNA” fraction increased in parallel with the endogenous activity.     

Seasonal pattern of glycosylation in frog liver

The circannual behaviour of glycosylation and protein synthesis in frog liver slices was studied following the incorporation of³H-galactose and¹⁴C-glucosamine into glycolipids and glycoproteins and³H-leucine into proteins. The activity of two enzymes the galactosyl-transferase and the N-acetyl-glucosaminyl-1-P-transferase was determined. The incorporations of both sugars into the soluble fraction and into the lipid extract present a maximum during the spring-summer period. The incorporation into the protein fraction displays a different pattern:¹⁴C-Glucosamine and³H-leucine incorporation increases from winter to a maximum in autumn; the incorporation of³H-Galactose has a sharp peak during spring. The pattern of glycosyltransferase activities is similar to the pattern of incorporation of the two saccharides into proteins, indicating these enzymes as important control points for glycosylation in Anurae.     

Evidence pointing to the main role of lysosomes in mitochondrial proteolysis at neutral pH

Recombination experiments using radioactive mitochondria and mitoplasts, and nonradioactive lysosomes or digitonin-soluble fraction of mitochondria, show equal rates of proteolysis and of inactivation of carbamyl phosphate synthetase; the amount of lysosomal protein was equal in both cases on the basis of N-acetyl-beta-glucosaminidase activity. Therefore, lysosomes seem to be responsible for all the proteolytic activity exhibited by the digitonin soluble fraction of mitochondrial preparations. Since this fraction contains ca. 90% of the proteolytic activity present in mitochondrial preparations, most of the proteolysis can be attributed to lysosomal contamination. These findings and stability characteristics "in vitro" and "in vivo" of some matrix enzymes are presented and discussed in relation to protein turnover.     

Rapid axoplasmic transport of free leucine

Axoplasmic transport of free 3H-leucine has been studied in vivo in the pike olfactory nerve following application of labeled leucine to the olfactory mucosa. A considerable amount of free 3H-leucine is transported at constant velocity along the axon in the form of a distinct peak. The maximum transport velocity for free 3H-leucine is the same as for rapidly transported 3H-protein (130 and 135 mm/day, respectively, at 19 degrees C). Microtubule inhibitors block or significantly reduce the amount of free 3H-leucine transported, but do not influence the transport velocity. Disruption of the oxygen supply abolishes free 3H-leucine transport, so that this phenomenon cannot be explained by diffusion. The amount of free leucine in the rapidly moving peak decreases with time and distance along the axon and is not detectable after 5 h or more. The transported 3H-leucine is not derived from the circulation or from proteolysis of rapidly transported proteins. This study may help to resolve the controversy over the axoplasmic transport of free amino acids since it shows that free 3H-leucine is transported rapidly but does not travel by rapid axoplasmic transport to the end of axons longer than about 30 mm.     

Effect of testosterone on the rate of total protein synthesis in the reproductive tract of guinea pig embryos in vitro

The influence of testosterone on the rate of total protein synthesis in the reproductive tract tissues and muscles was studied in the guinea pig male embryos from 26 days of development until birth. The rate of protein synthesis was estimated by the intensity of 3H-leucine incorporation per 1 mg protein for 30 min. Testosterone increased the rate of protein synthesis at all studies stages. A higher rate of protein synthesis was revealed at the stages of active morphogenesis and growth of rudiments and organs of genital system and correlated with their ability of 3H-testosterone uptake (Ivanova, 1982).     

Proteolysis in mitochondrial preparations and in lysosomal preparations derived from rat liver

A method of preparing rat liver mitochondria with low residual contamination by lysosomal proteases is described. Preparations of mitochondria are divided into two equal portions, one of which is supplemented with a lysosomal fraction. The addition of the lysosomal fraction causes an increase in proteolysis of between 26- and 56-fold at pH 5.0 in four similar experiments. This increase matches the increase in the lysosomal marker beta-glucuronidase and indicates that all proteolysis at pH 5.0 is due to enzymes of the lysosomal fraction. Above pH 7.0, the addition of a lysosomal supplement increases proteolysis by 1.5- to 5-fold only, suggesting that in the absence of a lysosomal supplement very little of the observed proteolysis is due to enzymes of lysosomal origin. A method of calculating the contribution to total proteolysis of enzymes of the lysosomal fraction or of the mitochondrial fraction is described. The calculations show that at pH 7.0 and above, more than 93% of the observed proteolysis is due to enzymes originating in the mitochondrial fraction. The results support the view of other workers that rat liver mitochondria contain an endogenous neutral proteolytic system capable of degrading mitochondrial proteins to acid-soluble products.     

Mitochondrial protein synthesis in a mammalian cell-line with a temperature-sensitive leucyl-tRNA synthetase.

The temperature-sensitive Chinese hamster ovary cell mutant tsH1, has been shown previously to contain a temperature-sensitive leucyl-tRNA synthetase. At the non-permissive temperature of 40 degrees C cytosolic protein synthesis is rapidly inhibited. The protein synthesis which continues at 40 degrees C appears to be mitochondrial, since: (a) whole-cell protein synthesis at the permissive temperature of 34 degrees C is not inhibied by tevenel, the sulfamoyl analogue of chloramphenicol and a specific inhibitor of mitochondrial protein synthesis; however, whole-cell protein synthesis at 40 degrees C is inhibited by tevenel, (b) Protein synthesis by isolated mitochondria from tsH1 cells is not significantly inhibited at 40 degrees C. (c) At 40 degrees C [14C]leucine is incorporated predominantly into the mitochondrial fraction of tsH1 cells. (d) The incorporation of [14C]leucine at 40 degrees C into mitochondrial proteins of tsH1 cells is inh-bited by tevenel but not by cycloheximide. These results suggest that the mitochondria of tsH1 cells contain a leucyl-tRNA synthetase which is different from the cytosolic enzyme. The inhibition of cytosolic, but not of mitochondrial protein synthesis in tsH1 cells at 40 degrees C allows the selective labelling of mitochondrial translation products in the absence of inhibitors. The mitochondrial translation products labelled in tsH1 cells at 40 degrees C and at 34 degrees C in the presence of cycloheximide have been compared by sodium dodecylsulphate-polyacrylamide gel electrophoresis. Both conditions of labelling give similar profiles. The mitochondrial translation products are resolved into two components, one with an apparent molecular weight range from 40,000 to 20,000 and a second with an apparent molecular weight range from 20,000 to 10,000.     

Effect of parvalbumin and S-100 protein on protein synthesis in rabbit reticulocyte lysate.

The effect of two high affinity Ca+ binding acidic proteins, parvalbumin and S-100 protein on protein synthesis in rabbit reticulocyte lysates (RRL), was investigated. Nuclease-treated RRL, supplemented with yeast mRNA, and 3H-leucine were incubated at 37 degrees C, and incorporation of 3H-leucine into protein was determined for 24 min. At 20 micrograms/100 microliters lysate concentration, both parvalbumin and S-100 protein caused a marked inhibition of protein synthesis compared with the control lysate. At a lower concentration parvalbumin was less inhibitory than histone H1; the effect of S-100 protein was not significant. The combined inhibitory effect of parvalbumin and H1 was not additive probably due to strong interaction between them as was evidenced by the enhanced absorbance of parvalbumin-H1 mixture. Spectrophotometric profiles of parvalbumin-tRNA mixture indicated that, unlike H1, parvalbumin did not inhibit protein synthesis by binding with nucleic acids. These results suggest an important role for parvalbumin in translational regulation.     

Effect of testosterone on the rate of total protein synthesis in the reproductive tract of rabbit embryos in vitro

The total protein synthesis rate in the reproductive tract and muscles of rabbit fetuses was evaluated at the 18th-22nd day of development by the intensity of 3H-leucine incorporation in experiments in vitro. Testosterone increased the total protein synthesis rate in the tissues of the reproductive tract at all the developmental stages under study. The highest rate of protein synthesis was recorded on the 18th day of development.     

ConA induced changes in energy metabolism of rat thymocytes

The influence of ConA on the energy metabolism of quiescent rat thymocytes was investigated by measuring the effects of inhibitors of protein synthesis, proteolysis, RNA/DNA synthesis, Na⁺K⁺-ATPase, Ca²⁺-ATPase and mitochondrial ATP synthesis on respiration. Only about 50% of the coupled oxygen consumption of quiescent thymocytes could be assigned to specific processes using two different media. Under these conditions the oxygen is mainly used to drive mitochondrial proton leak and to provide ATP for protein synthesis and cation transport, whereas oxygen consumption to provide ATP for RNA/DNA synthesis and ATP-dependent proteolysis was not measurable. The mitogen ConA produced a persistent increase in oxygen consumption by about 30% within seconds. After stimulation more than 80% of respiration could be assigned to specific processes. The major oxygen consuming processes of ConA-stimulated thymocytes are mitochondrial proton leak, protein synthesis and Na⁺K⁺-ATPase with about 20% each of total oxygen consumption, while Ca²⁺-ATPase and RNA/DNA synthesis contribute about 10% each. Quiescent thymocytes resemble resting hepatocytes in that most of the oxygen consumption remains unexplained. In contrast, the pattern of energy metabolism in stimulated thymocytes is similar to that described for Ehrlich Ascites tumour cells and splenocytes, which may also be in an activated state. Most of the oxygen consumption is accounted for, so the unexplained process(es) in unstimulated cells shut(s) off on stimulation.     

ConA induced changes in energy metabolism of rat thymocytes

The influence of ConA on the energy metabolism of quiescent rat thymocytes was investigated by measuring the effects of inhibitors of protein synthesis, proteolysis, RNA/DNA synthesis, Na⁺K⁺-ATPase, Ca²⁺-ATPase and mitochondrial ATP synthesis on respiration. Only about 50% of the coupled oxygen consumption of quiescent thymocytes could be assigned to specific processes using two different media. Under these conditions the oxygen is mainly used to drive mitochondrial proton leak and to provide ATP for protein synthesis and cation transport, whereas oxygen consumption to provide ATP for RNA/DNA synthesis and ATP-dependent proteolysis was not measurable. The mitogen ConA produced a persistent increase in oxygen consumption by about 30% within seconds. After stimulation more than 80% of respiration could be assigned to specific processes. The major oxygen consuming processes of ConA-stimulated thymocytes are mitochondrial proton leak, protein synthesis and Na⁺K⁺-ATPase with about 20% each of total oxygen consumption, while Ca²⁺-ATPase and RNA/DNA synthesis contribute about 10% each. Quiescent thymocytes resemble resting hepatocytes in that most of the oxygen consumption remains unexplained. In constrast, the pattern of energy metabolism in stimulated thymocytes is similar to that described for Ehrlich Ascites tumour cells and splenocytes, which may also be in an activated state. Most of the oxygen consumption is accounted for, so the unexplained process(es) in unstimulated cells shut(s) off on stimulation.     

Degradation of a cAMP-binding protein is inhibited by human c-Ha-ras gene products

Incubation of the particulate fraction of cell extract prepared from NIH3T3 mouse fibroblasts resulted in preferential proteolytic degradation of a cAMP-binding protein. The proteolysis was inhibited by human c-Ha-ras gene products produced by Escherichia coli. The proteolysis was observed at pH 6 to 7, and inhibited by antipain and leupeptin. These results suggest that cAMP-binding proteins might be cleaved by thiol proteinases. In fact, c-Ha-ras gene products were proved to inhibit the cathepsin B-like activity present in the particulate fraction.     

The Effect of Estradiol Dipropionate on the Rate of Protein Synthesis in the Testicle of the Sea Urchin Strongylocentrotus nudus

The effect of estradiol dipropionate on the rate of protein synthesis in the testicle of the mature sea urchin Strongylocentrotus nuduswas studied. The injection of this estrogen considerably intensified ³H-leucine incorporation into the gonad. The change in the level of cell synthetic activity is assumed to be associated with a rise in effective incorporation of ³H-leucine into proteins following an increase in its intracellular level, and with an increase in the protein synthesis rate. The participation of sex hormones in the regulation of protein synthesis in the S. nudusgonad is discussed.