scholarly journals Aminoacyltransferase I-catalysed binding of phenylalanyl-transfer ribonucleic acid to muscle ribosomes from normal and diabetic rats

1971 ◽  
Vol 124 (3) ◽  
pp. 537-541 ◽  
Author(s):  
D. P. Leader ◽  
I. G. Wool ◽  
J. J. Castles
Keyword(s):  
Escherichia Coli ◽  
Skeletal Muscle ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Ribonucleic Acid ◽  
Diabetic Rats ◽  
The Difference ◽  
Escherichia Coli B

The aminoacyltransferase I-catalysed binding of phenylalanyl-tRNA (unfractionated Escherichia coli B tRNA acylated with radioactive phenylalanine and 19 non-radioactive amino acids) to skeletal-muscle ribosomes from diabetic rats was less than that to ribosomes from normal rats when the Mg2+ concentration was low (7.5mm); whereas just the reverse was true when the concentration of the cation was higher (15mm). Thus the Mg2+ dependency of aminoacyltransferase I-catalysed binding of phenylalanyl-tRNA to ribosomes from normal and diabetic rats paralleled the effect of Mg2+ concentration on synthesis of polyphenylalanine reported before. During incubation at 7.5mm-Mg2+ phenylalanyl-tRNA was bound only to ribosomes bearing nascent peptidyl-tRNA. There are fewer such ribosomes in a preparation from the muscle of diabetic animals because diabetic animals synthesize less protein in vivo. Thus the difference in polyphenylalanine synthesis in vitro is adequately explained by the difference in enzyme-catalysed binding of phenylalanyl-tRNA to ribosomes, however, the basis of the difference in protein synthesis in vivo is still unknown.

Mechanism of action of an inhibitor from Proteus vulgaris on cell-free protein synthesis by Escherichia coli B

1969 ◽  
Vol 15 (2) ◽  
pp. 159-164
Author(s):  
J. J. McEvoy ◽  
W. E. Inniss
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Mechanism Of Action ◽  
Ribonucleic Acid ◽  
Proteus Vulgaris ◽  
Flash Evaporation ◽  
Free System ◽  
Messenger Ribonucleic Acid ◽  
Escherichia Coli B

An inhibitory substance(s) has been found in S-30 fractions from Proteus vulgaris which prevented an Escherichia coli B cell-free system from incorporating a mixture of 14C-amino acids, L-phenylalanine-14C, or L-lysine-14C into protein, as directed by natural messenger ribonucleic acid, polyuridylic acid, or polyadenylic acid respectively. Similar results were obtained when the inhibitor was isolated from S-100 fractions by using dialysis, concentration of the dialysate by flash evaporation, hydrolysis, evaporation to dryness, dissolution to the original volume in distilled water, and neutralization. The effect of the inhibitor on the various individual reactions involved in protein synthesis was examined. No effect was found on the activation of amino acids as determined by the formation of L-phenylalanine-14C hydroxamate isolated chromatographically or by adenosine triphosphate – pyrophosphate exchange. Also no inhibition of L-phenylalanine-14C attachment to transfer ribonucleic acid occurred. However, ribosome-dependent reactions were markedly inhibited. The mechanism of action of the inhibitor appeared to be the prevention of binding of phenylalanyl-transfer ribonucleic acid to the ribosomes.

scholarly journals Identification in skeletal muscle of a distinct extracellular pool of amino acids, and its role in protein synthesis

1971 ◽  
Vol 121 (5) ◽  
pp. 817-827 ◽  
Author(s):  
R. C. Hider ◽  
E. B. Fern ◽  
D. R. London
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Incubation Medium ◽  
Extensor Digitorum Longus ◽  
Extensor Digitorum ◽  
Longus Muscle ◽  
Kinetics Of

1. The kinetics of radioactive labelling of extra- and intra-cellular amino acid pools and protein of the extensor digitorum longus muscle were studied after incubations with radioactive amino acids in vitro. 2. The results indicated that an extracellular pool could be defined, the contents of which were different from those of the incubation medium. 3. It was concluded that amino acids from the extracellular pool, as defined in this study, were incorporated directly into protein.

Effect of Cycloheximide on In Vitro and In Vivo Protein Synthesis by Certain Tissues of Rats and Pigeons with Special Reference to Muscle

1973 ◽  
Vol 51 (12) ◽  
pp. 933-941 ◽  
Author(s):  
Njanoor Narayanan ◽  
Jacob Eapen
Keyword(s):  
Amino Acids ◽  
Body Weight ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Incorporation ◽  
Contrast Administration ◽  
Acid Incorporation ◽  
Tissue Homogenates

The effect of cycloheximide in vitro and in vivo on the incorporation of labelled amino acids into protein by muscles, liver, kidneys, and brain of rats and pigeons was studied. In vitro incorporation of amino acids into protein by muscle microsomes, myofibrils, and myofibrillar ribosomes was not affected by cycloheximide. In contrast, administration of the antibiotic into intact animals at a concentration of 1 mg/kg body weight resulted in considerable inhibition of amino acid incorporation into protein by muscles, liver, kidneys, and brain. This inhibition was observed in all the subcellular fractions of these tissues during a period of 10–40 min after the administration of the precursor. Tissue homogenates derived from in vivo cycloheximide-treated animals did not show significant alteration in in vitro amino acid incorporation with the exception of brain, which showed a small but significant enhancement.

scholarly journals The rate-limiting step of protein synthesis in vivo and in vitro and the distribution of growing peptides between the puromycinlabile and puromycin-non-labile sites on polyribosomes

1971 ◽  
Vol 122 (3) ◽  
pp. 267-276 ◽  
Author(s):  
D. C. N. Earl ◽  
Susan T. Hindley
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Donor Site ◽  
Peptide Bond ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Donor And Acceptor ◽  
Rate Limiting

1. At 3 min after an intravenous injection of radioactive amino acids into the rat, the bulk of radioactivity associated with liver polyribosomes can be interpreted as growing peptides. 2. In an attempt to identify the rate-limiting step of protein synthesis in vivo and in vitro, use was made of the action of puromycin at 0°C, in releasing growing peptides only from the donor site, to study the distribution of growing peptides between the donor and acceptor sites. 3. Evidence is presented that all growing peptides in a population of liver polyribosomes labelled in vivo are similarly distributed between the donor and acceptor sites, and that the proportion released by puromycin is not an artifact of methodology. 4. The proportion released by puromycin is about 50% for both liver and muscle polyribosomes labelled in vivo, suggesting that neither the availability nor binding of aminoacyl-tRNA nor peptide bond synthesis nor translocation can limit the rate of protein synthesis in vivo. Attempts to alter this by starvation, hypophysectomy, growth hormone, alloxan, insulin and partial hepatectomy were unsuccessful. 5. Growing peptides on liver polyribosomes labelled in a cell-free system in vitro or by incubating hemidiaphragms in vitro were largely in the donor site, suggesting that either the availability or binding of aminoacyl-tRNA, or peptide bond synthesis, must be rate limiting in vitro and that the rate-limiting step differs from that in vivo. 6. Neither in vivo nor in the hemidiaphragm system in vitro was a correlation found between the proportion of growing peptides in the donor site and changes in the rate of incorporation of radioactivity into protein. This could indicate that the intracellular concentration of amino acids or aminoacyl-tRNA limits the rate of protein synthesis and that the increased incorporation results from a rise to a higher but still suboptimum concentration.

scholarly journals Amino acid regulation of synthesis of ribonucleic acid and protein in the liver of rats

1971 ◽  
Vol 124 (2) ◽  
pp. 385-392 ◽  
Author(s):  
R. W. Wannemacher ◽  
C. F. Wannemacher ◽  
M. B. Yatvin
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Free Amino Acids ◽  
Free Amino ◽  
Cellular Protein ◽  
Deficient Diet ◽  
Cellular Protein Content ◽  
Regulate Protein

Weanling (23-day-old) rats were fed on either a low-protein diet (6% casein) or a diet containing an adequate amount of protein (18% casein) for 28 days. Hepatic cells from animals fed on the deficient diet were characterized by markedly lower concentrations of protein and RNA in all cellular fractions as compared with cells from control rats. The bound rRNA fraction was decreased to the greatest degree, whereas the free ribosomal concentrations were only slightly less than in control animals. A good correlation was observed between the rate of hepatic protein synthesis in vivo and the cellular protein content of the liver. Rates of protein synthesis both in vivo and in vitro were directly correlated with the hepatic concentration of individual free amino acids that are essential for protein synthesis. The decreased protein-synthetic ability of the ribosomes from the liver of protein-deprived rats was related to a decrease in the number of active ribosomes and heavy polyribosomes. The lower ribosomal content of the hepatocytes was correlated with the decreased concentration of essential free amino acids. In the protein-deprived rats, the rate of accumulation of newly synthesized cytoplasmic rRNA was markedly decreased compared with control animals. From these results it was concluded that amino acids regulate protein synthesis (1) by affecting the number of ribosomes that actively synthesize protein and (2) by inhibiting the rate of synthesis of new ribosomes. Both of these processes may involve the synthesis of proteins with a rapid rate of turnover.

Regulation of eukaryotic initiation factor-2B activity in muscle of diabetic rats

1993 ◽  
Vol 264 (1) ◽  
pp. E101-E108 ◽  
Author(s):  
A. M. Karinch ◽  
S. R. Kimball ◽  
T. C. Vary ◽  
L. S. Jefferson
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Cardiac Muscle ◽  
Casein Kinase Ii ◽  
Diabetic Rats ◽  
Casein Kinase ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Fast Twitch

Peptide-chain initiation is inhibited in fast-twitch skeletal muscle, but not heart, of diabetic rats. We have investigated mechanisms that might maintain eukaryotic initiation factor (eIF)-2B activity, preventing loss of efficiency of protein synthesis in heart of diabetic rats but not in fast-twitch skeletal muscle. There was no change in the amount or phosphorylation state of eIF-2 in skeletal or cardiac muscle during diabetes. In contrast, eIF-2B activity was decreased in fast-twitch but not slow-twitch muscle from diabetic animals. NADP+ inhibited partially purified eIF-2B in vitro, but addition of equimolar NADPH reversed the inhibition. The NADPH-to-NADP+ ratio was unchanged in fast-twitch muscle after induction of diabetes but was increased in heart of diabetic rats, suggesting that NADPH also prevents inhibition of eIF-2B in vivo. The activity of casein kinase II, which can phosphorylate and activate eIF-2B in vitro, was significantly lower in extracts of fast-twitch, but not cardiac muscle, of diabetic rats compared with controls. The results presented here demonstrate that changes in eIF-2 alpha phosphorylation are not responsible for the effect of diabetes on eIF-2B activity in fast-twitch skeletal muscle. Modulation of casein kinase II activity may be a factor in the regulation of protein synthesis in muscle during acute diabetes. The activity of eIF-2B in heart might be maintained by the increased NADPH/NADP+.

Effects of food deprivation on protein synthesis and degradation in rat skeletal muscles

1976 ◽  
Vol 231 (2) ◽  
pp. 441-448 ◽  
Author(s):  
JB Li ◽  
AL Goldberg
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Food Deprivation ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Rna Content ◽  
Fasted Rats ◽  
Synthesis And Degradation

The effects of food deprivation on protein turnover in rat soleus and extensor digitorum longus (EDL) were investigated. Muscles were removed from fed or fasted growing rats, and protein synthesis and breakdown were measured during incubation in vitro. Rates of synthesis and degradation were higher in the dark soleus than in the pale EDL. One day after food removal protein synthesis and RNA content in the EDL decreased. On the 2nd day of fasting, rates of protein catabolism in this muscle increased. Little or no change in synthesis and degradation occurred in the soleus. Consequently, during fasting the soleus lost much less weight than the EDL and other rat muscles. In unsupplemented buffer or in medium containing amino acids, glucose, and insulin, the muscles of fasted rats showed a lower rate of protein synthesis expressed per milligram of tissue but not per microgram of RNA. Thus the decrease in muscle RNA on fasting was responsible for the reduced synthesis observed under controlled in vitro conditions. In vivo the reduction in muscle protein synthesis on fasting results both from a lower RNA content and lower rate of synthesis per microgram of RNA. Reduced supply of glucose, insulin, and amino acids may account for the lower rate of synthesis per microgram of RNA demonstrable in vivo.

Amino acid fluxes in rat thin limb segments of Henle’s loop during in vitro microperfusion

1999 ◽  
Vol 277 (2) ◽  
pp. F204-F210 ◽  
Author(s):  
Olga H. Brokl ◽  
William H. Dantzler
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Apparent Movement ◽  
Transepithelial Transport ◽  
Acid Transport ◽  
Henle's Loop ◽  
Vasa Recta ◽  
The Difference

Amino acids are apparently recycled between loops of Henle and vasa recta in the rat papilla in vivo. To examine more closely papillary amino acid transport, we measured transepithelial fluxes ofl-[14C]alanine and [14C]taurine in thin limbs of Henle’s loops isolated from rat papilla and perfused in vitro. In descending thin limbs (DTL) in vitro, unidirectional bath-to-lumen fluxes tended to exceed unidirectional lumen-to-bath fluxes for both radiolabeled amino acids, although the difference was statistically significant only for taurine. In ascending thin limbs (ATL) in vitro, unidirectional lumen-to-bath fluxes tended to exceed unidirectional bath-to-lumen fluxes, although the difference was again statistically significant only for taurine. These results are compatible with apparent directional movements of amino acids in vivo. However, none of the unidirectional fluxes was saturable or inhibitable, an observation compatible with apparent reabsorption from the ATL in vivo but not compatible with apparent movement from vasa recta to DTL in vivo. There was no evidence of net active transepithelial transport when concentrations of radiolabeled amino acids were matched on both sides of perfused tubule segments. These data suggest that regulation of amino acid movement in vivo may involve the vasa recta, not the DTL of Henle’s loops. The data also suggest that transepithelial movement of amino acids in thin limbs of Henle’s loop may occur via a paracellular route.

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