scholarly journals The effect of inhibitors in vivo on protein synthesis and the amino acid pool in the sheep blowfly, Lucilia cuprina

1975 ◽  
Vol 150 (2) ◽  
pp. 227-234 ◽  
Author(s):  
A J Campbell ◽  
L M Birt
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Actinomycin D ◽  
Pool Size ◽  
Dynamic State ◽  
Amino Acid Pool ◽  
Pronounced Increase ◽  
Acid Pool ◽  
Quantitative Estimates

1. The rates of detoxification of cycloheximide (33 μg/g fresh wt.), puromycin (167 μg/g fresh wt.) and actinomycin D (1 μg/g fresh wt.) were assessed in vivo on the basis of acid-insoluble [14C]leucine incorporation in the sheep blowfly, Lucilla cuprina; these were compared with quantitative estimates which took account not only of incorporation data but also of leucine pool size and turnover. Quantitatively, cycloheximide and puromycin were still at least 50% effective in inhibiting protein synthesis after 6.5 and 24.5h of exposure respectively, whereas values based only on incorporation data suggested that cycloheximide was 83% effective and puromycin completely ineffective after the respective periods. Quantitative estimates also showed that actinomycin D effectiveness increased with increasing exposure over 24.5h, in contrast with values based only on incorporation data, which suggested that it was completely ineffective after 24h.2. All inhibitors affected the dynamic state of the amino acid pool; there was a marked decrease in the rate of leucine-pool turnover as well as an increase in the half-life of leucine in the pool. 3. Inhibition of protein synthesis resulted in changes in leucine-pool size; the most pronounced increase occurred with cycloheximide and puromycin and the most pronounced decreases with actinomycin D. 4. Evidence is presented which suggests that proteolysis is functionally linked to protein synthesis, which determines its rate indirectly.

scholarly journals The specific radioactivity of the tissue free amino acid pool as a basis for measuring the rate of protein synthesis in the rat in vivo

1974 ◽  
Vol 142 (2) ◽  
pp. 413-419 ◽  
Author(s):  
Edward B. Fern ◽  
Peter J. Garlick
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Free Amino Acid ◽  
Free Amino ◽  
Gastrocnemius Muscle ◽  
Specific Radioactivity ◽  
Amino Acid Pool ◽  
Free Amino Acid Pool ◽  
Acid Pool

1. Rats were infused in vivo with [U-14C]glycine for periods of 2–6h, during which time the specific radioactivity of the free glycine in plasma and tissue approached a constant value. 2. Free serine also became labelled. The ratio of specific radioactivity of serine to that of glycine in the protein of liver, kidney, brain, jejunum, heart, diaphragm and gastrocnemius muscle was closer to the ratio in the free amino acid pool of the tissue than that of the plasma. 3. The kinetics of incorporation of [14C]glycine and [14C]serine into the protein of gastrocnemius muscle further suggested that the plasma free amino acids were not the immediate precursors of protein. 4. Infusion of rats with [U-14C]serine resulted in labelling of free glycine. The ratio of specific radioactivity of glycine to serine in the protein of liver, kidney, brain, jejunum and heart again suggested incorporation from a pool similar to the free amino acid pool of the tissue. 5. Rates of tissue protein synthesis calculated from the incorporation into protein of both radioactive glycine and serine, either infused or derived, were very similar when the precursor specific radioactivity was taken to be that in the total free amino acids of the tissue. Except for gastrocnemius muscle and diaphragm during the infusion of radioactive serine, the rates of tissue protein synthesis calculated from the specific radioactivity of the free glycine and serine in plasma differed markedly.

scholarly journals Regulation of enzyme turnover during tissue differentiation. Interactions of insulin, prolactin and cortisol in controlling the turnover of fatty acid synthetase in rabbit mammary gland in organ culture

1976 ◽  
Vol 154 (2) ◽  
pp. 359-370 ◽  
Author(s):  
Brian K. Speake ◽  
Raymond Dils ◽  
R. John Mayer
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Fatty Acid ◽  
Amino Acid ◽  
Culture Medium ◽  
Fatty Acid Synthetase ◽  
Amino Acid Pool ◽  
Acid Pool ◽  
Intracellular Amino Acid ◽  
Enzyme Degradation

1. Explants of mammary gland from mid-pregnant rabbits were cultured in Medium 199 containing combinations of insulin, prolactin and cortisol. With hormone combinations which included prolactin, a sustained increase in the apparent rate of synthesis and in the amount of fatty acid synthetase was measurable immunologically. Maximum increase was produced with insulin, prolactin and cortisol present together. 2. With prolactin present alone, synthetase activity in the explants decreased to undetectable values after 1 day in culture, whereas the incorporation of l-[U-14C]leucine into immunodetectable material increased. Prolactin may therefore direct the synthesis of immunologically cross-reactive precursors of fatty acid synthetase which are enzymically inactive. 3. Culture with dibutyryl cyclic AMP plus theophylline in the presence of insulin, prolactin and cortisol delayed the increase in the rate of synthesis and accumulation of the synthetase. These compounds may also prevent the apparent decrease in the rate of degradation of the synthetase which occurs on day 2 of culture. 4. A large decrease in the apparent rate of degradation of the synthetase on day 2 of culture occurs during culture with hormone combinations which include prolactin. The protein obtained by centrifugation of explant homogenates for 6min at 14000gav. is degraded continuously throughout the culture period. 5. This decrease in the apparent rate of degradation of the synthetase was measured by radio-immunological precipitation. It is probably part of a regulated programme of enzyme degradation and not a reflexion of the reutilization of radioactive amino acids for the following reasons. (a) The calculated increase in the amount of the synthetase in explants on day 2 of culture with insulin, prolactin and cortisol was approximately equal to the measured increase of the enzyme complex which accumulates in the explants. This suggests little or no enzyme degradation has occurred. (b) Explants were cultured for 24h with insulin, prolactin and cortisol. They were then incubated with l-[U-14C]leucine, washed and incubated again for up to 4½h. l-[U-14C]Leucine rapidly equilibrated with the intracellular amino acid pool. Within 10min of incubation after washing explants to remove endogenous l-[U-14C]leucine the previously linear incorporation of l-[U-14C]-leucine into total explant protein ceased. This suggests that protein is synthesized from an amino acid pool which rapidly equilibrates with amino acids in the culture medium. (c) Explants were cultured for 24h as described in (b) but after washing they were cultured with insulin, prolactin and cortisol for 24h. Approx. 90% of the radioactivity lost from the ‘free’ intracellular amino acid pool and from amino acids derived from the degradation of explant protein in this period was detected in the culture medium. This suggests that the ‘free’ intracellular amino acids and amino acids derived from protein degradation can equilibrate with amino acids in the medium. A residual ‘free’ radioactive amino acid pool was present in the tissue. (d) Casein represents approx. 20% of the protein synthesized after 1 day in culture with insulin, prolactin and cortisol. Histological evidence suggests that on day 2 of culture, casein is unlikely to be degraded in the tissue. No increase in the radioactivity incorporated into casein can be measured in the 23h after incubation of explants with l-[U-14C]leucine as described in (b). This suggests that the incorporation of radioactivity into proteins during culture after incubation with l-[U-14C]leucine is minimal. (e) Inhibition of protein synthesis in explants by cycloheximide after incubation with l-[U-14C]leucine does not reveal a latent continuous degradation of fatty acid synthetase on day 2 of culture which might have been masked by the high rates of protein synthesis and therefore the accumulation of the enzyme. 6. The conclusion is discussed that there is a real decrease (or even cessation) in the rate of degradation of fatty acid synthetase during the period when the enzyme accumulates in explants cultured with hormone combinations which contain prolactin.

Liver Amino Acid Pool may be Homogeneous with Respect to Protein Synthesis

1971 ◽  
Vol 70 (1) ◽  
pp. 173-174 ◽  
Author(s):  
Kinji TSUKADA ◽  
Takako MORIYAMA ◽  
Irving LIEBERMAN
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Pool ◽  
Acid Pool
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