Measurement of instant rates of protein degradation in the livers of intact mice by the accumulation of bestatin-induced peptides.
Bestatin induces the accumulation of di- and tripeptide intermediates in cellular protein breakdown. In liver, a single set of bestatin-sensitive cytosolic peptidases are involved in the degradation to amino acids of the major classes of cellular proteins. Accumulation of bestatin-induced peptides, in isolated hepatocytes, is proportional to the rate of protein degradation (Botbol, V., and Scornik, O. A. (1989) J. Biol. Chem. 264, 13504-13509). Injection of 1 mg of bestatin into mice results in detectable amounts of hepatic intermediates in 15 min. We propose to use the accumulation of these peptides as a relative measurement of liver protein degradation. There is at present no other way to determine transient changes in protein breakdown in the tissues of intact animals. As an example of the applications of this procedure, we present the effects of a single meal on hepatic protein metabolism. Protein synthesis was estimated by the incorporation into liver protein of a massive dose of radioactive leucine (Scornik, O. A. (1974) J. Biol. Chem. 249, 3876-3883) and degradation of long-lived or short-lived proteins by the accumulation of bestatin-induced peptides, labeled in carboxy-C of their Leu or Arg moieties, 1 day or 1 h beforehand. A single meal resulted in an 18% increase in liver protein in 8 h, a 45% increase in the rate of hepatic protein synthesis, and a 3-fold decrease in the rate of breakdown of long-lived proteins. Short-lived proteins were not affected. To establish the efficiency with which bestatin-induced peptides accumulate in the livers of fasting mice, we compared them with the disappearance, in 1 day, of protein-bound 14C-guanidino-Arg residues, labeled by previous injection of 14C-bicarbonate (Swick, R. W., and Ip, M. M. (1974) J. Biol. Chem. 249, 6836-6841). From this comparison, we estimated that bestatin-induced Leu-labeled intermediates, accumulating in 15 min, represented 39% of the hepatic proteins degraded in that interval. For Arg-labeled intermediates the value was 55%. Correcting for these efficiencies, we estimate that in 4 h a meal decreased the rate of degradation of long-lived Arg-labeled proteins from 2.02 to 0.73%/h. For Leu-labeled proteins the estimated rates were 1.76 and 0.66%/h, respectively. Although a transient slowdown of liver protein degradation after a single meal had been suggested before, this is the first time that acute changes such as this can be determined directly in intact animals.(ABSTRACT TRUNCATED AT 400 WORDS)
Botbol
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V
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756.
Scornik
O A
OA
D000626:Aminopeptidases / Q000037:antagonists & inhibitors*; D000818:Animals; D002940:Circadian Rhythm; D004044:Dietary Proteins / Q000378:metabolism; D004435:Eating; D005215:Fasting; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008099:Liver / Q000378:metabolism*; D008297:Male; D051379:Mice; D009584:Nitrogen / Q000652:urine; D009929:Organ Size; D010455:Peptides / Q000378:metabolism*; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism*
D004044:Dietary Proteins; D010455:Peptides; D011506:Proteins; D000626:Aminopeptidases; D007930:Leucine; C012211:ubenimex; D009584:Nitrogen