Cell-free translation of carbamyl phosphate synthetase I and ornithine transcarbamylase messenger RNAs of rat liver. Effect of dietary protein and fasting on translatable mRNA levels.

Abstract

Total RNA of rat liver was translated in a cell-free system derived from rabbit reticulocytes, and synthesized precursor forms of carbamyl phosphate synthetase I (EC 6.3.4.16) and ornithine transcarbamylase (EC 2.1.3.2) were isolated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Synthesis of both enzyme precursors was optimal with 90 to 180 m M potassium ion, 1.2 m M magnesium, and 50 pg/ml of rat liver tRNA.Enzyme synthesis increased with total RNA up to 1.2 mg/ml and with time up to 90 min at 25 "C.Translatable levels of hepatic mRNAs for carbamyl phosphate synthetase I and ornithine transcarbamylase were 4.2-and 2.2-fold higher, respectively, in the case of rats fed a 60% casein diet as compared with rats fed a 5% casein diet.The differences in mRNA levels were slightly higher than the differences in the levels of the enzyme activities (3.3-and 1.9fold, respectively) and of the enzyme proteins (3.3-and 2.l-fold, respectively).The results indicate that the dietary protein-dependent changes in the levels of carbamyl phosphate synthetase I and ornithine transcarbamylase are due mainly to changes in levels of translatable mRNAs for the enzymes.The hepatic levels of the two enzymes expressed per g wet weight of liver were also increased 1.7-and 1.3-fold, respectively, during a 7-day fasting over those of control animals kept on a 24% protein diet.However, the levels of translatable mRNAs for carbamyl phosphate synthetase I and ornithine transcarbamylase were decreased to 54 and 6796, respectively, of those of the control animals.Thus, the increase of the two enzyme levels during fasting appears to be the result of a decreased rate of degradation of the enzymes.Carbamyl phosphate synthetase I (EC 6.3.4.16) and ornithine transcarbamylase (EC 2.1.3.2) catalyze the first two steps of urea synthesis in the liver of ureotelic animals.T h e former enzyme consists of two identical subunits of M , = 156,000-160,000 (1-4), is located in the mitochondrial matrix ( 5 ) , and constitutes 20-30% of total matrix protein (2, 6).Ornithine transcarbamylase is a trimer of identical subunits