Measurement of phenylalanine hydroxylase turnover in cultured hepatoma cells.
A substantially new method has been developed to measure protein turnover. Its basis is the notion that in labeling experiments a secreted protein can be used to determine the specific radioactivity of the intracellular amino acid precursor pool. To measure protein turnover in the Reuber hepatoma H4 cell line, cultures were labeled with [3H]leucine for specified periods after which phenylalanine hydroxylase was isolated and its leucine specific radioactivity determined. Serum albumin secreted by the cultures was also isolated and used to estimate the leucine precursor pool specific radioactivity. The protein half-life of phenylalanine hydroxylase could them be calculated. Experiments performed at long and short labeling times and with high and low concentrations of leucine in the medium yielded equivalent results. Phenylalanine hydroxylase half-life in the H4 cells was investigated under both normal and hydrocortisone-induced growth conditions. Average half-lives of 7.4 and 8.2 h were found for induced and uninduced cultures, respectively. Although these measured enzyme half-lives were not essentially different, the steady state level of phenylalanine hydroxylase was increased 6.2-fold upon hydrocortisone induction, from 0.076 to 0.47 microgram/10(6) cells. The results demonstrated that hydrocortisone induces phenylalanine hydroxylase in the H4 cells by causing an increase in the rate of enzyme synthesis.
D000818:Animals; D002460:Cell Line; D006854:Hydrocortisone / Q000494:pharmacology; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D010651:Phenylalanine Hydroxylase / Q000096:biosynthesis / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000187:drug effects; D051381:Rats; D012709:Serum Albumin / Q000096:biosynthesis
D012709:Serum Albumin; D010651:Phenylalanine Hydroxylase; D007930:Leucine; D006854:Hydrocortisone