The turnover of L-type pyruvate kinase in cultured rat hepatocytes.
Hepatocytes were isolated by collagenase perfusion of livers from rats that had been allowed access to a carbohydrate-rich diet or laboratory chow or had been deprived of food 48h before use. By incubation with l-[4,5-(3)H]leucine and precipitation with anti-(L-type pyruvate kinase) sera the rates of synthesis and degradation of L-type pyruvate kinase were measured in freshly prepared cells and hepatocytes maintained in monolayer culture for up to 5 days. Hepatocytes from carbohydrate-rich-diet-fed rats synthesized more L-type pyruvate kinase than did cells from chow-fed animals, which in turn synthesized more than cells from 48h-starved rats. Hepatocytes maintained in culture for up to 5 days synthesized L-type pyruvate kinase at similar rates to freshly prepared cells. The degradation of [(3)H]leucine-labelled L-type pyruvate kinase was shown to be biphasic. A phase with t((1/2)) (half-time) 4.9h and a duration of 8-10h was followed by a phase with t((1/2)) 79.2h. Cells from chow-fed and carbohydrate-rich-diet-fed rats showed similar patterns of degradation of L-type pyruvate kinase. The addition of 2mm-fructose and 0.1mum-insulin to the culture medium increased the t((1/2)) of the rapid phase to 12h in cells isolated from carbohydrate-rich-diet-fed rats, but not in cells from chow-fed rats. The secondary, slower, phase of degradation remained unaffected. The degradation of fructose 1,6-bisphosphatase and total cell protein followed first-order kinetics. The half-life of fructose 1,6-bisphosphatase was 41.0h in cells from chow-fed animals and 48.5h in cells from carbohydrate-rich-diet-fed donors. Fructose and insulin did not affect the rate of enzyme degradation. We propose that there is a role for protein catabolism in the short-term and long-term control of L-type pyruvate kinase concentration.
D000818:Animals; D002478:Cells, Cultured; D004040:Dietary Carbohydrates / Q000494:pharmacology; D006597:Fructose-Bisphosphatase / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D011770:Pyruvate Kinase / Q000096:biosynthesis / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D004040:Dietary Carbohydrates; D007527:Isoenzymes; D011770:Pyruvate Kinase; D006597:Fructose-Bisphosphatase; D007930:Leucine