Turnover of Carbamyl-phosphate Synthase, of Other Mitochondrial Enzymes and of Rat Tissues. Effect of Diet and of Thyroidectomy

Abstract

The relative rate constant of protein degradation for a number of enzymes as well as for rat liver fractions and for ‘soluble’ and ‘insoluble’ protein of rat tissues has been measured by the double-isotope technique. This technique has been evaluated and found reliable for measurements involving half-lives of about 2–8 days and therefore generally useful for liver, kidney and possibly heart but not for brain and skeletal muscle. Carbamyl-phosphate synthase represents the major component (∼ 20%) of the protein of rat liver mitoplasts. Particular attention was given to determine its turnover, for obviously this enzyme should be a good ‘marker’ for comparative studies with other mitochondrial proteins. The also hitherto unknown half-lives of malate dehydrogenase and of glutamate dehydrogenase from rat liver mitochondria have been measured. The rate constants of degradation for rats fed ‘basal’ diet were: ‘homogenate’, kd= 0.20 day−1; ‘mitochondria’, kd= 0.10 day−1; ‘microsomes’, kd= 0.28 day−1; ‘cytosol’, kd= 0.20 day−1; malate dehydrogenase, kd= 0.27 day−1; carbamyl-phosphate synthase, kd= 0.09 day−1; heart proteins: ‘insoluble’, kd= 0.086 day−1; ‘soluble’, kd= 0.175 day−1; kidney proteins: ‘insoluble’, kd= 0.28 day−1; ‘soluble’, kd= 0.245 day−1. Contrary to reports showing faster degradation of long polypeptide chains than those of smaller size, the apparent turnover rate of carbamyl-phosphate synthase is slower than those of other mitochondrial enzymes with smaller subunits. Similar measurements were carried out with tissues from rats fed ‘high-protein’ and ‘protein-free’ diets and after thyroidectomy. Rats fed a high-protein diet showed a general increase of the rate constant of protein degradation in all the liver fractions studied when compared with data from animals on the basal diet. Feeding a protein-free diet produced an increase of the rate constant of degradation of liver mitochondria and fractions thereof. Thyroidectomy decreased the rate constant of degradation in all the rat liver fractions and enzymes thereof. Kidney and heart insoluble and soluble proteins behaved in the same way as those from rat liver. This is the first time that a direct correlation of the often-mentioned, but hitherto not directly measured, effect on protein turnover has been determined. Small-scale purification of carbamyl-phosphate synthase, malate dehydrogenase and glutamate dehydrogenase from a single rat liver is described. The preparations showed a high degree of homogeneity by disc gel electrophoresis and by sodium dodecyl sulfate electrophoresis. Small-scale purification, when applicable, should minimize uncertainties in regard to homogeneity of enzymes used for antibody production in protein turnover studies. Moreover, it is more economical in time.