Studies on the biosynthesis of hepatic pyruvate kinase and its correlation with enhanced hepatic lipogenesis in meal-trained rats.
Metabolic and enzymic changes were measured in meal-trained rats fed on high-carbohydrate diet. Rates of hepatic fatty acid synthesis are probably greater than rates of gluconeogenesis throughout the 24 h day provided that animals are fed. The daily enhancement of fatty acid synthesis on meal feeding coincided with the maximum activation of hepatic pyruvate kinase. Maximum activation of this enzyme was reflected in increased total catalytic activity (Vmax.), increased activity at 0.5 MM-phosphoenolpyruvate (V0.5), decreased Vmax./V0.5 ratio and a decrease in co-operativity of phosphoenolpyruvate binding as measured by the Hill coefficient (h). The latter changes are consistent with a decrease in enzyme phosphorylation during activation of the enzyme. To estimate changes in enzyme protein, quantitative enzyme precipitation with rabbit antisera was used. Giving a high-carbohydrate diet to meal-trained animals induced enzyme synthesis within a few hours. Adaptations in diet that enhanced fatty acid synthesis (chow to high carbohydrate; starved to high carbohydrate) led to an increased steady-state concentration of pyruvate kinase protein. An approximate estimate of the half-life of hepatic pyruvate kinase was 56 h. Whenever pyruvate kinase specific activity was measured in liver tissue extracts it was always considerably less (20--100 mumol/min per mg of protein, depending on dietary status) than the specific activity of pure pyruvate kinase (200 mumol/min per mg of protein). Antigenically active, catalytically inactive protein was removed during enzyme purification from cytosol at the stage of (NH4)2SO4 fractionation. The fraction precipitated by 30--45%-satd. (NH4)2SO4 was enzymically active, antigenically reacting protein was identified in the remaining (NH4)2SO4 fractions (0--30%- and 45--85%-satd.) and this contained no enzyme activity. These may correspond to inactive proteolytic fragments of pyruvate kinase. The rate-determining step in adjusting enzyme concentration seems to be proteolysis.
D000818:Animals; D000906:Antibodies; D002940:Circadian Rhythm; D003600:Cytosol / Q000201:enzymology; D004040:Dietary Carbohydrates / Q000494:pharmacology; D004435:Eating; D005227:Fatty Acids / Q000096:biosynthesis*; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D010728:Phosphoenolpyruvate / Q000378:metabolism; D011770:Pyruvate Kinase / Q000096:biosynthesis* / Q000276:immunology; D051381:Rats
D000906:Antibodies; D004040:Dietary Carbohydrates; D005227:Fatty Acids; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase