Erythrocyte metabolism: kinetic and electrophoretic analyses of pig red cell hexokinase.
The mature erythrocyte of the pig has been observed to possess the slowest metabolic rate of any mammalian cell type. Previous studies in this laboratory suggested that the hexokinase isolated from these cells was inhibited by glucose in concentrations in excess of 0.2 mM. In the present study, the enzyme was isolated by utilizing DEAE-Sephadex A-50, ammonium sulfate precipitation, DEAE-cellulose (DE-52), and Sephadex G-100 gel-filtration. Studies on the hexokinase isolated from the pig mature erythrocyte by the above procedures revealed two distinct isozymes of hexokinase that do not behave kinetically and electrophoretically as those previously found in other mammalian red blood cells. The isozyme isolated from the erythrocyte of the young adult pig (less than six months of age) migrated at a slower electrophoretic rate than the one isolated from the adult pig (more than six months of age). Coupled with the observed difference in electrophoretic mobilities were changes in the apparent Km values as well as Vmax as a function of substrate concentration. In spite of the changes observed in relation to glucose, the apparent Km for Mg-ATP-2 was not altered during development. Diphosphoglycerate (DPG) was observed to be a "linear-mixed" inhibitor of both isozymes with respect to Mg-ATP-2. An experimental designed to determined the type of inhibition by DPG on the type I isozyme isolated from the horse erythrocyte revealed competitive inhibition with the Mg-ATP-2 site. Free Mg activated both isozymes in low concentrations (less than 2.5 mM) but inhibited the enzymatic activity as the concentration was elevated. The data suggest that both the young adult and the adult pig erythrocyte possess two distinct type III isozymes of hexokinase.
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D004163:Diphosphoglyceric Acids / Q000494:pharmacology; D004592:Electrophoresis, Starch Gel; D004912:Erythrocytes / Q000201:enzymology*; D006593:Hexokinase / Q000097:blood*; D007527:Isoenzymes / Q000097:blood*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D013552:Swine / Q000097:blood* / Q000254:growth & development
D004163:Diphosphoglyceric Acids; D007527:Isoenzymes; D000255:Adenosine Triphosphate; D006593:Hexokinase; D008274:Magnesium