Pig red blood cell hexokinase: evidence for the presence of hexokinase types II and III, and their purification and characterization.

Pig erythrocytes, in contrast to red blood cells from other mammals (M. Magnani, V. Stocchi, F. Canestrari, M. Dachà, and G. Fornaini (1982) Biochem. Int. 4, 673), have been shown to contain hexokinase (EC 2.7.1.1) types II and III. Hexokinase type III is the predominant form, accounts for 98% of the total glucose phosphorylating activity, and has been purified 290,000-fold by a combination of ion-exchange chromatography and affinity chromatography on Sepharose-N-hexanoylglucosamine. The enzyme was shown to be homogeneous by polyacrylamide and sodium dodecyl sulfate-gel electrophoresis. The highest specific activity obtained was 190 units/mg protein with a yield of 60%. Because the amount of hexokinase II was small, it was only partially purified by ion-exchange chromatography. The native proteins have the same molecular weight of 100,000 by gel filtration on Ultrogel AcA44. The apparent isoelectric point of hexokinase type II was shown to be 4.8 and 4.9 pH units, whereas hexokinase type III was shown to have a pI of 4.3 to 4.4 pH units by isoelectric focusing. Both hexokinases are able to phosphorylate several hexoses. However, while hexokinase II shows an apparent Km for glucose of 1.5 X 10(-4) M with negative cooperativity (nH = 0.4), hexokinase III shows an apparent Km for glucose of 1.5 X 10(-5) M and a positive cooperative effect (nH = 1.5). Furthermore, glucose at concentrations higher than 0.4 mM becomes an inhibitor of hexokinase III. Amino acid analysis of hexokinase type III revealed a low number of the aromatic residues Phe, Tyr, and Trp; this is in agreement with the low extinction coefficient of E1%280nm = 12.5.