ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 226, No. 1, October 1, pp. 365-3’76. 1933 Pig Red Blood Cell Hexokinase: Evidence for the Presence of Hexokinase Types II and Ill, and Their Purification and Characterization’ VILBERTO STOCCHI, MAURO MAGNANI, GIUSEPPE NOVELLI, MARINA DACHA, AND GIORGIO FORNAINI’ Istituto di Chimica Biologica, Universitci degli Studi di Urbino, via Sa&, 2410.29 U&no, Italy Received February 14, 1983, and in revised form May 17, 1933 Pig erythrocytes, in contrast to red blood cells from other mammals (M. Magnani, V. Stocchi, F. Canestrari, M. Dacha, and G. Fornaini (1982) Biochewa. ht. 4,6’73), 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 electro- phoresis. 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. 1O-4 M with negative cooperativity (nn = 0.4), hexokinase III shows an apparent Km for glucose of 1.5. 10m5 M and a positive cooperative effect (nn = 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 EL&,, = 12.5. Hexokinase in mammalian tissues exists as four isoenzymes with distinct kinetic properties and tissue distribution (1, 2). These isozymes are also readily distin- guished by their Km values for glucose (3) which are lop5 M for hexokinase I, lop4 M for hexokinase II, 10e5 M for type III, and lo-’ M for glucokinase (type IV), while, with the exception of the latter, they show the same molecular weight. We have previ- ously reported (4-7) that mammalian red ’ This work was supported by CNR, Minister0 della Pubblica Istruzione and Cassa di Risparmio di Pesaro. ’ To whom correspondence should be addressed. blood cells contain mainly hexokinase type I or its subtypes. In the course of our stud- ies we also showed that pig erythrocytes contain two different glucose-phosphory- lating activities, which differ in glucose af- finity and electrophoretic mobility (8). More recently, Dixon and Wilson (9) have obtained similar results. In this paper we present evidence that these two glucose-phosphorylating activ- ities correspond to hexokinase type II and type III (in the nomenclature of Katzen and Schimke (10)). We also describe the procedure for the purification to homo- geneity of hexokinase type III, which, in pig erythrocytes, represents 98% of the to- 365 0003-9861/83 $3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.