PURIFICATION AND PROPERTIES OF BUFFALO (BUBALUS-BUBALIS) ERYTHROCYTE HEXOKINASE

Buffalo erythrocytes contain one isozyme of hexokinase that apparently lacks microheterogeneity as shown by chromatographic properties. A si ngle protein band was detected by means of Western blotting using an a ntibody raised in rabbits against homogeneous rat brain hexokinase I. The native protein has a molecular weight of 200,000 +/- 2880 by gel f iltration. Partial purification of erythrocyte hexokinase by a combina tion of several procedures, including affinity chromatography, which w as previously applied successfully to the purifica tion of other mamma lian type I hexokinases, produced a partially purified enzyme that sho wed several contami nants after SDS-polyacrylamide gel electrophoresis . The affinity of buffalo erythrocyte hexokinase for glucose (K-m = 0. 012 +/- 0.001 mM) is lower than most other mammal hexokinases type I. It phosphorylates other sugars, with considerably higher K-m values. T his isozyme is able to use MgATP but does not use MgGTP, MgCTP or MgUT P. We used inhibition patterns, obtained with products to elucidate en zyme sequential mechanisms. Our results are clearly in agreement with a random sequential mechanism and in disagreement with an ordered sequ ential mechanism with either glucose or ATP as the obligatory first su bstrates. The ADP inhibition was of mixed type with both ATP and gluco se as substrates. (C) 1997 Elsevier Science Inc.