MICROHETEROGENEITY OF CYTOSOLIC AND MEMBRANE-BOUND HEXOKINASE-II IN MORRIS HEPATOMA-3924A

Phosphorylation of glucose by hexokinase is the key step in glucose an d energy metabolism of the cell. In the Morris hepatoma 3924A, hexokin ase II is the predominant hexokinase isoenzyme and occurs in the cytos ol as well as bound to membranes. Hexokinase II was isolated by DEAE-c ellulose chromatography from both the cytosolic and the mitochondria-e nriched fractions and further resolved by hydrophobic-interaction chro matography on phenyl-Sepharose into two components designated hexokina se IIa and IIb. In both the soluble and the mitochondria-enriched frac tions, type IIb was the predominant form, but the IIb/IIa ratio was hi gher in the particulate (6-8) as compared with the cytosolic fraction (1.5-2.0). Binding of the isolated forms of the enzyme to rat liver mi tochondria resulted in a 2-10-fold activation of both subtypes. Bioche mical characterization showed that both subtypes are closely related t o the isoenzyme commonly referred to as hexokinase II, and that the mi croheterogeneity was not a consequence of contamination with hexokinas e I or III. Both subtypes had a molecular mass of 110 kDa, they were i nhibited by P-i at concentrations higher than 5 mM, and activated by t he detergent CHAPS. The two subtypes differed in electrophoretic mobil ity (IIa > IIb), in K-m values for glucose (IIa, 0.109 mM; IIb, 0.216 mM), in K-i values for glucose 6-phosphate (IIa, 25 mu M; IIb, 0.106 m M), and in K-i values for glucose 1,6-bisphosphate (IIa, 12.2 mu M; II b, 5.5 mu M), An artificial proteolytic cleavage as cause of the hexok inase II microheterogeneity can be excluded, since both subtypes show the same molecular mass and the ability to bind to mitochondria and ph enyl-Sepharose. In addition, the relative proportions of the two subty pes did not vary markedly between several enzyme preparations. Norther n-blot analysis with a hexokinase II-specific cDNA probe revealed two distinct mRNA transcripts of 5.2 and 6.3 kb in length, which offers th e possibility that hexokinase II microheterogeneity is due to differen tial RNA transcription and/or processing.