Previous studies from this laboratory have shown that mitochondrial bound
hexokinase is markedly elevated in highly glycolytic
hepatoma cells (Parry, D. M., and Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912). A pore-forming
protein,
porin, within the outer membrane appears to comprise at least part of the receptor site (Nakashima, R.A., Mangan, P.S., Colombini, M., and Pedersen, P.L. (1986). Biochemistry 25, 1015-1021). In studies reported here experiments were carried out to assess the functional significance of mitochondrial bound
tumor hexokinase. Two approaches were used to determine whether the bound
enzyme has preferred access to mitochondrially generated
ATP relative to cytosolic
ATP. The first approach compared the time course of
glucose 6-phosphate formation by AS-30D
hepatoma mitochondria under conditions where
ATP was regenerated endogenously via oxidative phosphorylation or exogenously by added
pyruvate kinase and
phosphoenolpyruvate. The second approach involved the measurement of the specific radioactivity of
glucose 6-phosphate formed following the addition of [gamma-32P]
ATP to either phosphorylating or nonphosphorylating AS-30D mitochondria. Both approaches provided results which show that the source of
ATP for bound
hexokinase is derived preferentially from the
ATP synthase residing within the inner mitochondrial membrane compartment rather than from the medium (i.e. from the cytosolic compartment). These results provide the first direct demonstration that the exceptionally high level of
hexokinase bound to mitochondria of highly glycolytic
tumor cells has preferred access to mitochondrially generated
ATP, a finding that may have rather profound metabolic significance for such
tumors.