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Purification and Characterization of a Bindable Form of Mitochondrial Bound Hexokinase from the Highly Glycolytic AS-30D Rat Hepatoma Cell Line¹

Laboratory for Molecular and Cellular Bioenergetics, Department of Biological Chemistry, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205

Recent studies from this laboratory have demonstrated that a form of bexokinase characteristic of rapidly growing, highly glycolytic tumor cells is bound to an outer mitochondrial membrane receptor complex containing a M_r 35,000 pore protein (D. M. Parry and P. L. Pedersen, J. Biol. Chem., 258: 10904–10912, 1983; R. A. Nakashima, et al., Biochemistry, 25: 1015–1021, 1986). In new studies reported here the specificity of this receptor complex for binding hexokinase is defined, and a purification scheme is described which leads to a homogeneous and bindable form of the tumor hexokinase.

In the AS-30D hepatoma, hexokinase activity is elevated more than 100-fold relative to liver tissue. The relative increase in hexokinase activity is 8 times greater than that of any other glycolytic enzyme. Hexokinase is the only glycolytic enzyme of AS-30D cells to exhibit a mitochondrial/cytoplasmic specific activity ratio greater than 1, showing a 3.5-fold elevation in the mitochondrial fraction.

Purification of hexokinase is accomplished by preferential solubilization of the mitochondrial bound enzyme with glucose-6-phosphate, followed by high-performance liquid chromatography on gel permeation and anion exchange columns. The final fraction has a specific activity of 144 units per mg of protein, with a K_m for glucose of 0.13 mM and for ATP of 1.4 mM. The purified tumor enzyme migrates as a single species upon sodium dodecyl sulfate: polyacrylamide gel electrophoresis with an apparent molecular weight of 98,000. Significantly, the purified tumor enzyme retains its activity for mitochondrial binding.

Additional results derived from chromatographic, polyclonal antibody, and amino acid analysis studies indicate that the predominant rat hepatoma hexokinase species is related most closely to isozymic form(s) of the enzyme commonly referred to as type II, and least related to the liver type IV isozyme (glucokinase).

¹ This work was supported by NIH Grant CA 32742 to P. L. P. R. A. N. and M. G. P. were supported, respectively, by a fellowship from the Leukemia Society of America and by CNR Grants 85.02159.44 and AIRC/86 from the Associazione Italiana per la Ricerca sul Cancro.

² Permanent address: Istituto Regina Elena per lo Studio e la Cura dei Tumori, V.le Regina Elena 291, 00161 Rome, Italy.

³ To whom requests for reprints should be addressed.

Received 6/17/87. Revised 10/22/87. Accepted 11/18/87.

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