In rapidly growing, highly glycolytic hepatoma cells as much as 65% of the total cell hexokinase is bound to the outer mitochondrial membrane [Parry, D.M., & Pedersen, P.L. (1983) J. Biol. Chem. 258, 10904-10912]. In this paper, we describe the purification to apparent homogeneity of a mitochondrial pore-forming protein from the highly glycolytic AS-30D rat hepatoma cell line. The purified protein shows a single 35 000-dalton band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an amino acid composition slightly more hydrophobic than that of the rat liver pore protein (also known as VDAC or mitochondrial porin), and a channel-forming activity of 136 channels min-1 (microgram of protein)-1. In addition to displaying the properties characteristic of VDAC (single-channel conductance, voltage dependence, and preference for anions), we observe that the AS-30D VDAC protein is one of only three mitochondrial proteins that bind [14C]dicyclohexylcarbodiimide (DCCD) at relatively low dosages (2 nmol of DCCD/mg of mitochondrial protein). Significantly, treatment of intact mitochondria isolated from either rat liver or the AS-30D hepatoma with DCCD results in an almost complete inhibition of their ability to binding hexokinase. Fifty percent inhibition of binding occurs at less than 2 nmol of DCCD/mg of mitochondrial protein. In contrast to DCCD, water-soluble carbodiimides are without effect on hexokinase binding. These results suggest that the pore-forming protein of tumor mitochondria forms at least part of the hexokinase receptor complex. In addition, they indicate that a carboxyl residue located within a hydrophobic region of the receptor complex may play a critical role in hexokinase binding.

The outer mitochondrial membrane receptor for hexokinase binding has been identified as the VDAC protein, also known as mitochondrial porin. The ability of the receptor to bind hexokinase is inhibited by pretreatment with dicyclohexylcarbodiimide (DCCD). At low concentrations, DCCD inhibits hexokinase binding by covalently labeling the VDAC protein, with no apparent effect on VDAC channel-forming activity. The stoichiometry of [14C]-DCCD labeling is consistent with one to two high-affinity DCCD-binding sites per VDAC monomer. A comparison between the sequence of yeast VDAC and a conserved sequence found at DCCD-binding sites of several membrane proteins showed two sites where the yeast VDAC amino acid sequence appears to be very similar to the conserved DCCD-binding sequence. Both of these sites are located near the C-terminal end of yeast VDAC (residues 257-265 and 275-283). These results are consistent with a model in which the C-terminal end of VDAC is involved in binding to the N-terminal end of hexokinase.

Undergraduate biochemistry laboratories traditionally expose students to biochemical techniques through a series of independent and usually unrelated laboratory exercises. Efforts to reorganize and update the undergraduate biochemistry laboratory at Texas Tech University have centered upon the development of a series of laboratory experiments that focus on a single biological system, the complex 11 (succinate:ubiquinone oxidoreductase) of Escherichia coli. Students are provided a computer-aided research environment in which to gain practical training in molecular biology, protein purification and enzyme kinetics. The laboratory schedule includes exercises on the succinate dehydrogenase operon (sdh) DNA sequence, and experiments that deal with isolation and characterization of sdh operon DNA, extraction and purification of complex 11 and characterization of complex 11 subunit structure and kinetic parameters. This unified approach to the biochemistry teaching laboratory is specifically designed to impact undergraduate student preparation for future studies, providing exposure to fundamental techniques of biochemistry experimentation and simulating the focused, single system, environment of a research laboratory.

We have compared Su~bern blots of rat he~ioma DNA probed with Types i, I1 md III hexokinase cDNAs is&tad from normal rat tissue.Hybridization patterns show several fragments recognized by both the Type I and 11 clones while no resemblance is observed between the Type iii probe and the other two isozymes. It therefore appars that the Type I-like and Type II-like hepatoma tizymes arc coded far by similar yet separate genes. while a dissimilar third gene codes for the Type Ill-like isozyme. In addition. a loss of heterozygosity was detected at the Tvpe f fl locus in the AS-3DD hepatoma when compared to normal tissue-As only the Type IMike isayme is highly expressed in highly glycolytic tumtrrs. these data have implications for dilTcrcntia1 gene regulation between the tumor isoqmes.

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