Hexokinase receptor complex in hepatoma mitochondria: evidence from N,N'-dicyclohexylcarbodiimide-labeling studies for the involvement of the pore-forming protein VDAC.
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.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002234:Carbodiimides / Q000494:pharmacology*; D002460:Cell Line; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology*; D005260:Female; D006019:Glycolysis; D006593:Hexokinase / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D008970:Molecular Weight; D018272:Porins; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D050994:Voltage-Dependent Anion Channels
D000596:Amino Acids; D002234:Carbodiimides; D008565:Membrane Proteins; D018272:Porins; D011956:Receptors, Cell Surface; D050994:Voltage-Dependent Anion Channels; D004024:Dicyclohexylcarbodiimide; D006593:Hexokinase