Hexokinase Receptor Complex in Hepatoma Mitochondria: Evidence from N,N’-Dicyclohexylcarbodiimide-Labeling Studies for the Involvement of the Pore-Forming Protein VDAC

Richard A. Nakashima, Peter L. Pedersen, Patrick S. Mangan, Marco Colombini

Research output: Contribution to journalArticle

Abstract

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(μg 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.

Original languageEnglish (US)
Pages (from-to)1015-1021
Number of pages7
JournalBiochemistry
Volume25
Issue number5
DOIs
StatePublished - Mar 1986

ASJC Scopus subject areas

  • Biochemistry

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