Hexokinase receptor complex in hepatoma mitochondria: evidence from N,N'-dicyclohexylcarbodiimide-labeling studies for the involvement of the pore-forming protein VDAC.

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 (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.

• Authors: R A Nakashima, P S Mangan, M Colombini, P L Pedersen
• Journal: Biochemistry (Biochemistry) Vol. 25 Issue 5 Pg. 1015-21 (Mar 11 1986) ISSN: 0006-2960 [Print] United States
• PMID: 3008816 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Amino Acids
• Carbodiimides
• Membrane Proteins
• Porins
• Receptors, Cell Surface
• Voltage-Dependent Anion Channels
• Dicyclohexylcarbodiimide
• Hexokinase

Topics

• Amino Acids (analysis)
• Animals
• Carbodiimides (pharmacology)
• Cell Line
• Dicyclohexylcarbodiimide (pharmacology)
• Female
• Glycolysis
• Hexokinase (isolation & purification, metabolism)
• Kinetics
• Liver Neoplasms, Experimental (enzymology)
• Membrane Proteins (isolation & purification, metabolism)
• Mitochondria (enzymology)
• Molecular Weight
• Porins
• Rats
• Rats, Inbred Strains
• Receptors, Cell Surface (isolation & purification, metabolism)
• Voltage-Dependent Anion Channels