Glucose phosphorylation in tumor cells: Cloning, sequencing, and overexpression in active form of a full-length cDNA encoding a mitochondrial bindable form of hexokinase

Krishan K. Arora, Maurizio Fanciulli, Peter L. Pedersen

Research output: Contribution to journalArticlepeer-review

Abstract

In rapidly growing tumor cells exhibiting high glucose catabolic rates, the enzyme hexokinase is markedly elevated and bound in large amounts (50-80% of the total cell activity) to the outer mitochondrial membrane (Arora, K. K., and Pedersen, P. L. (1988) J. Biol. Chem. 263, 17422-17428; Parry, D. M., and Pedersen, P. L. (1983) J. Biol. Chem. 258, 10904-10912). In extending these studies, we have isolated a cDNA clone of hexokinase from a λgt11 library of the highly glycolytic, c37 mouse hepatoma cell line. This clone, comprising 4,198 base pairs, contains a single open reading frame of 2,754 nucleotides which encode a 918-amino acid hexokinase with a mass of 102,272 daltons. This enzyme exhibits, respectively, 68 and 32 amino acid differences, including several charge differences, from the recently sequenced human kidney and rat brain enzymes. The putative glucose and ATP binding domains present in the latter two enzymes and in rat liver glucokinase are conserved in the tumor enzyme. At its N-terminal region, tumor hexokinase has a 12-amino acid hydrophobic stretch which is present in the rat brain enzyme but absent in rat liver glucokinase, a cytoplasmic enzyme. The mature tumor hexokinase protein has been overexpressed in active form in Escherichia coli and purified 9-fold. The overexpressed enzyme binds to rat liver mitochondria in the presence of MgCl2. This is the first report describing the cloning and sequencing of a tumor hexokinase, and the first report documenting the overexpression of any hexokinase type in E. coli. Questions pertinent to the enzyme's mechanism, regulation, binding to mitochondria, and its marked elevation in tumor cells can now be addressed.

Original languageEnglish
Pages (from-to)6481-6488
Number of pages8
JournalJournal of Biological Chemistry
Volume265
Issue number11
Publication statusPublished - 1990

ASJC Scopus subject areas

  • Biochemistry

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