Molecular and Cellular Biochemistry

, Volume 155, Issue 1, pp 23–29

Purification and characterization of the carboxyl-domain of human hexokinase type III expressed as fusion protein

Authors

  • Francesco Palma
    • Istituto di Chimica Biologica ‘Giorgio Fornaini’Università di Urbino
  • Deborah Agostini
    • Istituto di Chimica Biologica ‘Giorgio Fornaini’Università di Urbino
  • Philip Mason
    • Haematology DepartmentHammersmith Hospital
  • Marina Dachá
    • Istituto di Chimica Biologica ‘Giorgio Fornaini’Università di Urbino
  • Giovanni Piccoli
    • Istituto di Chimica Biologica ‘Giorgio Fornaini’Università di Urbino
  • Beatrice Biagiarelli
    • Istituto di Chimica Biologica ‘Giorgio Fornaini’Università di Urbino
  • Mara Fiorani
    • Istituto di Chimica Biologica ‘Giorgio Fornaini’Università di Urbino
  • Vilberto Stocchi
    • Istituto di Chimica Biologica ‘Giorgio Fornaini’Università di Urbino
Article

DOI: 10.1007/BF00714329

Cite this article as:
Palma, F., Agostini, D., Mason, P. et al. Mol Cell Biochem (1996) 155: 23. doi:10.1007/BF00714329

Abstract

In mammalian tissues hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) exists as four isoenzymes encoded by distinct genes. These proteins are homologous and are organized in two homologous domains, with the exception of hexokinase type IV which has only one. This organization is believed to be the result of a duplication and tandem fusion event involving the gene encoding for the ancestral hexokinase. In this study we cloned the carboxyl-domain of human hexokinase type III and expressed it in Escherichia coli as a glutathione S-transferase fusion protein, using the pGEX-2T expression vector. The recombinant protein showed catalytic activity. A comparative study of the kinetic properties of the expressed carboxyl-domain and the enzyme partially purified from human lymphocytes is also shown. The results now allow a better understanding of the role of the carboxyl-domain in determining the catalytic properties of the enzyme.

Key words

hexokinase type IIIpGEX-2T vectorfusion proteinglutathione S-transferaseD-glucose

Abbreviations

SSPE

0.15 mol/l NaCl/ 10 mmol/l Na-phosphate/ 1 mmol/l EDTA pH 7.4

Denhardt's

0.02 g/l BSA Fractions V/ 0.02 g/l Ficoll 400/ 0.02 g/l polyvinylpyrollidone

SSC

0.15 mol/l NaCl/ 15 mmol/l Na3-citrate pH 7.0

IPTG

isopropyl β-D-thiogalactopyranoside

PMSF

phenylmethylsulfonyl fluoride

Glc-6-P

glucose 6-phosphate

PAGE

polyacrylamide gel electrophoresis

Copyright information

© KluwerAcademic Publishers 1996