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

  • Francesco Palma
  • Deborah Agostini
  • Philip Mason
  • Marina Dachá
  • Giovanni Piccoli
  • Beatrice Biagiarelli
  • Mara Fiorani
  • Vilberto Stocchi
Article

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 III pGEX-2T vector fusion protein glutathione S-transferase D-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

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Copyright information

© KluwerAcademic Publishers 1996

Authors and Affiliations

  • Francesco Palma
    • 1
  • Deborah Agostini
    • 1
  • Philip Mason
    • 2
  • Marina Dachá
    • 1
  • Giovanni Piccoli
    • 1
  • Beatrice Biagiarelli
    • 1
  • Mara Fiorani
    • 1
  • Vilberto Stocchi
    • 1
  1. 1.Istituto di Chimica Biologica ‘Giorgio Fornaini’Università di UrbinoUrbinoItaly
  2. 2.Haematology DepartmentHammersmith HospitalLondonUK

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