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Biochemistry (Moscow)

, Volume 75, Issue 7, pp 873–880 | Cite as

Isolation and properties of human transketolase

  • L. E. MeshalkinaEmail author
  • O. N. Solovjeva
  • Yu. A. Khodak
  • V. L. Drutsa
  • G. A. Kochetov
Article

Abstract

Recombinant human (His)6-transketolase (hTK) was obtained in preparative amounts by heterologous expression of the gene encoding human transketolase in Escherichia coli cells. The enzyme, isolated in the form of a holoenzyme, was homogeneous by SDS-PAGE; a method for obtaining the apoenzyme was also developed. The amount of active transketolase in the isolated protein preparation was correlated with the content of thiamine diphosphate (ThDP) determined in the same preparation. Induced optical activity, facilitating studies of ThDP binding by the apoenzyme and measurement of the transketolase reaction at each stage, was detected by circular dichroism spectroscopy. A single-substrate reaction was characterized, catalyzed by hTK in the presence of the donor substrate and in the absence of the acceptor substrate. The values of the Michaelis constant were determined for ThDP and a pair of physiological substrates of the enzyme (xylulose 5-phosphate and ribose 5-phosphate).

Key words

human transketolase thiamine diphosphate induced optical activity circular dichroism spectroscopy 

Abbreviations

DHEThDP

α,β-dihydroxyethylthiamine diphosphate

DTT

dithiothreitol

GA

glycolaldehyde

3-GAP

glyceraldehyde 3-phosphate

GAPD

glyceraldehyde-3-phosphate dehydrogenase

hTK

transketolase from normal human tissues

R-5-P

ribose 5-phosphate

ThDP

thiamine diphosphate

TK

transketolase

TKTL1

transketolase from human tumor tissues

X-5-P

xylulose 5-phosphate

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Copyright information

© Pleiades Publishing, Ltd. 2010

Authors and Affiliations

  • L. E. Meshalkina
    • 1
    Email author
  • O. N. Solovjeva
    • 1
  • Yu. A. Khodak
    • 1
  • V. L. Drutsa
    • 1
  • G. A. Kochetov
    • 1
  1. 1.Belozersky Institute of Physico-Chemical BiologyLomonosov Moscow State UniversityMoscowRussia

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