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Purification and partial biochemical–genetic characterization of trehalose 6-phosphate synthase from muscles of adult female Ascaris suum

Published online by Cambridge University Press:  09 May 2012

M. Dmitryjuk ,
M. Dopieralska ,
E. Łopieńska-Biernat  and
R.J. Frączek
M. Dmitryjuk*
Affiliation:
Department of Biochemistry, Faculty of Biology, University of Warmia and Mazury, Oczapowskiego 1A, 10-957 Olsztyn, Poland
M. Dopieralska
Affiliation:
Department of Biochemistry, Faculty of Biology, University of Warmia and Mazury, Oczapowskiego 1A, 10-957 Olsztyn, Poland
E. Łopieńska-Biernat
Affiliation:
Department of Biochemistry, Faculty of Biology, University of Warmia and Mazury, Oczapowskiego 1A, 10-957 Olsztyn, Poland
R.J. Frączek
Affiliation:
Department of Biochemistry, Faculty of Biology, University of Warmia and Mazury, Oczapowskiego 1A, 10-957 Olsztyn, Poland
*
*Fax: (48 89) 535 20 15 E-mail: [email protected]
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Abstract

Trehalose 6-phosphate (T6P) synthase (TPS; EC 2.4.1.15) was isolated from muscles of Ascaris suum by ammonium sulphate fractionation, ion-exchange DEAE SEPHACELTM anion exchanger column chromatography and Sepharose 6B gel filtration. On sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), 265-fold purified TPS exhibited a molecular weight of 66 kDa. The optimum pH and temperature of the purified enzyme were 3.8–4.2 and 35°C, respectively. The isoelectric point (pI) of TPS was pH 5.4. The studied TPS was not absolutely substrate specific. Besides glucose 6-phosphate, the enzyme was able to use fructose 6-phosphate as an acceptor of glucose. TPS was activated by 10 mM MgCl2, 10 mM CaCl2 and 10 mM NaCl. In addition, it was inhibited by ethylenediaminetetra-acetic acid (EDTA), KCl, FeCl3 and ZnCl2. Two genes encoding TPS were isolated and sequenced from muscles of the parasite. Complete coding sequences for tps1 (JF412033.2) and tps2 (JF412034.2) were 3917 bp and 3976 bp, respectively. Translation products (AEX60788.1 and AEX60787.1) showed expression to the glucosyltransferase-GTB-type superfamily.

Type
Research Papers
Information
Journal of Helminthology , Volume 87 , Issue 2 , June 2013 , pp. 212 - 221
Copyright
Copyright © Cambridge University Press 2012 

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