Purification and characterization of the major isotypes of apyrase from the cytoskeleton fraction in Pisum sativum

  • Abe S
  • Moustafa M
  • Shibata K
 et al. 
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Abstract

We isolated isotypes of the 49-kDa apyrase from the cytoskeleton fraction of pea (Pisum sativum L. var. Alaska) stems, separated them using heparin affinity and anion exchange column chromatography, and investigated the enzymatic activities of each isotype. When potassium acetate gradients at constant pH were employed, there was poor separation between isotypes. However, when a pH gradient of 6.7-8.5 was used in conjunction with a potassium acetate gradient from 0 to 1 M, five peaks were identifiable, eluting between 0.35 and 0.65 M potassium acetate, and termed P0, P1, P2, P3, and P4. 2D-Polyacrylamide gel electrophoresis showed that each of these peaks was highly enriched for an individual isotype, and the isoelectric points of these isotypes were 5.82, 6.05, 6.30, 6.55, and 6.80 in fractions P0, P1, P2, P3, and P4, respectively. The isotypes of pI 6.05, 6.30, and 6.55 were the most abundant, and the more acidic isotypes had slightly higher molecular mass than other isotypes. Based on their partial amino acid sequences, their capability to hydrolyze both nucleoside tri- and di-phosphates into their respective mono-phosphates, and their similar hydrolyzing activity towards ADP, we presume they are all isotypes of the 49-kDa apyrase (EC 3.6.1.5). Since the calculated isoelectric point of apyrase based upon its amino acid sequence is 7.11, these results indicate that the enzyme is modified in various ways (most likely including phosphorylation) to furnish different isoforms with different activities over different substrates. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Author-supplied keywords

  • 2D-PAGE
  • Apyrase
  • Cytoskeleton
  • Hydrolysis
  • Isoelectric point
  • Phosphorylation
  • Pisum sativum

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Authors

  • Shunnosuke Abe

  • Mahmoud F.M. Moustafa

  • Koichi Shibata

  • Motohito Yoneda

  • Eric Davies

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