Purification and kinetic studies of adenine phosphoribosyltransferase from human erythrocytes

Satish Srivastava, Ernest Beutler

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Abstract

Adenine phosphoribosyltransferase was purified 2200-fold from human erythrocytes. The purification steps involved (NH4)2SO4 fractionation followed by Sephadex-G75 column chromatography, DEAE-Sephadex chromatography, and (NH4)2SO4 precipitation. Partially purified enzyme was stabilized by 0.3 m (NH4)2SO4, which also protected the enzyme from heat denaturation. The enzyme was partially inhibited by sodium ions which were partially antagonized by magnesium. Divalent cation is an absolute requirement for high enzyme activity. Ca2+, Mn2+, and Mg2+ supported high initial rates and Hg2+ completely inhibited the enzyme. The kinetic data with initial velocity and the product inhibition studies are consistent with a mechanism in which the synthesis of nucleotide proceeds as an ordered sequential reaction.

Original languageEnglish (US)
Pages (from-to)426-434
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume142
Issue number2
DOIs
StatePublished - 1971
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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