Immobilized E. coli Alkaline Phosphatase - Its properties, stability, and utility in studying the dephosphorylation of proteins

Khaja Basheeruddin, Vicki Rothman, Simeon Margolis

Research output: Contribution to journalArticle

Abstract

We have immobilized E. coli alkaline phosphatase (EC 3.1.3.1) by linking it covalently to sepharose 4B. This preparation has several advantages over the soluble enzyme. The immobilized enzyme is easily separable from other constituents in incubation mixtures. The immobilized enzyme can be reused repeatedly and is more stable than the soluble enzyme to heat treatment in the presence of 10 mM Mg2+. The insoluble and soluble phosphatases removed 75 and 77%, respectively, of the inorganic phosphorus from casein. The immobilized enzyme inactivated two enzymes believed to be active in the phosphorylated state, acyl-CoA : cholesterol acyltransferase (ACAT) by 39% and NADPH-cytochrome P-450 reductase by 89%. The utility of immobilized alkaline phosphatase for studying the phosphorylation and dephosphorylation of soluble or membrane-bound enzymes and proteins is discussed.

Original languageEnglish (US)
Pages (from-to)133-140
Number of pages8
JournalApplied Biochemistry and Biotechnology
Volume11
Issue number2
DOIs
StatePublished - Apr 1 1985

Keywords

  • Acyl-CoA: cholesterol acyltransferase
  • Alkaline phosphatase, immobilized
  • casein
  • dephosphorylation, immobilized alkaline phosphatase and
  • immobilized alkaline phosphatase, from E. coli
  • protein dephosphorylation
  • stability, of immobilized alkaline phosphatase

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology

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