The interaction of phosphofructokinase with erythrocyte membranes

T. Higashi, C. S. Richards, K. Uyeda

Research output: Contribution to journalArticle

69 Scopus citations

Abstract

The interaction of muscle phosphofructokinase with human erythrocyte ghost membranes was investigated. Scatchard analysis of the binding reveals only a single class of binding sites numbering approximately 4 x 105 sites/ghost with an association constant of 2 x 107 M-1. Maximum binding is observed below pH 6.8 and is complete within 30 min at 4°, 24°, and 37°C; binding is inhibited by high ionic strength. Studies on the effect of ligands on the binding show that fructose-1,6-P2, Mg2+, and ATP favor binding, while ADP and 2,3-P2-glycerate do not. The nature of the phosphofructokinase binding site on the membrane was investigated using membranes which had been washed with saline, EDTA, and NaOH to remove various peripheral membrane proteins. The results show that the removal of peripheral proteins from the membrane does not diminish the binding capacity of the membranes, suggesting that an integral membrane protein is the binding site. Phosphofructokinase binding is inhibited by aldolase and glyceraldehyde-3-P dehydrogenase, which have been reported to bind to Band 3 protein. Furthermore, both aldolase and glyceraldehyde-3-P dehydrogenase also mediate the dissociation of phosphofructokinase from these membranes; the amount of phosphofructokinase released is proportional to the amount of aldolase bound. Neither spectrin (0.1 mg/ml) nor hemoglobin (0.1 to 50 mg/ml) influences the binding of phosphofructokinase. The results demonstrate that phosphofructokinase binds to a specific site, probably Band 3 protein, on the inner surface of human erythrocyte membranes. A model which explains the relationship of the binding sites of these enzymes is presented.

Original languageEnglish (US)
Pages (from-to)9542-9550
Number of pages9
JournalJournal of Biological Chemistry
Volume254
Issue number19
StatePublished - Dec 1 1979

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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