Membrane binding of phospholipases C-beta 1 and C-beta 2 is independent of phosphatidylinositol 4,5-bisphosphate and the alpha and beta gamma subunits of G proteins

Biochemistry. 1996 Dec 24;35(51):16824-32. doi: 10.1021/bi961606w.

Abstract

We have measured the membrane binding affinities of purified phosphatidylinositol-specific phospholipases C-beta 1 and C-beta 2 to membranes of varying lipid composition using fluorescence methods. Our studies show that these proteins bind with affinities of 10(-5)-10(-4) M, with a small dependence on lipid type. Binding was relatively insensitive to the presence of phosphatidylinositol-specific phospholipases C-beta s' major physiological substrate, phosphatidylinositiol 4,5-bisphosphate, as well as the presence of Ca2+, which is required for activity. The presence of purified GTP gamma S-activated alpha 11 subunits of heterotrimeric guanine nucleotide binding proteins (G proteins) did not alter the membrane binding affinity of phosphatidylinositol-specific phospholipases C-beta 1, even though alpha 11 is a potent activator of this protein. Similarly, the presence of purified beta gamma subunits of G proteins did not alter the membrane association of phosphatidylinositol-specific phospholipases C-beta 2 even though these subunits strongly activate this isoform. These results argue against a recruitment model for PLC-beta activation by G proteins, negatively charged lipids, Ca2+, or substrate, and suggest that activation occurs through association of the membrane-bound species.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Energy Transfer
  • Erythrocyte Membrane / metabolism
  • GTP-Binding Proteins / chemistry
  • GTP-Binding Proteins / metabolism
  • Humans
  • In Vitro Techniques
  • Isoenzymes / chemistry
  • Isoenzymes / genetics
  • Isoenzymes / metabolism*
  • Membrane Lipids / metabolism
  • Molecular Sequence Data
  • Phosphatidylinositol 4,5-Diphosphate / metabolism
  • Phospholipase C beta
  • Protein Binding
  • Protein Conformation
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Spectrometry, Fluorescence
  • Type C Phospholipases / chemistry
  • Type C Phospholipases / genetics
  • Type C Phospholipases / metabolism*

Substances

  • Isoenzymes
  • Membrane Lipids
  • Phosphatidylinositol 4,5-Diphosphate
  • Recombinant Proteins
  • Type C Phospholipases
  • Phospholipase C beta
  • GTP-Binding Proteins