Binding of phosphatidylinositol-specific phospholipase C to phospholipid interfaces, determined by fluorescence resonance energy transfer.
Dissociation constants for binding of phosphatidylinositol-specific phospholipase C from Bacillus cereus (bcPI-PLC) and the mammalian phosphatidylinositol-specific phospholipase C-delta(1) to lipid interfaces containing phosphoinositol, phosphocholine, and phosphomethanol head groups were determined by fluorescence resonance energy transfer. Dansyl-labeled lipid probes were used as acceptors, with intrinsic tryptophan of the enzyme as the donor. Titration of protein into lipid provided data from which K(d) and N, the limiting number of lipid molecules per protein bound, were calculated by non-linear regression analysis of exact binding equations. These results were compared with apparent K(m) values from kinetic data. K(d) values in the low microM range in terms of lipid monomers or low nM range in terms of binding sites were calculated with good fits of experimental data to theoretical binding curves. bcPI-PLC binds with high affinity to PI interfaces, slightly lower affinity to PC interfaces, and much lower affinity to PM interfaces. The mammalian enzyme also binds with high affinity to PI interfaces, but shows little or no binding with PC interfaces under similar concentration conditions. These K(d) values correlate reasonably with apparent K(m) values from kinetic experiments.
Hendrickson
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HS
Department of Chemistry, University of Washington, P.O. Box 351700, Seattle, WA 98195-1700, USA. hend@u.washington.edu
Hendrickson
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EK
D001409:Bacillus cereus / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008823:Micelles; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D008823:Micelles; D010743:Phospholipids; D011994:Recombinant Proteins; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase