Proteolytic fragments of phosphoinositide-specific phospholipase C-delta 1. Catalytic and membrane binding properties.
Active proteolytic fragments of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta 1) were generated by trypsin digestion of the native protein. Brief proteolysis produced a 77-kDa fragment that contained the highly conserved X and Y regions but lacked the amino-terminal domain (amino acids 1-60). Prolonged digestion of PLC-delta 1 produced two fragments, one of 45 kDa that contained the entire X region and another of 32 kDa that consisted of the entire Y region and COOH-terminal domain. The 45- and 32-kDa fragments were isolated as an active heterodimeric complex. The 77-kDa fragment and the complex catalyzed calcium-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in detergent/phospholipid mixed micelles. When compared with the native enzyme, both the 77-kDa fragment and the complex exhibited a reduced capacity to processively hydrolyze PIP2; increasing the mole fraction of PIP2 in the mixed micelle surface greatly increased the rate of PIP2 hydrolysis catalyzed by the native enzyme but not the fragments. Both fragments also exhibited a reduced affinity for substrate; the native enzyme bound to bilayer vesicles consisting of phosphatidylcholine and PIP2 with high affinity (Ka approximately 10(6) M-1), whereas the fragments bound weakly (Ka < 10(4) M-1). These results demonstrate that the X, Y, and COOH-terminal regions form a calcium-dependent catalytic core that is resistant to proteolysis. The amino-terminal domain appears to be essential for high affinity binding to PIP2 but not catalysis. These observations are consistent with the idea that the amino-terminal domain forms part of a PIP2 binding site, which anchors PLC-delta 1 to the membrane surface during processive hydrolysis of its substrate.
Cifuentes
M E
ME
Department of Physiology and Biophysics, State University of New York, Stony Brook 11794.
Honkanen
L
L
Rebecchi
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D003600:Cytosol / Q000201:enzymology; D000939:Epitopes / Q000032:analysis; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D008433:Mathematics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D019269:Phosphatidylinositol 4,5-Diphosphate; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010716:Phosphatidylinositols / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000906:Antibodies; D000939:Epitopes; D007527:Isoenzymes; D008051:Lipid Bilayers; D010446:Peptide Fragments; D010455:Peptides; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium