Phosphatidylinositol-specific phospholipase C-gamma1 undergoes pH-induced activation and conformational change.
Phospholipase C-gamma1 displayed sigmoidal kinetics with a S(0.5) value of 0.17 mole fraction PIP(2) when assayed at pH 6.8 using detergent:lipid mixed micelles. The pH optimum for hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C-gamma1 was dependent on the mole fraction of substrate in the micelle. The pH optimum was 5.5 when the enzyme was assayed below the S(0.5). The pH optima shifted to a pH range of 6.0-6.3 when the enzyme was assayed above the S(0.5). The kinetic parameters for phospholipase C-gamma1 assayed at various pH values from pH 7.0 to 5.0 yielded similar n values (n=4), but the constant, K', decreased from 1x10(-2) (mole fraction)(2) at pH 7.0 to 1x10(-5) (mole fraction)(2) at pH 5.0. Maximum enzyme specificity occurred at pH values below pH 6.0 as determined by the plot of logk(cat)/S(0.5) versus pH. Intrinsic fluorescence spectroscopy revealed that at a pH value above 7.0 or below 6.3, tryptophan quenching occurred. Fluorescence quenching experiments performed with acrylamide determined phospholipase C-gamma1 incubated at pH 5.0 had a larger collisional quenching constant than enzyme incubated at pH 7.0. Lowering the pH to 5.0 apparently resulted in interior tryptophans becoming more solvent accessible. These data suggest that pH may activate phospholipase C-gamma1 by disrupting ionizable groups leading to a conformational change.
Jones
G A
GA
Department of Pharmacology, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA. gaj5m@virginia.edu
Lazarchic
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M
D020106:Acrylamide; D016367:Baculoviridae / Q000378:metabolism; D001665:Binding Sites; D002021:Buffers; D004789:Enzyme Activation; D005033:Ethylmaleimide; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology; D051966:Phospholipase C gamma; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D010738:Type C Phospholipases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D002021:Buffers; D007527:Isoenzymes; D019269:Phosphatidylinositol 4,5-Diphosphate; D020106:Acrylamide; D014443:Tyrosine; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D005033:Ethylmaleimide