Purification and characterization of phospholipase C preferentially hydrolysing phosphatidylcholine in Tetrahymena membranes.
A phospholipase C (PLC) activity that preferentially hydrolyses phosphatidylcholine to diacylglycerol and phosphorylcholine was found to be present in Tetrahymena pyriformis, strain W and most of its activity was recovered in the membrane fraction. This enzyme was extracted with 1% Triton X-100 from the membrane fraction and purified to apparent homogeneity by sequential chromatographies on Fast Q-Sepharose, hydroxyapatite HCA-100S, Mono Q and Superose 12 gel filtration columns. The purified enzyme had specific activity of 2083 nmol of diacylglycerol released/mg of protein/min for dipalmitoylphosphatidylcholine hydrolysis. Its apparent molecular mass was 128 kDa as determined by SDS-polyacrylamide gel electrophoresis and was 127 kDa by gel filtration chromatography, indicating that the enzyme is present in a monomeric form. The enzyme exhibited an optimum pH 7.0 and the apparent Km value was determined to be 166 microM for dipalmitoylphosphatidylcholine. A marked increase was observed in phosphatidylcholine hydrolytic activity in the presence of 0.05% (1.2 mM) deoxycholate. Ca2+ but not Mg2+ enhanced the activity at a concentration of 2 mM. This purified phospholipase C exhibited a preferential hydrolytic activity for phosphatidylcholine but much less activity was observed for phosphatidylinositol (approximately 9%) and phosphatidylethanolamine (approximately 2%).
Alam
S
S
Department of Biochemistry, Gifu University School of Medicine, Japan.
Banno
Y
Y
Nozawa
Y
Y
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D010713:Phosphatidylcholines / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D013379:Substrate Specificity; D013769:Tetrahymena pyriformis / Q000201:enzymology*; D010738:Type C Phospholipases / Q000302:isolation & purification* / Q000378:metabolism*
D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010716:Phosphatidylinositols; D015060:1,2-Dipalmitoylphosphatidylcholine; C020888:1,2-dielaidoylphosphatidylethanolamine; D017886:Durapatite; D010738:Type C Phospholipases; D008274:Magnesium; D002118:Calcium