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Purification and Characterization of Phospholipase C Preferentially Hydrolysing Phosphatidylcholine in Tetrahymena Membranes
Author(s) -
ALAM SHARIFUL,
BANNO YOSHIKO,
NOZAWA YOSHINORI
Publication year - 1993
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/j.1550-7408.1993.tb04473.x
Subject(s) - phosphatidylcholine , chromatography , dipalmitoylphosphatidylcholine , phospholipase , size exclusion chromatography , phosphatidylethanolamine , phospholipase c , diacylglycerol kinase , biochemistry , gel electrophoresis , biology , phospholipid , membrane , chemistry , enzyme , protein kinase c
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 K m value was determined to be 166 μM for dipalmitoylphosphatidylcholine. A marked increase was observed in phosphatidylcholine hydrolytic activity in the presence of 0.05% (1.2 mM) deoxycholate. Ca 2+ but not Mg 2+ 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 (∼ 9%) and phosphatidylethanolamine (∼ 2%).