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Purification of Phosphatidylinositol Synthetase from Rat Brain by CDP‐Diacylglycerol Affinity Chromatography and Properties of the Purified Enzyme
Author(s) -
Ghalayini Abdallah,
Eichberg Joseph
Publication year - 1985
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1985.tb07128.x
Subject(s) - diacylglycerol kinase , phosphatidylinositol , affinity chromatography , biochemistry , enzyme , specific activity , chemistry , chromatography , microsome , inositol , gel electrophoresis , phosphatidylcholine , phospholipid , membrane , protein kinase c , kinase , receptor
A purification procedure for rat brain phosphatidylinositol synthetase (PI synthetase; CDP‐1,2‐diacyl sn ‐glycerol: myo ‐inositol 3‐phosphatidyltransferase; EC 2.7.8.11) is described. The enzyme was purified 200–250‐fold from the homogenate by solubilization with Triton X‐100 from microsomal membranes and affinity chromatography on CDP‐diacylglycerol‐Sepharose. Elution of enzyme activity required the presence of Triton X‐100, CDP‐diacylglycerol, and either phosphatidylcholine or asolectin. The product that was obtained in 5–10% yield from whole brain and in 70% yield from the microsomal fraction contained three protein bands as determined by sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The final preparation contained levels of CDP‐diacylglycerol hydrolase and CDP‐diacylglycerol: sn ‐glycero‐3‐phosphate 3‐phosphatidyltransferase activities that were less than 1% of PI synthetase activity. The purified enzyme displayed a pH optimum of 8.5–9.0, required either Mg 2+ or Mn 2+ and exhibited a K m of 4.6 m M for myo ‐inositol.