The Purification And Properties Of A Phospholipase A2 From Bovine Brain Which Acts On Phosphatidylinositol

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Doctor of Philosophy


A phospholipase A(,2) with an alkaline pH optimum (pH 7.5) has been purified 1600 times from bovine brain grey matter microsomes (106,000g pellet obtained from a 30,000g supernatant), which shows a selectivity towards phosphatidylinositol. The enzyme was extracted (Triton X-100) and stabilized into a medium containing the non-ionic detergent Triton X-100, glycerol, asolecithin and (beta)-mercaptoethanol. The purification was carried out, keeping the enzyme in the above stabilizing medium where possible. The purification was achieved by ammonium sulphate fractionation, column chromatography (Sephadex G-200 and DEAE-Sephacel) and preparative polyacrylamide gel electrophoresis. The purified enzyme runs as a single band on a SDS-polyacrylamide gel and gives a molecular weight of 18,300; amino acid analysis gives the molecular weight to be 18,521.;The enzyme was found to be heat resistant (up to 70(DEGREES)C) and stimulated by Ca('2+) (2-5mM) and the non-ionic detergents, Triton X-100 (0.025%) and octyl glucoside (25mM). The thiol reagents, p-hydroxymercuribenzoate, iodoacetic acid and N-ethylmaleimide were all found to be inhibitory; however, dithiothreitol was found to protect the activity.;The kinetic parameters are: K(,m) = 0.52mM and V(,max) = 1440 nanomoles ('14)C-oleic acid released/min/mg protein, using phosphatidylinositol labelled with ('14)C-oleic acid in the 2-position.;Phosphatidylinositols labelled in the 2-position with different fatty acids, gave the following order of release with the purified enzyme: C(,16:0) > C(,18:0) > C(,18:1) > C(,20:4). The addition of these fatty acids (5(mu)M and 10(mu)M) caused an inhibition of fatty acid release in the reverse order to that noted above. There was little or no release from phosphatidylinositols labelled with fatty acid in the 1-position. These evaluations were based on ('14)C- or ('3)H- counts and gas liquid chromatography analysis of the fatty acids released. The enzyme showed the following order of activity towards phosphoglycerides containing ('14)C-oleic acid in the 2-position: phosphatidylinositol (set at 100%), phosphatidylcholine, 62.5%, phosphatidic acid, 32.6%, phosphatidylethanolamine, 25.1% and phosphatidylserine, 21.5%.;The purified phospholipase A(,2) from the 106,000g pellet of bovine brain has the appropriate properties required of an enzyme to function in a deacylation-reacylation cycle for phosphatidylinositol, and possibly for other phosphoglycerides.

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