Partial characterization of rat heart cytosolic phospholipase A1 and demonstration of essential sulfhydryl groups.
The cytosol of rat heart has been previously shown to contain phospholipase A activity in substantial amounts. This paper describes the isolation and partial purification of rat heart cytosolic phospholipase A. After homogenization of rat heart followed by centrifugation to remove membraneous material, the phospholipase A activity was isolated by ammonium sulfate precipitation and further purified by gel permeation chromatography with Sephadex G-100 in the presence of 5 mM taurodeoxycholate. Two peaks were isolated: a minor peak at the void volume and major peak corresponding to a molecular weight of 45,000. The molecular weight observed in HPLC gel permeation chromatography experiments was also Mr 45,000 and was not significantly affected by the nature of the detergent used. Phospholipase A was purified 77-fold over the crude cytosol. Further purification could not be attained due to lability of the phospholipase A activity. The enzyme is a phospholipase of the A1 type which does not require Ca2+ and lacks lipase or transacylase activity. It is unusual for the phospholipases A described to date, since it is inhibited by thiol reagents and is protected by beta-mercaptoethanol, suggesting the presence of essential sulfhydryl residues.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D003902:Detergents / Q000494:pharmacology; D006358:Hot Temperature; D008245:Lysophospholipase / Q000032:analysis; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054466:Phospholipases A1; D011489:Protein Denaturation; D051381:Rats; D011919:Rats, Inbred Strains; D013438:Sulfhydryl Compounds / Q000032:analysis*
D003902:Detergents; D013438:Sulfhydryl Compounds; D010740:Phospholipases; D010741:Phospholipases A; D054466:Phospholipases A1; D008245:Lysophospholipase