Studies on a phospholipase B from Penicillium notatum. Purification, properties, and mode of action.
1. Phospholipase B which hydrolyzes both the acyl ester bonds of diacylphospholipids (diacyl-hydrolase) and the acyl ester bond of monoacylphospholipids or lysophospholipids, [monoacyl-hydrolase or lysophospholipase, EC 3.1.1.5] was purified from Penicillium notatum about 2000-fold over the crude extract. The final preparation was homogeneous on disc electrophoresis. The apparent molecular weight, determined by gel filtration on Sephadex G-200, was about 116,000. The isoelectric point was pH 4.0. 2. The purified enzyme was a glycoprotein. The carbohydrate content was approximately 30%, consisting of mannose, glucose, and glucosamine. The amino acid composition was also determined. 3. The ratio of monoacyl-hydrolase to diacyl-hydrolase activities was influenced by the physical state of the substrate in the assay system. It was about 1 : 1 or 100 : 1 in the presence of absence of Triton X-100, respectively, and the latter value remained constant throughout the purification procedures. 4. Both enzyme activities had the same pH optimum, 4.0, and were heat-labile. None of the metals tested had any effect on either activity except for Fe2+ and Fe3+. Diisopropyl fluorophosphate at relatively high concentrations completely inhibited both enzyme activities. 5. The Michaelis-Menten constants (Km) of the enzyme for egg lecithin were about 1.5 and 25 mM in the absence and presence of Triton X-100, respectively. The Km value for dicaproyllecithin was 9.8 mM in the absence of Triton X-100. 6. Using a mixture of 1-[14C]stearoyl-lecithin and 2-[14C]oleoyl-lecithin in the presence of Triton X-100 as a substrate, it was found that the P. notatum phospholipase B attacked the acyl ester bonds sequentially, first the 2-acyl and then 1-acyl groups.
D000596:Amino Acids / Q000032:analysis; D003902:Detergents / Q000494:pharmacology; D004237:Diuron / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005944:Glucosamine / Q000032:analysis; D005947:Glucose / Q000032:analysis; D006023:Glycoproteins; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000494:pharmacology; D007700:Kinetics; D008358:Mannose / Q000032:analysis; D008970:Molecular Weight; D010407:Penicillium / Q000201:enzymology*; D010740:Phospholipases / Q000302:isolation & purification / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology
D000596:Amino Acids; D003902:Detergents; D006023:Glycoproteins; D011092:Polyethylene Glycols; D004492:Edetic Acid; D004237:Diuron; D007501:Iron; D010740:Phospholipases; D005947:Glucose; D005944:Glucosamine; D008358:Mannose