Purification and properties of a membrane-bound phospholipase A1 from Mycobacterium phlei.

A phospholipase A1 bound tightly to the membranes of Mycobacterium phlei cells was purified approximately 500fold to near homogeneity by extraction with Triton X-100, delipidation with organic solvents, solubilization with sodium dodecyl sulfate, column chromatographies on Sephadex G-200 in the presence of sodium dodecyl sulfate and on DEAE-cellulose in the presence of BRIJ 58, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Two active fractions with molecular weights of 27,000 and 45,000 were obtained. The enzyme hydrolyzes the I-acyl chains of phosphatidylethanolamine and phosphatidylcholine. It also attacks 1-acyland 2-acyl-glycerylphosphorylethanolamine at about the same rates. It hydrolyzes the two acyl ester linkages in diacylphospholipids stepwise, as formulated in Reactions I and II, accumulating 2-acyl-lysophospholipids as an intermediate. At acidic pH values the enzyme also hydrolyzes the acidic phospholipids, phosphatidylglycerol and cardiolipin, at comparable rates to neutral phospholipids, such as phosphatidylcholine and phosphatidylethanolamine. It does not hydrolyze triglycerides or diglycerides appreciably. Appropriate concentrations of Triton X-100 caused a ‘I-fold increase in activity for hydrolysis of phosphatidylethanolamine and a slight increase in activity for lysophosphatidylethanolamine, but at higher concentrations this detergent inhibited both activities. Other ionic detergents suppressed enzyme activity at all concentrations tested. The effects on this phospholipase A1 of various metal ions and enzyme inhibitors closely resembled their effects on the lysophospholipases so far reported. Thus, this phospholipase A1 may be regarded as a position nonspecific lysophospholipase with phospholipase A1 activity, or a kind of phospholipase B.