Detergent-resistant phospholipase A, which is tightly bound to the outer membranes of Escherichia coli K-12 cells, was purified approximately 2000-fold to near homogeneity by solubilization with sodium dodecylsulfate and butan-1 -01, acid precipitation, acetone fractionation and column chromatographies on Sephadex G-100 in the presence of sodium dodecylsulfate and on DEAEcellulose in the presence of Triton X-100. The final preparation showed a single band in the sodium dodecylsulfate gel system.The enzyme hydrolyzes both the 1-acyl and 2-acyl chains of phosphatidylethanolamine or phosphatidylcholine. It also attacks 1-acyl and 2-acylglycerylphosphorylethanolamine. Thus, this enzyme shows not only phospholipase A1 and lysophospholipase LI activities but also phospholipase A2 and lysophospholipase L2 activities.The enzyme lost its activity completely on incubation at 80 "C for 5 min at either pH 6.4 or pH 8.0. It was stable in 0.5 % sodium dodecylsulfate at below 40 "C. The enzyme was inactivated on incubation for 5 min at 90 "C in 1 % sodium dodecylsulfate/l % 2-mercaptoethanol/4 M urea.The native and inactivated enzymes showed different protein bands with RF values corresponding to M , 21 000 and M , 28 000 respectively, in a sodium dodecylsulfate gel system. Triton X-100 seemed to protect the enzyme from inactivation. The purified enzyme was fully active on phosphatidylethanolamine in the presence of 0.0002% or 0.05% Triton X-100. The enzyme requires Ca2+. From its properties this enzyme seems to be identical with the enzyme purified from crude extracts of Escherichia coli B by Scandella and Kornberg. However, it differs from the latter in its positional specificity and susceptibility to sodium dodecylsulfate. Possible explanation of the difference of positional specificity of the two preparations is also described.A phospholipase A of Escherichia coli, described by several investigators [1,3-lo], is now widely used as a marker of the outer membranes of this bacterium [ 1 1 -131. This enzyme was purified to near homogeneity by Scandella and Kornberg [14] and identified as phospholipase A1 with lysophospholipase activity. On the other hand, from studies on crude extracts of mutants which lack phospholipase A activity, Doi and Nojima [15,16] concluded that detergent-resistant phospholipase A of Escherichia coli K-12 has both phospholipase A1 and A2 and lysophospholipase activities. Therefore, further purification of the enzyme was necessary to decide whether a single enzyme shows both phospholipase A1 and A2 activities and lysophospholipase activities [17].

A crude preparation of membrane-bound phospholipase A (detergent-resistant) in Escherichia coli K-12 cells was found to be quite stable or even apparently activated on incubation at 100 degrees C, but became strikingly thermolabile when it was highly purified and Triton X-100 was removed from the purified enzyme preparation. The rate of inactivation showed a biphasic temperature dependence: inactivation was rapid at 37 degrees C and also above 70 degrees C. Inactivation above 70 degrees C changed the mobility of the enzyme on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, but inactivation at 37 degrees C did not affect the electrophoretic mobility. Triton X-100 effectively protected the enzyme against inactivation at 37 degrees C. The concentration required for the protection of the enzyme was more than its critical micelle concentration. Phospholipids, such as phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, phosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylcholine, also protected the enzyme against inactivation at 37 degrees C. These results suggest that the binding of hydrophobic compounds stabilizes the enzyme.

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