Detergent-resistant phospholipase A of Escherichia coli K-12. Purification and properties.
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-ol, acid precipitation, acetone fractionation and column chromatographies on Sephadex G-100 in the presence of sodium dodecylsulfate and on DEAE-cellulose 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 L1 activities but also phospholipase A2 and lysophospholipase L2 activities. The enzyme lost its activity completely on incubation at 80 degrees C for 5 min at either pH 6.4 or pH 8.0. It was stable in 0.5% sodium dodecylsulfate at below 40 degrees C. The enzyme was inactivated on incubation for 5 min at 90 degrees C in 1% sodium dodecylsulfate/1% 2-mercaptoethanol/4 M urea. The native and inactivated enzymes showed different protein bands with RF values corresponding to Mr 21 000 and Mr 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.
D004355:Drug Stability; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010740:Phospholipases / Q000302:isolation & purification / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D013696:Temperature
D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D011092:Polyethylene Glycols; D010740:Phospholipases