PURIFICATION AND PROPERTIES OF A MEMBRANE-BOUND PHOSPHOLIPASE-B FROM MYCOBACTERIUM-LEPRAEMURIUM

The phospholipid deacylating enzyme was solubilized from the particula te (membrane) fraction of Mycobacterium lepraemurium with Triton X-100 and sodium cholate, and purified 1100-fold to homogeneous state by 5 steps of column chromatography: DE-52, PL-Sepharose (phosphatidylserin e-attached sepharose), Mono P, heparin-Agarose and Mono Q column chrom atography. The purified enzyme was composed of single polypeptide chai n and molecular mass of 37 kDa was estimated for the protein by SDS-PA GE. The isoelectric point was determined about pH 4.6 and the protein was highly resistant to various kinds of proteolytic enzymes. The puri fied enzyme hydrolyzed both diacyl and monoacyl phospholipids showing that this enzyme was classified to phospholipase B (phospholipase A(1) /lysophospholipase). This phospholipase B had acidic pH optima and hyd rolyzed both neutral phospholipids such as phosphatidylcholine (PC), p hosphatidylethanolamine (PE) and acidic phospholipids such as phosphat idylserine (PS), phosphatidylinositol (PI) and phosphatidylglycerol (P G). Various fatty acids such as 12:0, 14:0, 16:0, 18:0 and 18:1 at sn- l position, and 18:1, 18:2, 18:3 and 16:0 at sn-2 position were libera ted from PC, suggesting no strict specificity toward the fatty acyl gr oups of phospholipids. From the comparison of degradation patterns of phosphatidylcholine with sn-1-[1-C-14]- and sn-2-[1-C-14]fatty acids, this enzyme was suggested to hydrolyze sn-1 position of phospholipid f irst and then sn-2 position, as the phospholipase B of M. phlei. This enzyme also attacked 1-acyl- and 2-acyl-lyso-PC at about same rates. T he K-m values for 1-acyl-2-oleoyl-PC and 2-oleoyl-lyso-PC were estimat ed 1.6 and 0.75 mM, respectively.