Purification and properties of a membrane-bound phospholipase B from Mycobacterium lepraemurium.
The phospholipid deacylating enzyme was solubilized from the particulate (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 (phosphatidylserine-attached sepharose), Mono P, heparin-Agarose and Mono Q column chromatography. The purified enzyme was composed of single polypeptide chain and molecular mass of 37 kDa was estimated for the protein by SDS-PAGE. The isoelectric point was determined about pH 4.6 and the protein was highly resistant to various kinds of proteolytic enzymes. The purified enzyme hydrolyzed both diacyl and monoacyl phospholipids showing that this enzyme was classified to phospholipase B (phospholipase A1/lysophospholipase). This phospholipase B had acidic pH optima and hydrolyzed both neutral phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) and acidic phospholipids such as phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylglycerol (PG). Various fatty acids such as 12:0, 14:0, 16:0, 18:0 and 18:1 at sn-1 position, and 18:1, 18:2, 18:3 and 16:0 at sn-2 position were liberated from PC, suggesting no strict specificity toward the fatty acyl groups of phospholipids. From the comparison of degradation patterns of phosphatidylcholine with sn-1-[1-14C]- and sn-2-[1-14C]fatty acids, this enzyme was suggested to hydrolyze sn-1 position of phospholipid first 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. The Km values for 1-acyl-2-oleoyl-PC and 2-oleoyl-lyso-PC were estimated 1.6 and 0.75 mM, respectively.
Maeda
S
S
National Institute for Leprosy Research, Tokyo, Japan.
Kashiwabara
Y
Y
D004795:Enzyme Stability; D006868:Hydrolysis; D007526:Isoelectric Point; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000378:metabolism; D008245:Lysophospholipase / Q000302:isolation & purification* / Q000378:metabolism*; D008970:Molecular Weight; D009167:Mycobacterium lepraemurium / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000378:metabolism; D013379:Substrate Specificity
D008244:Lysophosphatidylcholines; D010713:Phosphatidylcholines; D008245:Lysophospholipase