Purification and regiospecificity of multiple enzyme activities of phospholipase A(1) from bonito muscle.
Phospholipase A(1) (PLA(1)), which catalyzes the hydrolysis of the sn-1 ester bond of diacyl phospholipids, was purified from 100,000 x g supernatant of bonito muscle to homogeneity by ammonium-sulfate precipitation and four consecutive column chromatographies (DEAE anion-exchange, ether-Toyopeal, hydroxylapatite and Toyopeal HW 50S columns). The final preparation showed a single band above the 67-kDa molecular marker on SDS-PAGE, and the molecular mass was determined to be 71.5 kDa by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using bovine serum albumin as a standard for calibration. The N-terminal 8 amino residues were determined to be Ala-Pro-Ala-Glu-Lys-Val-Lys-Try. Regiospecificity of multiple enzyme activities of the PLA(1) was examined using positionally defined synthetic phosphatidylcholine (PC) and lysophosphatidylcholines (LPC). An acyl ester bond at the sn-1 position of PC was exclusively hydrolyzed by phospholipase activity, and 1-acyl LPC was cleaved to fatty acid and glycerophosphocholine by lysophospholipase (LPL) activity. However, the positional isomer, 2-acyl LPC was a poor substrate for LPL activity. PC/transacylation activity was also observed when excess 2-acyl LPC was supplied in the reaction mixture, and fatty acid at the sn-1 position of donor PC was transferred to the sn-1 position of acceptor LPC. These results demonstrate that the multiple enzyme activities of PLA(1), this is lysophospholipase, transacylase as well as phospholipase, have a strict regiospecificity at the sn-1 position of substrates.
Hirano
K
K
Department of Applied Biological Science, Faculty of Engineering, Fukuyama University, Fukuyama, Japan.
Okada
E
E
Tanaka
T
T
Satouchi
K
K
D000217:Acyltransferases / Q000378:metabolism; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005399:Fishes; D008244:Lysophosphatidylcholines / Q000378:metabolism; D008245:Lysophospholipase / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism; D009132:Muscles / Q000201:enzymology*; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D008244:Lysophosphatidylcholines; D009097:Multienzyme Complexes; D000217:Acyltransferases; D010741:Phospholipases A; D008245:Lysophospholipase; C021782:lysophospholipase-transacylase