Purification and characterization of 1-O-acylceramide synthase, a novel phospholipase A2 with transacylase activity

被引:79
作者
Abe, A [1 ]
Shayman, JA [1 ]
机构
[1] Univ Michigan, Dept Internal Med, Div Nephrol, Med Ctr, Ann Arbor, MI 48109 USA
关键词
D O I
10.1074/jbc.273.14.8467
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A novel pathway for ceramide metabolism, 1-O-acylceramide formation, was previously reported (Abe, A, Shayman, J. A., and Radin, N. S. (1996) J. Biol. Chem. 271, 14383-14389), In this pathway a fatty acid in the sn-2 position of phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl position of ceramide, An enzyme that catalyzes the esterification of N-acetylsphingosine was purified from the postmitochondrial supernatant of calf brain through consecutive steps, including ammonium sulfate fractionation, DEAE-Sephacel, phenyl-Sepharose, S-Sepharose, Sephadex G-75, concanavalin A-agarose, and heparin-Sepharose chromatography. The molecular mass of the enzyme was determined to be 40 kDa by gel filtration on Sephadex G-75, The enzyme bound to concanavalin A-agarose column was eluted with the buffer containing 500 mM alpha-methyl-D-mannopyranoside. Further purification by heparin-Sepharose chromatography resulted in separation of two peaks of enzyme activity, Coincidence between the transacylase activity and a stained protein of a molecular mass of 40 kDa was observed, as determined by SDS-polyacrylamide gel electrophoresis and recovery after separation over an acidic native gel, The second peak of activity from the heparin-Sepharose chromatography represented a purification of 193,000-fold, These results are consistent with the enzyme being a glycoprotein of a molecular mass of about 40 kDa with a single polypeptide chain. The purified enzyme had a pH optimum at pH 4.5, The divalent cations Ca2+ and Mg2+ enhanced but were not essential for the transacylase activity, Neither activation nor inactivation of the enzyme activity was observed in the presence of 2 mM ATP or 2 mM dithiothreitol, Preincubation of the enzyme with 1 mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride, or 3.1 mu M bromoenol lactone, a potent inhibitor of cytosolic Ca2+-independent phospholipase A(2), had no significant effect on the enzyme activity, The enzyme activity was completely abolished in the presence of greater than 773 mu M Triton X-100. Partial inhibition of the enzyme activity was observed in the presence of 10-100 mu g/ml heparin, In the absence of N-acetylsphingosine, the enzyme acted as a phospholipase A(2). These results strongly suggest that 1-O-acylceramide synthase is both a transacylase and a novel phospholipase A(2).
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页码:8467 / 8474
页数:8
相关论文
共 28 条
[1]   Induction of glucosylceramide synthase by synthase inhibitors and ceramide [J].
Abe, A ;
Radin, NS ;
Shayman, JA .
BIOCHIMICA ET BIOPHYSICA ACTA-LIPIDS AND LIPID METABOLISM, 1996, 1299 (03) :333-341
[2]   A novel enzyme that catalyzes the esterification of N-acetylsphingosine - Metabolism of C-2-ceramides [J].
Abe, A ;
Shayman, JA ;
Radin, NS .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (24) :14383-14389
[3]   INHIBITION OF MACROPHAGE CA2+-INDEPENDENT PHOSPHOLIPASE A(2) BY BROMOENOL LACTONE AND TRIFLUOROMETHYL KETONES [J].
ACKERMANN, EJ ;
CONDEFRIEBOES, K ;
DENNIS, EA .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (01) :445-450
[4]  
ACKERMANN EJ, 1994, J BIOL CHEM, V269, P9227
[5]   Function and inhibition of intracellular calcium-independent phospholipase A(2) [J].
Balsinde, J ;
Dennis, EA .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (26) :16069-16072
[6]   CHARACTERIZATION AND PARTIAL-PURIFICATION OF SOLUBLE, LYSOSOMAL PHOSPHOLIPASE(S)-A2 FROM ADRENAL-MEDULLA [J].
BARTOLF, M ;
FRANSON, RC .
BIOCHIMICA ET BIOPHYSICA ACTA, 1990, 1042 (02) :247-254
[7]   ASSAY OF PROTEINS IN PRESENCE OF INTERFERING MATERIALS [J].
BENSADOUN, A ;
WEINSTEIN, D .
ANALYTICAL BIOCHEMISTRY, 1976, 70 (01) :241-250
[8]  
CHEN J, 1994, J BIOL CHEM, V269, P23018
[9]  
DENNIS EA, 1994, J BIOL CHEM, V269, P13057
[10]   CHEMISTRY AND METABOLISM OF SPHINGOLIPIDS . 3-OXO DERIVATIVES OF N-ACETYLSPHINGOSINE AND N-ACETYLDIHYDROSPHINGOSINE [J].
GAVER, RC ;
SWEELEY, CC .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1966, 88 (15) :3643-&