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J Biol Chem, Vol. 273, Issue 14, 8467-8474, April 3, 1998
From the Division of Nephrology, Department of Internal Medicine,
University of Michigan Medical Center, Ann Arbor, Michigan 48109
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 
-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 µM bromoenol lactone, a potent inhibitor
of cytosolic Ca2+-independent phospholipase A2,
had no significant effect on the enzyme activity. The enzyme activity
was completely abolished in the presence of greater than 773 µM Triton X-100. Partial inhibition of the enzyme
activity was observed in the presence of 10-100 µg/ml heparin. In
the absence of N-acetylsphingosine, the enzyme acted as a
phospholipase A2. These results strongly suggest that 1-O-acylceramide synthase is both a transacylase and a
novel phospholipase A2.
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