Partial purification and characterization of sphingosine N-acyltransferase (ceramide synthase) from bovine liver mitochondrion-rich fraction.
Sphingosine N-acyltransferase (ceramide synthase, E.C. 2.3.1.24) was solubilized from bovine liver mitochondrion-rich fraction with n-octyl beta-D-thioglucoside as the detergent and partially purified by sequential chromatography on columns of DE-32, shingosine affinity, and Sepharose CL-6B. The partially purified preparation migrated on SDS-polyacrylamide gel electrophoresis as two major protein bands of 62 and 72 kDa. The molecular mass of the enzyme estimated by gel filtration was 240-260 kDa, suggesting that the partially purified enzyme is present in a subunit form or simply has an aggregative nature. The specific activity of the final preparation for the condensation of sphingosine with stearoyl-CoA increased by 98.7-fold compared with the starting material. The optimal pH value for the ceramide synthesis was 7.5. The partially purified enzyme had an apparent Km of 146 microM and a Vmax of 11.1 nmol/min/mg protein for stearoyl-CoA. The Km and Vmax values toward sphingosine were 171 microM and 11.3 nmol/min/mg protein, respectively. Interestingly, sphinganine was also a good substrate for this enzyme, and the Km and Vmax values were 144 microM and 8.5 nmol/min/mg protein, respectively.
Shimeno
H
H
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Fukuoka University, Japan.
Soeda
S
S
Sakamoto
M
M
Kouchi
T
T
Kowakame
T
T
Kihara
T
T
D000214:Acyl Coenzyme A; D000217:Acyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002518:Ceramides / Q000096:biosynthesis; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D012995:Solubility; D013110:Sphingosine / Q000031:analogs & derivatives; D051049:Sphingosine N-Acyltransferase; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D002518:Ceramides; C017725:stearoyl-coenzyme A; D000217:Acyltransferases; D051049:Sphingosine N-Acyltransferase; D013110:Sphingosine; C005682:safingol