Purification of the serine palmitoyltransferase complex responsible for sphingoid base synthesis by using affinity peptide chromatography techniques.
Serine palmitoyltransferase (SPT), a membrane-bound enzyme of the endoplasmic reticulum, catalyzes the condensation of palmitoyl coenzyme A (CoA) and L-serine to produce 3-ketodihydrosphingosine. This enzyme contains at least two different subunits, named the LCB1 and LCB2 proteins. In the present study, we expressed a FLAG- and His(6) peptide-tagged version of the hamster LCB1 protein in a Chinese hamster ovary cell mutant strain lacking the endogenous LCB1 subunit and purified SPT from the cells near to homogeneity by affinity peptide chromatography. The endogenous LCB2 protein was co-purified with the tagged LCB1 protein in purification of SPT. In various aspects, including optimum pH, acyl-CoA specificity, and sphingofungin sensitivity, the activity of purified SPT was consistent with the activity detected in lysates of wild-type Chinese hamster ovary cells. The optimum concentration of palmitoyl-CoA for 3-ketodihydrosphingosine formation by purified SPT was approximately 25 microM, and the apparent K(m) of L-serine was 0.28 mM. Competition analysis of the SPT reaction with various serine analogs showed that all of the amino, carboxyl, and hydroxyl groups of L-serine were responsible for the substrate recognition of the enzyme. SDS-polyacrylamide gel electrophoretic analysis of purified SPT, together with immunoprecipitation analysis of metabolically labeled LCB proteins, strongly suggested that the SPT enzyme consisted of the LCB1 and LCB2 proteins with a stoichiometry of 1:1.
Hanada
K
K
Department of Biochemistry and Cell Biology, Japan Science and Technology Corp., National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. hanadak@nih.go.jp
Hara
T
T
Nishijima
M
M
D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000187:drug effects; D000818:Animals; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010171:Palmitoyl Coenzyme A / Q000378:metabolism*; D010455:Peptides; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D051102:Serine C-Palmitoyltransferase; D013107:Sphingolipids / Q000096:biosynthesis; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D000596:Amino Acids; D009842:Oligopeptides; D010455:Peptides; D013107:Sphingolipids; C002882:ketodihydrosphingosine; D010171:Palmitoyl Coenzyme A; C085706:FLAG peptide; D000217:Acyltransferases; D051102:Serine C-Palmitoyltransferase; D013110:Sphingosine