Kinetic characterization of mammalian ceramide synthases: determination of K(m) values towards sphinganine.
Ceramide is a key metabolite in the pathway of sphingolipid biosynthesis. In mammals, ceramide is synthesized by N-acylation of a sphingoid long-chain base by a family of ceramide synthases (CerS), each of which displays a high specificity towards acyl CoAs of different chain lengths. We now optimize a previously-described assay for measuring CerS activity for use upon over-expression of mammalian CerS, and using these conditions, establish the K(m) value of each CerS towards sphinganine. Remarkably, the K(m) values towards sphinganine are all similar, ranging from 2 to 5microM, even for CerS proteins that are able to use more than one acyl CoA for ceramide synthesis (i.e. CerS4). The availability of this assay will permit further accurate characterization of the kinetic parameters of mammalian CerS proteins.
Lahiri
Sujoy
S
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Lee
Hyunmi
H
Mesicek
Judith
J
Fuks
Zvi
Z
Haimovitz-Friedman
Adriana
A
Kolesnick
Richard N
RN
Futerman
Anthony H
AH
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D007700:Kinetics; D051379:Mice; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D017931:DNA Primers; D011994:Recombinant Proteins; D010088:Oxidoreductases; C108138:dihydroceramide desaturase; D013110:Sphingosine; C005682:safingol