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Kinetic characterization of mammalian ceramide synthases: Determination of K m values towards sphinganine
Author(s) -
Lahiri Sujoy,
Lee Hyunmi,
Mesicek Judith,
Fuks Zvi,
Haimovitz-Friedman Adriana,
Kolesnick Richard N.,
Futerman Anthony H.
Publication year - 2007
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2007.10.018
Subject(s) - ceramide , characterization (materials science) , chemistry , stereochemistry , biochemistry , nanotechnology , materials science , apoptosis
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 5 μM, 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.