Further characterization of mammalian ceramide kinase: substrate delivery and (stereo)specificity, tissue distribution, and subcellular localization studies.
Recombinant human ceramide kinase (HsCERK) was analyzed with regard to dependence on divalent cations and to substrate delivery, spectrum, specificity, and stereoselectivity. Depending on the chain length of the ceramide, either albumin for short-chain ceramide or a mixed micellar form (octylglucoside/cardiolipin) for long-chain ceramide was preferred for the substrate delivery, the former resulting in higher activities. Bacterially expressed HsCERK was highly dependent on Mg2+ ions, much less on Ca2+ ions. A clear preference for the d-erythro isomer was seen. Various N-acylated amino alcohols were no substrate, but N-hexanoyl-1-O-hexadecyl-2-desoxy-2-amino-sn-glycerol and N-tetradecanoyl-2S-amino-1-butanol were phosphorylated, suggesting that the secondary hydroxy group is not required for recognition. The properties of HsCERK, expressed in CHO cells, were similar to those of the bacterially expressed protein, including the Mg2+ dependence. In mouse, the highest activities were found in testis and cerebellum, and upon subcellular fractionation the activity was recovered mainly in the microsomal fraction. This fits with the plasma membrane localization in CHO cells, which was mediated by the N-terminal putative pleckstrin domain. No evidence for phosphorylation of ceramide by the recently described multiple lipid kinase was found. The latter kinase is localized in the mitochondria, but no firm conclusions with regard to its substrate could be drawn.
Van Overloop
Helena
H
Katholieke Universiteit Leuven, Faculteit Geneeskunde, Departement Moleculaire Celbiologie, Afdeling Farmacologie, Leuven, Belgium.
Gijsbers
Sofie
S
Van Veldhoven
Paul P
PP
D000818:Animals; D001921:Brain / Q000201:enzymology / Q000378:metabolism; D016466:CHO Cells; D002413:Cations, Divalent / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007700:Kinetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology / Q000378:metabolism; D014162:Transfection
D002413:Cations, Divalent; D002518:Ceramides; D011994:Recombinant Proteins; C492429:AGK protein, human; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase