Abstract
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.
Highlights
Recombinant human ceramide kinase (HsCERK) was analyzed with regard to dependence on divalent cations and to substrate delivery, spectrum, specificity, and stereoselectivity
The other lipid kinase, HsLK2/multisubstrate lipid kinase (MuLK), was expressed in bacteria transformed with pSG004 and in CHO cells transfected with pSG005
Subcellular fractions enriched in nuclei, mitochondria, lysosomes and peroxisomes, microsomal vesicles, and cytosol were prepared from testis or brain tissue homogenates, made with Potter-Elvehjem homogenizers, as described previously for rat liver [31]
Summary
Sphinganine, sphingenine stereoisomers, and N-octyl-sphingenine were obtained from Avanti Polar Lipids, Sigma, or Toronto Research Chemicals. After adding 240 Al of tetrahydrofuran containing 11 Amol of 2,6-dichlorobenzoic acid, the solution was stirred for 2 h at room temperature followed by the addition of 660 Al of 146 mM triethylamine in dichloromethane containing sphingenine (16.5 Amol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (55 Amol). The lower phase was dried, and the ceramide produced was further purified by preparative TLC (silica G60; Merck) with chloroform-methanol-acetic acid (95:5:1, v/v) as solvent. Water (4 ml) and EtOH (4 ml) were added, and the amides were extracted into 2 Â 5 ml of diethylether. 5 ml of 1 M HCl was added and amides were extracted into diethylether. After a wash with chloroform-methanol (2:1 and 1:9, v/v), amides were eluted with methanol-acetic acid (95:5). TLC analysis was done in chloroform-methanol-acetic acid (80:18:2, v/v)
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