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 Mg 21 ions, much less on Ca 21 ions. A clear preference for the D-erythro isomer was seen. Various N-acylated amino alcohols were no substrate, but Nhexanoyl-1-O-hexadecyl-2-desoxy-2-amino-sn-glycerol and Ntetradecanoyl-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 Mg 21 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.

C(2)-ceramide (N-acetyl-sphingenine) is often used as an analog to study ceramide-mediated cellular processes. According to Lee et al. [J. Biol. Chem. 271 (1996), 209-217], C(2)-ceramide is formed by an acetyl transfer from platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to sphingenine. To substantiate these unconfirmed findings, we (i) developed a method to quantify C(2)-ceramide and (ii) analyzed C(2)-ceramide levels in Pex5(-/-) mice, a model for Zellweger syndrome, in which the synthesis of ether lipids such as PAF is impaired. The presence of C(2)-ceramide could be established in brain (+/-10 pmol/g) and liver (+/-25 pmol/g) from control mice, and was approximately 5000-fold less than the main long-chain ceramide species. In Pex5(-/-) mice, C(2)-ceramide levels did not differ significantly compared to control tissues. Given the presence of a ceramide kinase in mammals, phosphorylation of C(2)-ceramide by human ceramide kinase (HsCERK) was tested. C(2)-ceramide appears to be a good substrate when albumin is used as carrier. In CHO cells overexpressing HsCERK, phosphorylation of exogenously added C(2)-ceramide could also be demonstrated. Our data indicate that C(2)-ceramide is present in mammalian tissues and can be converted to C(2)-ceramide-1-phosphate, in addition to other documented metabolic alterations, but does not seem to be linked to ether lipid metabolism.

Upon exposure of truncated ceramides, such as N -acetyl-sphingenine, and long-chain ceramides to moderate acidic conditions, three derivatives are formed. Two of them turned out to be O -acylated sphingenine, 1-O -and 3-O -acylsphingenine, and the other was identified as sphingenine. Truncated dihydroceramides (e.g., N -acetyl-and N -hexanoylsphinganine) also show this type of rearrangement, which is therefore not related to the presence of the allylic hydroxy group or to the length of the N -acyl chain. Of particular concern is the fact that the O -acylated compounds, which can be considered sphingoid base analogs, can be formed in chloroform or chloroform-methanol mixtures upon storage. For longterm storage, methanol or dichloromethane is the preferred solvent. A procedure to document the presence/formation of such O -acylated sphingoid bases in ceramide solutions in the picomole range, based on reversed-phase chromatography after derivatization of their amino group with 6-aminoquinolyl-N -hydroxysuccinimidyl carbamate, is presented. When analyzing lipid kinase activity in bacterial lysates using labeled truncated ceramide analogs, the presence of unknown labeled compounds was noticed in the organic extracts. With N -[ 14 C]acetyl-sphingenine, prepared as described previously (1), as substrate, two additional radioactive spots, one with a lower (derivative X) and one with a higher relative mobility (R f ; derivative Y) than the substrate, were observed after TLC in an acidic solvent ( Fig. 1A ). These lipids did not comigrate with any known commercially available sphingolipid. As the formation of these derivatives was not dependent on the presence of bacterial proteins (Fig. 1A), the reaction and extraction conditions were investigated. This revealed that the acidification of the assay mixture with HCl, to halt the reaction and to improve the extraction of the ceramide phosphate in the organic phase, was to blame. This was confirmed using unlabeled N -acetyl-sphingenine. After treatment of this lipid with 0.5 N HCl, three extra iodine-reactive spots were formed (Fig. 1B). One derivative comigrated with sphingenine; the others resembled derivatives X and Y with regard to R f values. All three stained with ninhydrin, suggesting that a migration of the acetyl group from the amino to one of the two adjacent hydroxy groups of the sphingoid base gave rise to the unknown ceramide derivatives X and Y ( Fig. 2 ). The conversions were concentration dependent and were even noticed when using 0.1 N HCl (Fig. 1B, C; Table 1 ). Other acids, such as H 2 SO 4 and HClO 4 , in equivalent normality as 0.5 N HCl, also affected N -acetylsphingenine, but 5% acetic acid and 0.5 N H 3 PO 4 did not (Fig. 1B, C; data not shown).Attempts to isolate the unknown lipids by column chromatography or to characterize them by gas chromatography-mass spectrometry were not successful, probably because of (some) reversions during these steps. However, mass spectral analysis (positive mode, LCQ-duo MSn Iontrap; ThermoFinnigan) of the spo...

Ceramide kinase (CERK) has been implicated in important cellular processes such as inflammation and apoptosis. Its activity is usually measured using radiolabeled ceramide or [γ-32P]-ATP, followed by extraction, thin-layer chromatography, and detection of the formed labeled ceramide-1-phosphate. To eliminate the use of radioactivity, we developed similarly but independently from the approach by Don and Rosen (2008), a fluorescence-based ceramide kinase assay, using N-[7-(4-nitrobenz-2-oxa-1,3-diazole)]-6-aminohexanoyl-sphingenine (NBD-C6-ceramide) as substrate. Its K m value (4 μM) was comparable to that of N-hexanoyl-sphingenine (C6-ceramide). The produced fluorescent NBD-C6-ceramide-1-phosphate was captured by means of solid-phase extraction on an aminopropyl phase, resulting in a fast and sensitive CERK measurement. By performing this assay in a 96-well format, it is also suitable for high-throughput screening (HTS) to search for CERK modulators. A limited screen revealed that some protein kinase inhibitors (e.g., U-0126; IC50 4 μM) and ceramide analogues (e.g., fenretinide, AMG-9810; IC50 1.1 μM) affect CERK in vitro.

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