PubMed · 13505821
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H S SISE, S M LAVELLE, R BECKER. 1957. A method for assay of Stuart factor.. https://doi.org/10.3181/00379727-96-23570
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Palladium(II)-coproporphyrin label and a set of corresponding monofunctional labeling reagents with different linker arms were evaluated for labeling of oligonucleotides and subsequent use in hybridization assays. The properties of resulting oligonucleotide probes including phosphorescence spectra, quantum yields, lifetimes, and labeling yields were examined as functions of the label and oligonucleotide structures. Upon hybridization with complementary sequences bearing dabcyl, QSY-7, and rhodamine green dyes, the probes displayed strong quenching due to close proximity effects. Intensity and lifetime changes of the phosphorescence, distance, and temperature dependences were investigated in detail. The potential of the new label and probes for sensitive and separation-free hybridization assays was discussed.
Human ceramide kinase was recently cloned and characterized. Recombinant ceramide kinase is highly active and ceramide is the only lipid that it phosphorylates, indicating that it should be useful for the measurement of ceramide levels in biological samples by conversion to ceramide-1-phosphate, in a manner analogous to that of the widely used Escherichia coli diacylglycerol kinase method. Using recombinant ceramide kinase, we have now developed a rapid and specific enzymatic method to quantify mass levels of long-chain ceramides in cellular lipid extracts. This new ceramide kinase assay is more specific than the commonly used diacylglycerol kinase method because the ubiquitous lipid diacylglycerol, the preferred substrate for diacyglycerol kinase which is usually present at higher concentrations than ceramide and can interfere with ceramide phosphorylation, is completely inactive with ceramide kinase. Moreover, this high specificity eliminates the need for analysis of the lipid product by thin-layer chromatography since ceramide-1-phosphate is the only radiolabeled lipid in organic solvent extracts of ceramide kinase reactions.