Short chain ceramides as substrates for glucocerebroside synthetase. Differences between liver and brain enzymes

Abstract

In order to increase the sensitivity of the assay for ceramide: UDPGlc glucosyltransferase, the enzyme that makes glucocerebroside, we synthesized a variety of ceramide homologues that might be better substrates than the naturally occurring ceramides. N-Octanoyl sphingosine proved to be the best lipid tested in liver and brain. It could be added to the tissue homogenate in the dry form, as a thin layer coated on Celite, or in liposomes, prepared from lecithin and cerebroside sulfate. The liposomal form produced better replication of assay values. It is suggested that the addition of cerebroside sulfate to liposomal preparations might be a good, and more physiological, replacement for the commonly used dicetyl phosphate. A new homologue of DL-sphinganine, decasphinganine, was synthesized by an efficient series of steps and acylated with different fatty acids to form ceramide homologues. The best substrate in this series was the lauroyl amide and it is suggested that this lipid be used in cerebroside synthetase assays because of the convenience of preparing it, even though it is not as good as octanoyl sphingosine. Both compounds are distinctly better than natural ceramide or DL-sphinganine amides. From comparisons of enzyme activity under various conditions, the tentative conclusion is drawn that the enzymes in liver and brain have different properties, and that liver has two different synthetases.