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.

Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis.

“…Properties of the enzyme, however, have not been studied satisfactorily because of the difficulty in assaying and purifying the enzyme. Thus far, only limited data have been published on GlcT-1: successful solubilization of the enzyme from rat Golgi fraction (4), the different properties of liver and brain enzymes (5), and a specific inhibitor of the enzyme (6). Papers from several laboratories have demonstrated that the synthesis of GlcCer occurs at the cytosolic surface of the Golgi apparatus, whereas other glycosylation reactions of GSL synthesis take place at the lumenal side of the organelle (7)(8)(9).…”

Sphingosine 1-Phosphate Induces Platelet Activation through an Extracellular Action and Shares a Platelet Surface Receptor with Lysophosphatidic Acid

“…C 2 -ceramide (Cer) (28) and N,N-dimethylsphingosine (29) were synthesized as described previously. [3-3 H]Sph-1-P was prepared by ATP-dependent phosphorylation of [3- …”

Sphingosine‐1‐phosphate inhibits actin nucleation and pseudopodium formation to control cell motility of mouse melanoma cells

“…N,N.dimethyisphingosine-l-phosphate (DMS-I-P) and N,N,N.trimethylsphingosine.l-phosphate (TMS-I-P) were synthesized [17]. Dihydro-Sph-l-P [15,18] and C2-Cer [19] were synthesized as previously described. Sphingosylphosphorylcholine (SPC) was purchased from Sigma Chemical Co. (St. Louis, MO) and purified by HPLC with CHCIalCHaOH/NH4OH (3:3:1).…”