The effect of six different structurally modified sphingosine analogues on biosynthesis of sphingolipids was studied in primary cultured murine cerebellar neurons. Treatment of cells with cis-4-methylsphingosine at micromolar levels resulted in a markedly decreased sphingolipid biosynthesis, whereas the other compounds examined, trans-4-methylsphingosine, cis-5-methylsphingosine, trans-5-methylsphingosine, cis-sphingosine, and 1-deoxysphingosine, inhibited sphingolipid biosynthesis less efficiently. The inhibition of sphingolipid biosynthesis by the various compounds was paralleled by a decrease of serine palmitoyltransferase activity in situ. For cis-4-methylsphingosine the inhibitory effect on serine palmitoyltransferase activity was shown to be concentration-and time-dependent. Half-maximal reduction of enzyme activity occurred after 24 h of treatment with 10 M of the compound. The activity of other enzymes of sphingolipid biosynthesis as well as phospholipid and protein biosynthesis was not affected.Analysis of the sphingoid moiety of cellular sphingolipids suggests that the sphingosine analogues listed above were subject to degradation rather than being utilized as precursors for sphingolipid biosynthesis by cultured neurons. Except of 1-deoxysphingosine, the other five sphingosine analogues were shown to be substrates for sphingosine kinase in vitro. After 24 h of treatment of primary cerebellar neurons with the various sphingosine analogues the relative percentage of the respective intracellular 1-phosphate derivatives paralleled exactly the inhibitory effect on serine palmitoyltransferase activity observed when cells were treated with the unphosphorylated compounds. In contrast to the respective 1-phosphate derivatives of the other methyl-branched sphingosine analogues examined, cis-4-methylsphingosine 1-phosphate showed an intracellular accumulation suggesting a delayed turnover rate in cultured murine neurons for this compound. These results suggest that the inhibitory effect of the sphingosine analogues on serine palmitoyltransferase is mediated by their respective 1-phosphate derivatives and that the pronounced effect of cis-4-methylsphingosine is caused by a high intracellular concentration of cis-4-methylsphingosine 1-phosphate. cis-4-Methylsphingosine, in addition, caused drastic changes in cell morphology of primary cerebellar neurons, which were not observed when these cells were treated with one of the other sphingosine analogues examined.

Two exo-P-galactosidases are involved in the lysosomal degradation of glycosphingolipids : GM1-P-galactosidase (EC 3.2.1.23) and galactosylceramidase (EC 3.2.1.46). Analyses were performed with both enzymes, using lactosylceramides with varying acyl chain lengths as substrates that were inserted into unilamellar liposomes and naturally occurring sphingolipid activator proteins sup-B and sup-C, rather than detergents, to stimulate the reaction.While sup-B was a better activator for the reaction catalyzed by GM1-P-galactosidase, sup-C preferentially stimulated lactosylceramide hydrolysis by galactosylceramidase. The enzymic hydrolysis of liposome-integrated lactosylceramides was significantly dependent on the structure of the lipophilic aglycon moiety of the lactosylceramide decreasing with increasing length of its fatty acyl chain (C, > C, > C, > C, > Clo> C,J. However, in the presence of detergents the degradation rates were independent of the acyl chain length. Hydrolysis of liposomal lactosylceramide was compared with sup-B-stimulated hydrolysis of liposomal ganglioside GM1 by GM1-P-galactosidase and sup-C-stimulated degradation of liposomal galactosylceramide by galactosylceramidase.Kinetic and dilution experiments indicated that sup-B forms water-soluble complexes with both lactosylceramide and GM1. These complexes were recognized by GM1-P-galactosidase as optimal substrates in the same mode, as postulated for the hydrolysis of sulfatides by arylsulfatase A [Fischer, G. and Jatzkewitz, H. (1977) Biochim. Biophys. A m 481,561 -5721. GM1-P-galactosidase was more active on these complexes than on glycolipids (GM1 and lactosylceramides) still residing in liposomal membranes. On the other hand, dilution experiments indicated that degradation of galactosylceramide and lactosylceramide by galactosylceramidase proceeds almost exclusively on liposomal surfaces : both activators, sup-C and sup-B, stimulated the hydrolysis of lactosylceramide analogues with long acyl chains more than the hydrolysis of lactosylceramides with short acyl chains.Glycosphingolipids are primarily components of the outer leaflet of plasma membranes. Their biosynthesis occurs in the endoplasmic reticulum and Golgi compartment, their degradation mainly in lysosomes [l]. A recent hypothesis on Correspondence to K. Sandhoff, Institut fur Organische Chemie und Biochemie, Universitat Bonn, Gerhard-Domagk-Stral3e 1, D-53121 Bonn, GermanyAbbreviations. Cer, ceramide ; LacCer, lactosylceramide, Galbl4GlcP1-1 Cer ; C,-LacCer, N-acetyl-(1-0-lactosyl) sphingosine; C,-LacCer, N-butanoyl-(1-0-lactosyl) sphingosine ; C,-LacCer, N-hexanoyl-(1-0-lactosyl) sphingosine; C,-LacCer, N-octanoyl-(1-0-lactosyl) sphingosine; C,,-LacCer, N-decanoyl-(1-0-lactosyl) sphingosine; C,,-LacCer, N-octadecanoyL(1-0-lactosyl) sphingosine; GalCer, galactosylceramide, Galbl-1 Cer ; GM1, Galj?l-3GalNAcj?l4Gal(3-2aNeuAc)~1-4Glc/?l-l Cer ; sup-B, sulfatide activator protein or SAP-1 or saposin B, sphingolipid activator protein B; sup-C, co-p-glucosidase or SAP-2 or saposin C, sphing...