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At least 19 recordsLinked to original sources
PubMed1994

Hydrolysis of lactosylceramide by human galactosylceramidase and GM1-beta-galactosidase in a detergent-free system and its stimulation by sphingolipid activator proteins, sap-B and sap-C. Activator proteins stimulate lactosylceramide hydrolysis.

Two exo-beta-galactosidases are involved in the lysosomal degradation of glycosphingolipids: GM1-beta-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 sap-B and sap-C, rather than detergents, to stimulate the reaction. While sap-B was a better activator for the reaction catalyzed by GM1-beta-galactosidase, sap-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 (C2 > C4 > C6 > C8 > C10 > C18). However, in the presence of detergents the degradation rates were independent of the acyl chain length. Hydrolysis of liposomal lactosylceramide was compared with sap-B-stimulated hydrolysis of liposomal ganglioside GM1 by GM1-beta-galactosidase and sap-C-stimulated degradation of liposomal galactosylceramide by galactosylceramidase. Kinetic and dilution experiments indicated that sap-B forms water-soluble complexes with both lactosylceramide and GM1. These complexes were recognized by GM1-beta-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. Acta 481, 561-572]. GM1-beta-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, sap-C and sap-B, stimulated the hydrolysis of lactosylceramide analogues with long acyl chains more than the hydrolysis of lactosylceramides with short acyl chains.

Antigens, CD↗
PubMed1994

Mechanism of GM3 ganglioside synthesis. Kinetic study of rat liver CMP-N-neuraminate:lactosylceramide alpha 2,3-sialyltransferase employing 19 molecular species of lactosylceramide.

The apparent Km and Vmax of CMP-N-acetylneuraminate:lactosylceramide alpha 2,3-sialyltransferase (LacCer-alpha 2,3-ST) for lactosylceramide and CMP-N-acetylneuraminic acid were determined using 19 molecular species of lactosylceramide. The Km for lactosylceramide varied 6-fold among these molecular species of lactosylceramide, but there was a poor correlation between the Km for a particular molecular species and the activity of Lac-Cer alpha 2,3-ST for that molecular species. The Km for CMP-N-acetylneuraminic acid also varied depending on the molecular species of lactosylceramide used as substrate, and there was a good correlation between the Km of Lac-Cer alpha 2,3-ST for CMP-N-acetylneuraminic acid and the activity of the enzyme. Kinetic studies indicate that the reaction mechanism of LacCer alpha 2,3-ST is a sequential, Ordered Bi Bi system. From considerations of the effects of the structure of the lactosylceramide molecular species on the Vmax and Km for CMP-N-acetylneuraminic acid, it is likely that LacCer alpha 2,3-ST first binds lactosylceramide and then CMP-N-acetylneuraminic acid and that the rate-limiting step in the reaction is the release of the product GM3.

Animals↗
PubMed1995

Relationship of membrane phospholipid composition, lactosylceramide molecular species, and the specificity of CMP-N-acetylneuraminate:lactosylceramide alpha 2,3-sialyltransferase to the molecular species composition of GM3 ganglioside.

The ceramide molecular species specificity of rat brain neuron CMP-N-acetylneuraminate:lactosylceramide alpha 2,3-sialyltransferase (LacCer alpha 2,3-ST) was determined using 19 molecular species of lactosylceramide incorporated into liposomes prepared with purified rat brain phospholipids. The neuron enzyme displayed a distinct molecular species specificity (which was different than the specificity of liver LacCer alpha 2,3-ST) based on both the long-chain base and the fatty acid composition of the lactosylceramide. Specifically, compared to the liver enzyme, relatively high activities were obtained with d18:1-16:0, d18:1-22:1, and d18:0-18:0 lactosylceramide molecular species. When the lipid composition of the neuron microsomal membranes was altered to resemble that of rat liver Golgi membrane lipids, the activities towards d18:1-16:0, d18:1-22:1, and d18:0-18:0 lactosylceramide molecular species were significantly (P < 0.01) reduced and the molecular species specificity of the neuron enzyme resembled that of liver LacCer alpha 2,3-ST. In the reciprocal experiment in which the lipid composition of the rat liver Golgi membranes was altered to resemble neuron microsomal membrane lipids, the molecular species specificity of liver LacCer alpha 2,3-ST was virtually identical to the specificity obtained with the native neuron enzyme. Analysis of the molecular species composition of lactosylceramide and GM3 in rat liver Golgi membranes revealed that the molecular species composition of rat liver Golgi membrane GM3 was precisely what would be expected based on the molecular species specificity of LacCer alpha 2,3-ST and the molecular species composition of lactosylceramide in the Golgi membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Lactosylceramide molecular species specificity of rat liver CMP-N-acetylneuraminate:lactosylceramide sialyltransferase.

Six naturally occurring and three synthetic molecular species of lactosylceramide (LacCer) were used to examine the molecular species specificity of CMP-N-acetylneuraminate:lactosylceramide alpha 2,3-sialyltransferase in a Golgi-rich fraction of rat liver. The enzyme molecular species specificity was determined either in the presence of nonspecific lipid transfer protein or in the presence of detergents. Assays performed in the presence of transfer protein showed that for those lactosylceramide molecular species with either d18:1 or d18:0 long chain base the enzyme activity decreased linearly as the effective carbon number of the fatty acid increased. An increase in the carbon number of the long chain base decreased the activity of the enzyme twice as much as a corresponding increase in the carbon number of the fatty acid. On the other hand, when the enzyme activity was assayed in the presence of detergents, there was no significant difference in activity among the various molecular species of lactosylceramide based upon the carbon number of the fatty acid or on the presence of a double bond in the long chain base. However, the decrease in enzyme activity with an increase in the carbon number of the long chain base persisted. These results demonstrate that sialyltransferase has binding specificity with respect to the long chain base, but not the fatty acid. The apparent molecular species towards the fatty acid is related to the aqueous solubility of the various LacCer molecular species.

Animals↗
PubMed1993

Effect of membrane lipids on the lactosylceramide molecular species specificity of CMP-N-acetylneuraminate:lactosylceramide sialyltransferase.

It has previously been shown that when the molecular species specificity of rat liver Golgi CMP-N-acetylneuraminate:lactosylceramide alpha 2,3-sialyltransferase was determined, using as the substrate lactosylceramide (LacCer) incorporated into liposomes prepared with rat liver Golgi lipids, the enzyme showed a pronounced variation in activity towards the various molecular species of LacCer (J. Lipid Res. 1989. 30: 1789-1797). In this paper, -the LacCer molecular species specificity of sialyltransferase from neuroblastoma NB2a cells was examined using five naturally occurring and three synthetic molecular species of LacCer. The enzyme activity was determined by following the formation of [14C]GM3 from CMP-[14C]neuraminic acid and individual molecular species of LacCer incorporated into liposomes. Nonspecific lipid transfer protein was included in the enzyme assay to facilitate the transfer of LacCer and other lipids between the liposomes and the membrane where sialyltransferase is located. In these enzyme assays the liposomes contained approximately 10 times more lipid phosphorus than either the microsomal fraction of NB2a cells or the Golgi fraction of rat liver. Thus, in the presence of nonspecific lipid transfer protein, the lipid composition of the membrane where sialyltransferase is located was modified to resemble the lipid composition of the liposomes. When the molecular species specificity of NB2a cell sialyltransferase was determined with LacCer incorporated into liposomes prepared with NB2a cell lipids, the enzyme showed no specificity towards the various molecular species of LacCer. However, when the molecular species specificity of NB2a cell sialyltransferase was determined with LacCer incorporated into liposomes prepared with rat liver Golgi lipids, the enzyme showed a variation in activity towards the various LacCer molecular species similar to that observed with the liver Golgi enzyme using liposomes prepared with liver Golgi lipids. Likewise, when the molecular species specificity of rat liver Golgi sialyltransferase was determined with LacCer incorporated into liposomes prepared with NB2a cell lipids, the liver enzyme then showed no specificity towards the various molecular species of LacCer. These results indicate that the lipid environment of the membrane can alter the molecular species specificity of sialyltransferase towards its lipid substrate, LacCer.

Animals↗
PubMed1992

Total metabolic flow of glycosphingolipid biosynthesis is regulated by UDP-GlcNAc:lactosylceramide beta 1-->3N-acetylglucosaminyltransferase and CMP-NeuAc:lactosylceramide alpha 2-->3 sialyltransferase in human hematopoietic cell line HL-60 during differentiation.

We have previously reported that ganglioside GM3 was remarkably increased during monocytoid differentiation of human myelogenous leukemia cell line HL-60 cells and that neolacto series gangliosides (NeuAc-nLc) were enriched during granulocytoid differentiation. In addition, HL-60 was differentiated into monocytic lineage by exogenous GM3 and into granulocytoid by NeuAc-nLc. In the present report, the enzymatic bases of glycosphingolipid biosynthesis in HL-60 during differentiation induced by 12-O-tetradecanoylphorbol-13-acetate and all-trans-retinoic acid were investigated. The following results were of particular interest. (i) Lactosylceramide alpha 2-->3 sialyltransferase (GM3 synthase) was remarkably up-regulated during monocyte differentiation, while the GM3 synthase level did not change in granulocytic differentiation. (ii) By contrast, lactosylceramide beta 1-->3N-acetylglucosaminyltransferase (Lc3Cer synthase) was down-regulated during monocytic differentiation, while the activity of Lc3Cer synthase was found to increase in granulocytic differentiation. (iii) The activities of four downstream glycosyltransferases (for synthesis of NeuAc-nLc) were found to increase or to remain unchanged during monocytic and granulocytic differentiation. These results strongly suggested the following. The dramatic GM3 increase and the decrease of NeuAc-nLc during monocytic differentiation are the consequences of the up-regulation of GM3 synthase and the down-regulation of Lc3Cer synthase, although the downstream enzymes are ready to catalyze their enzyme reactions. The notable increase of NeuAc-nLc and the relative decrease of GM3 during granulocytic differentiation are the results of the unchanged level of GM3 synthase and the up-regulation of Lc3Cer synthase together with the activation of the downstream glycosyltransferases. These results suggest that these two key upstream glycosyltransferases, GM3 synthase and Lc3Cer synthase, play critical roles in regulating the glycosphingolipid biosynthesis in HL-60 cells during differentiation. This switching mechanism of these two glycosyltransferases, together with our previous findings, might be one of the most important parts of the determining system of differentiation direction in human myeloid cells into monocytic or granulocytic lineages.

Antibodies, Monoclonal↗
PubMed1994

Preparation of defined molecular species of lactosylceramide by chemical deacylation and reacylation with N-succinimidyl fatty acid esters.

A procedure for the preparation of specific molecular species of D-erythro-lactosylceramide involving deacylation and reacylation of lactosylceramide prepared from bovine brain gangliosides is described. Lactosylceramide was N-deacylated by alkaline hydrolysis and the resulting four lysolactosylceramides, which contained d18:1, d20:1, d18:0 and d20:0 long-chain bases, were simultaneously re-N-acylated with the N-succinimidyl ester of either 16:0, 18:0, 20:0, 22:0, 24:0, 20:1, 22:1 or 24:1 fatty acid. The resulting lactosylceramide contained four molecular species of lactosylceramides, i.e., d18:1, d20:1, d18:0 and d20:0 long-chain bases coupled with the fatty acid that was introduced. Lactosylceramides prepared in this manner were separated into four individual molecular species by high-performance liquid chromatography (HPLC). Each of the purified molecular species of lactosylceramide was quantitated by HPLC after derivatization with benzoylchloride and was characterized by mass spectrometry. The yields of reacylated lactosylceramide were 38-58% relative to the starting lactosylceramide; the purity of each of the molecular species of lactosylceramide was greater than 95%.

Acetylation↗
PubMed1998

The lactosylceramide binding specificity of Helicobacter pylori.

The possible role of glycosphingolipids as adhesion receptors for the human gastric pathogen Helicobacter pylori was examined by use of radiolabeled bacteria, or protein extracts from the bacterial cell surface, in the thin-layer chromatogram binding assay. Of several binding specificities found, the binding to lactosylceramide is described in detail here, the others being reported elsewhere. By autoradiography a preferential binding to lactosylceramide having sphingosine/phytosphingosine and 2-D hydroxy fatty acids was detected, whereas lactosylceramide having sphingosine and nonhydroxy fatty acids was consistently nonbinding. A selective binding of H. pylori to lactosylceramide with phytosphingosine and 2-D hydroxy fatty acid was obtained when the different lactosylceramide species were incorporated into liposomes, but only in the presence of cholesterol, suggesting that this selectivity may be present also in vivo . Importantly, lactosylceramide with sphingosine and hydroxy fatty acids does not bind in this assay. Furthermore, a lactosylceramide-based binding pattern obtained for different trisaccharide glycosphingolipids is consistent with the assumption that this selectivity is due to binding of a conformation of lactosylceramide in which the oxygen of the 2-D fatty acid hydroxyl group forms a hydrogen bond with the Glc hydroxy methyl group, yielding an epitope presentation different from other possible conformers. An alternative conformation that may come into consideration corresponds to the crystal structure found for cerebroside, in which the fatty acid hydroxyl group is free to interact directly with the adhesin. By isolating glycosphingolipids from epithelial cells of human stomach from seven individuals, a binding of H.pylori to the diglycosylceramide region of the non-acid fraction could be demonstrated in one of these cases. Mass spectrometry showed that the binding-active sample contained diglycosylceramides with phytosphingosine and 2-D hydroxy fatty acids with 16-24 carbon atoms in agreement with the results related above.

Animals↗
PubMedDate not supplied

Lactosylceramide-induced apoptosis in primary amnion cells and amnion-derived WISH cells.

Amnion apoptosis is part of a programmed process of fetal membrane remodeling leading to weakening and rupture. The apoptotic agent lactosylceramide is elevated in amniotic fluid of premature infants with rupture of membranes. We have shown that apoptosis in WISH cells, induced by staurosporine, cycloheximide, or actinomycin D, can be blocked by cyclooxygenase inhibitors, suggesting a relationship between prostaglandin production and apoptosis. Cyclic adenosine monophosphate (cAMP) is known to inhibit prostaglandin release in amnion and WISH cells. This study was undertaken to determine the apoptotic potential of lactosylceramide and the effect of cyclooxygenase inhibitors and cAMP activators on lactosylceramide-induced apoptosis in primary amnion and WISH cells. Primary amnion cells and WISH cells were incubated with lactosylceramide to determine apoptosis and prostaglandin E(2) (PGE(2)) release. Apoptosis was confirmed by agarose gel electrophoretic DNA fragmentation analysis, nuclear matrix protein (NMP), and nucleosome enzyme-linked immunosorbent assay. In some studies, cells were preincubated with cyclooxygenase inhibitors or cAMP activators. Lactosylceramide induced a 20-fold increase in NMP (measure of cell death) in both cell types. Apoptosis was confirmed by the studies listed in methods. Lactosylceramide increased PGE(2) release in parallel with apoptosis. Cyclooxygenase inhibitors as well as cAMP activators inhibited both PGE(2) release and apoptosis. Lactosylceramide-induced apoptosis in both amnion and WISH cells. Parallel PGE(2) release was demonstrated with apoptosis. Cyclooxygenase inhibitors and cAMP activators blocked both processes.

Amnion↗
PubMed1998

A novel carbohydrate-glycosphingolipid interaction between a beta-(1-3)-glucan immunomodulator, PGG-glucan, and lactosylceramide of human leukocytes.

The immunomodulator Betafectin(R) PGG-glucan is a homopolymer of glucose derived from yeast cell walls which has been demonstrated to enhance leukocyte anti-infective activity in vitro and in vivo, without the induction of proinflammatory cytokines. We report here the purification of a PGG-glucan-binding element from human leukocytes and its identification as lactosylceramide, a major glycosphingolipid of neutrophils, which includes the CDw17 epitope. The binding of radiolabeled PGG-glucan to purified lactosylceramide was saturable, specific, and time- and temperature-dependent. Lactosylceramides from human leukocytes were fractionated by high performance liquid chromatography in order to analyze the effect of ceramide structure on binding. A variety of fatty acid chain lengths with varying degrees of unsaturation were found to support binding to radiolabeled PGG-glucan. However, DL-lactosylceramides containing dihydrosphingosine did not bind. Radiolabeled PGG-glucan bound several other neutral glycosphingolipids with a terminal galactose, including galactosylceramide, globotriaosylceramide, and gangliotetraosylceramide. The binding of radiolabeled PGG-glucan to lactosylceramide was not inhibited by glycogen, dextran, mannan, pustulan, laminarin, or a low molecular weight beta-(1-3)-glucan, but was inhibited by high molecular weight beta-(1-3)-glucans and by a monoclonal antibody to lactosylceramide. Although this glycosphingolipid has been shown in numerous reports to bind various microorganisms, this represents the first report of lactosylceramide binding to a macromolecular carbohydrate.

Adjuvants, Immunologic↗
PubMed1990

Cryptococcus neoformans, Candida albicans, and other fungi bind specifically to the glycosphingolipid lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), a possible adhesion receptor for yeasts.

The role of glycosphingolipids as adhesion receptors for yeasts was examined. Cryptococcus neoformans, Candida albicans, and Saccharomyces cerevisiae, as well as Histoplasma capsulatum and Sporotrichum schenckii (in their yeast phases), bound specifically to lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), as measured by overlaying glycosphingolipid chromatograms with 125I-labeled organisms. An unsubstituted galactosyl residue was required for binding, because the yeasts did not bind to glucosylceramide (Glc beta 1-1Cer) derived from lactosylceramide by treatment with beta-galactosidase or to other neutral or acidic glycosphingolipids tested that contained internal lactosyl residues. Interestingly, the yeasts preferentially bound to the upper band of the lactosylceramide doublet in human lung and bovine erythrocytes, suggesting that the ceramide structure also affects binding. Active metabolism of the yeasts was required for binding to lactosylceramide, as binding was maximal in buffer containing glucose and was almost completely abolished in nutrient-deficient medium. C. neoformans also bound to human glioma brain cells grown in monolayers, and this binding was inhibited by liposomes containing lactosylceramide but not by liposomes containing glucosylceramide. Lactosylceramide is a major glycosphingolipid in these cells and the only one to which the yeasts bound. As lactosylceramide is widely distributed in epithelial tissues, this glycosphingolipid may be the receptor for yeast colonization and disseminated disease in humans.

Antigens, CD↗
PubMed1979

Enzymatic sulfation of galactosyl- and lactosylceramides in cell lines derived from renal tubules.

1. The renal cell lines, JTC-12 and MDCK, not only synthesize galactosylceramide 3-sulfate and lactosylceramide 3'-sulfate in vivo, but also contain enzymes that catalyze the transfer of sulfate to galactosylceramide and lactosylceramide in vitro. 2. Concentration of cations necessary for maximum sulfotransferase activity occurred at 40 mM Ca2+ with galactosylceramide and 15 mM Ca2+ with lactosylceramide as the substrate. Na+ was also found to stimulate the sulfation of galactosylceramide, but was slightly inhibitory for the sulfation of lactosylceramide. 3. The products of the in vitro assay mixture were characterized as galactosylceramide 3-sulfate and lactosylceramide 3'-sulfate by a variety of TLC separations. 4. The apparent Km of JTC-12 cells for galactosylceramide was 17 microM, while that for lactosylceramide was 82 microM. The Km values of MDCK cells were comparable to those of JTC-12 cells. Competition studies suggested that galactosylceramide and lactosylceramide were sulfated by a single enzyme in both cell lines.

Adenosine Triphosphate↗
PubMed1996

Role of lactosylceramide and MAP kinase in the proliferation of proximal tubular cells in human polycystic kidney disease.

Polycystic kidney disease (PKD) is a common genetic disease characterized by the proliferation of epithelial cells, formation of cysts, and the progression of renal deficiency. We have investigated a possible role of glycosphingolipids in the proliferation of human kidney cells in this disease. The levels of glucosylceramide and lactosylceramide and the activity of glucosylceramide synthase (GlcT-1) and lactosylceramide synthase (GalT-2) were elevated 2-fold and 3-fold, respectively, in the PKD tissue compared to control. Lactosylceramide, but not glucosylceramide (10 microM) derived from PKD exerted a 4-fold stimulation in the proliferation of these cells. However, at a concentration of 40 microM, lactosylceramide and glucosylceramide both stimulated cell proliferation on the order of 10-fold and 2.5-fold, respectively, as compared to control. This phenomenon may be due to the enrichment of lactosylceramide containing shorter chain fatty acids (C16:0-C18:0). Lactosylceramide, but not glucosylceramide exerted a time-dependent stimulation in the phosphorylation of mitogen-activated protein kinase (p44 MAPK) in normal human kidney proximal tubular cells. Moreover, the kidneys and cultured cells from the PKD patients contained higher levels of the p44 MAPK as compared to normal human kidneys. In sum, our studies indicate that lactosylceramide present in the PKD kidney may stimulate cell proliferation via activation of the p44 MAPK, and contribute to the pathophysiology in this disease.

Adult↗
PubMed2005

Isolation and mass spectrometry characterization of molecular species of lactosylceramides using liquid chromatography-electrospray ion trap mass spectrometry.

Reverse-phase liquid chromatography/electrospray ion trap mass spectrometry (LC-ESI-MSn) was established for identification of the molecular species of lactosylceramides. Lactosylceramides derived from porcine blood cells were separated on a CapcellPak C8 column using a mixture of methanol and 1 mM ammonium formate from the C16 to C26 fatty acyl chains based on the length of total carbon chains and the nature of sphingoid bases (w'') and fatty acyl chains (Y0'-w'') was identified by MS3 as their [M+H]+ ions. The same number of fatty acyl moieties appeared in the order of unsaturated, (2-)hydroxylated, and saturated components. The molecular species of lactosylceramides derived from porcine blood cells totaled more than 33 and included mainly C24:0-d18:1, Ch24:0-d18:1, Ch24:1-d18:1, C24:1-d18:1, and C22:0-d18:1 in addition to 28 minor species from C16:0 to C26:0 fatty acyl moieties. The molecular species of lactosylceramides in the membrane microdomain fraction of HL-60 cells (70% were differentiated into macrophage-lineage cells) were identified as C24:0-d18:1, C24:1-d18:1, C22:0-d18:1, C16:0-d18:1, and more than 21 other minor species. Our results suggest that reverse-phase LC-ESI-MSn is a useful and simple method for identification of lactosylceramide molecular species.

Animals↗
PubMed1999

cDNA cloning and expression of human lactosylceramide synthase.

Lactosylceramide synthase is an enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycolipids in mammals. We have isolated and sequenced the cDNA clone encoding human lactosylceramide synthase. The deduced amino acid sequence of the human lactosylceramide synthase showed 94.2% identity with rat lactosylceramide synthase. Northern blotting analysis revealed that lactosylceramide synthase mRNA was expressed in various tissues, with the highest level in brain and adrenal gland.

Amino Acid Sequence↗
PubMed1976

GM1-ganglioside and lactosylceramide beta-galactosidase from rabbit brain: inhibitor and substrate competition studies.

A rabbit brain beta-galactosidase catalyzes the hydrolysis of synthetic substrates and the natural substrates Gm1-ganglioside, lactosylceramide, and asialo-Gm1-ganglioside. gamma-D-Galactonolactone competitively inhibited hydrolysis of Gm1-ganglioside, lactosylceramide, and MU-galactoside with Ki values of 0.26 mM, 0.13 mM, and 0.77 mM, respectively. From activity plots comparing the degree of inhibition to the inhibitor concentration, a single binding site for each substrate was found. NP-Galactoside inhibited the hydrolysis of Gm1-ganglioside and lactosylceramide, where as Gm1-ganglioside inhibited lactosylceramide hydrolysis. At low substrate concentrations (less than 1 mM), Gm1-ganglioside was hydrolyzed effectively in the presence of NP-galactoside, but at higher concentrations hydrolysis of the latter was preferred. Chloromercuriphenylsulfonic acid and iodoacetate were effective inhibitors of the enzyme, but N-ethylmaleimide was not. The degree of inhibition with chloromercuriphenylsulfonic acid was different for each substrate. At 0.5 mugM chloromercuriphenylsulfonic acid, all activity towards NP-galactoside, 75% towards lactosylceramide, and 25% of the Gm1-ganglioside activity was lost. Two possible models are presented to explain these results. The data favour the presence of multiple active sites in the enzyme.

Animals↗
PubMed1975

Lactosylceramide beta-galactosidase in human sphingolipidoses. Evidence for two genetically distinct enzymes.

In view of recent conflicting reports from two laboratories, activities of lactosylceramide beta-galactosidase were reinvestigated in detail in brains and livers of normal individuals and of patients with globoid cell leukodystrophy or GM1-gangliosidosis. Both sets of the apparently totally contradictory results were readily reproduced simply by using the different assay systems of the respective laboratories. With our own assay system, hepatic lactosylceramide beta-galactosidase appeared deficient only in Gm1-gangliosidosis, while it appeared deficient only in globoid cell leukodystrophy when the assay system of Wenger et al. (Wenger, D.A., Sattler, M., Clark, D., and McKelvey, H. (1974) Clin. Chim. Acta 56, 199-206) was used. Analyses of individual constitutents in the two assay systems revealed their complex effects on measured activities of the enzyme. The findings were strongly indicative of the existence of two genetically distinct lactosylceramide-cleaving enzymes. One enzyme (lactosylceramidase I) may be identical with galactosylceramide betal-galactosidase, and the other (lactosylceramidase II) is closely related to nonspecific 4-methylumbelliferyl beta-galactosidase. Normal human brain contains mostly lactosylceramidase I, while normal liver contains predominantly lactosylceramidase II. Lactosylceramidase I is genetically lacking globoid cell leukodystrophy, and lactosylceramidase II in GM1-gangliosidosis. Lactosylceramidase I is activated by either pure or crude taurocholate and by oleic acid and is only slightly activated by chloride ions. Lactosylceramidase II is activated by crude taurocholate but not by pure taurocholate. As activators, oleic acid is less effective and chloride more effective than for lactosylceramidase I. Citrate-phosphate buffer is more favorable to lactosylceramidase I than citrate buffer, while lactosylceramidase II responds in reverse. The standard assay system used by Wenger et al. determines almost exclusively lactosylceramidase I, while our own standard system is optimal for lactosylceramidase II and is less favorable for lactosylceramidase I. With a highly purified human hepatic beta-galactosidase preparation, exxentially free of galactosylceramide beta-galactosidase activity, lactosylceramide-cleaving activity determined by the Wenger system was less than 2 per cent of that determined by our system. If lactosylceramide beta-balactosidase assays are to be used for diagnosis of globoid cell leukodystrophy, it is absolutely essential to use an appropriate assay system in order to avoid errors of serious consequences.

Brain↗