Asialo Derivative Research Articles

Ethylenedioxy-PIP2 Oxalate Reduces Ganglioside Storage in Juvenile Sandhoff Disease Mice

Sandhoff disease is an incurable neurodegenerative disorder caused by mutations in the lysosomal hydrolase β-hexosaminidase. Deficiency in this enzyme leads to excessive accumulation of ganglioside GM2 and its asialo derivative, GA2, in brain and visceral tissues. Small molecule inhibitors of ceramide-specific glucosyltransferase, the first committed step in ganglioside biosynthesis, reduce storage of GM2 and GA2. Limited brain access or adverse effects have hampered the therapeutic efficacy of the clinically approved substrate reduction molecules, eliglustat tartrate and the imino sugar NB-DNJ (Miglustat). The novel eliglustat tartrate analog, 2-(2,3-dihydro-1H-inden-2-yl)-N-((1R,2R)-1-(2,3-dihydrobenzo[b][1, 4]dioxin-6-yl)-1-hydroxy-3-(pyrrolidin-1-yl)propan-2-yl)acetamide (EtDO-PIP2, CCG-203586 or "3h"), was recently reported to reduce glucosylceramide in murine brain. Here we assessed the therapeutic efficacy of 3h in juvenile Sandhoff (Hexb-/-) mice. Sandhoff mice received intraperitoneal injections of phosphate buffered saline (PBS) or 3h (60 mg/kg/day) from postnatal day 9 (p-9) to postnatal day 15 (p-15). Brain weight and brain water content was similar in 3h and PBS-treated mice. 3h significantly reduced total ganglioside sialic acid, GM2, and GA2 content in cerebrum, cerebellum and liver of Sandhoff mice. Data from the liver showed that 3h reduced the key upstream ganglioside precursor (glucosylceramide), providing evidence for an on target mechanism of action. No significant differences were seen in the distribution of cholesterol or of neutral and acidic phospholipids. These data suggest that 3h can be an effective alternative to existing substrate reduction molecules for ganglioside storage diseases.

Substrate reduction reduces gangliosides in postnatal cerebrum-brainstem and cerebellum in GM1 gangliosidosis mice

II3NeuAc-GgOse4Cer (GM1) gangliosidosis is an incurable lysosomal storage disease caused by a deficiency in acid beta-galactosidase (beta-gal), resulting in the accumulation of ganglioside GM1 and its asialo derivative GgOse4Cer (GA1) in the central nervous system, primarily in the brain. In this study, we investigated the effects of N-butyldeoxygalacto-nojirimycin (N B-DGJ), an imino sugar that inhibits ganglioside biosynthesis, in normal C57BL/6J mice and in beta-gal knockout (beta-gal-/-) mice from postnatal day 9 (p-9) to p-15. This is a period of active cerebellar development and central nervous system (CNS) myelinogenesis in the mouse and would be comparable to late-stage embryonic and early neonatal development in humans. N B-DGJ significantly reduced total ganglioside and GM1 content in cerebrum-brainstem (C-BS) and in cerebellum of normal and beta-gal-/- mice. N B-DGJ had no adverse effects on body weight or C-BS/cerebellar weight, water content, or thickness of the external cerebellar granule cell layer. Sphingomyelin was increased in C-BS and cerebellum, but no changes were found for cerebroside (a myelin-enriched glycosphingolipid), neutral phospholipids, or GA1 in the treated mice. Our findings indicate that the effects of N B-DGJ in the postnatal CNS are largely specific to gangliosides and suggest that N B-DGJ may be an effective early intervention therapy for GM1 gangliosidosis and other ganglioside storage disorders.

Blocking of fimbria-mediated adherence of Haemophilus influenzae by sialyl gangliosides

The structure of the receptor for the fimbriae of Haemophilus influenzae on human oropharyngeal epithelial cells and erythrocytes was determined in inhibition experiments with various sugars, glycolipids, and glycoproteins. Of 30 monosaccharides and disaccharides at a concentration of 0.1 M and of 3 polysaccharides at a concentration of 1 mg/ml, none inhibited fimbria-specific adherence and hemagglutination. Inhibition was obtained with gangliosides GM1, GM2, GM3, and GD1a in nanomolar concentrations, whereas the asialo derivative of GM1, sialyl-lactose, and sialoglycoproteins were poor inhibitors. These findings indicate that sialyl-lactosylceramide (GM3) is the minimal structure for the fimbria-dependent binding of H. influenzae to its receptor on oropharyngeal epithelial cells and erythrocytes. As is the case with GM2, substitution of GM3 with N-acetylgalactosamine makes the molecule a 10-fold-better receptor analog.

Hypoglycemic neurotoxicity in vitro: Involvement of excitatory amino acid receptors and attenuation by monosialoganglioside GM1

Rat cerebellar granule cells, when subjected to a glucose-free environment for 4 h, developed extensive degeneration of neuronal cell bodies and their associated neurite network over the following 24 h. This neuronal damage was quantitated with a colorimetric assay using the metabolic dye 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide. Hypoglycemie neuronal injury could be markedly reduced by the presence of both competitive (3-(±)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid) and non-competitive (phencyclidine) N- methyl- d- aspartate receptor antagonists, but not by kainate/ quisqualate preferring antagonists 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione. Glucose deprivation neuronal injury was also reduced by adding glutamate-degrading enzymes to the incubation medium. Monosialoganglioside GM1, but not its asialo derivative (lacking sialic acid), was also effective in protecting against hypoglycemie neurodegeneration when included during the period of glucose deprivation. These results suggest that the neuronal injury to cerebellar granule cells resulting from glucose deprivation is mediated predominantly by activation of the N- methyl- d- aspartate type of excitatory amino acid receptor, perhaps through the action of endogenously released glutamate. Furthermore, the monosialoganglioside GM1, a member of a class of naturally occurring sialoglycosphingolipids, is able to attenuate this neuronal injury—as already observed for glutamate neurotoxicity and anoxic neuronal death in cerebellar granule cells. Gangliosides may thus prove to be of therapeutic utility in excitatory amino acid-associated neuropathologies.

Identification of α-Galactose (α-Fucose)-asialo-GM1 Glycolipid Expressed by Subsets of Rat Dorsal Root Ganglion Neurons

Abstract Distinct cell-surface glycoconjugates are expressed on specific subsets of dorsal root ganglion (DRG) neurons and DRG terminals projecting to the superficial dorsal horn of rat spinal cord (Dodd, J., and Jessell, T. M. (1985) J. Neurosci. 5, 3278-3294). Carbohydrate antigens detected by monoclonal antibodies (mAbs) TC6, KH10, and LD2 are restricted to about 20% of DRG neurons projecting to lamina IIB (dorsal), whereas antigens recognized by mAb LA4 are expressed by about 50% of DRG neurons projecting to lamina IIB (ventral). These mAbs were generated against rat pancreatic acinar cell line AR4-2J antigens. The glycolipid antigens in AR4-2J cells reacting with these mAbs have been structurally characterized by sequential hydrolysis with various exoglycosidases, immunochemical tests, linkage analysis of permethylated alditol acetates, capillary gas liquid chromatography-mass spectrometry, mass spectrometry of permethylated compounds, and by fast atom bombardment mass spectrometry of the native antigens. The structure of the major antigen (IA) in AR4-2J cells was determined to be: (formula; see text) The asialo derivative of IA and the novel disialo form of IA (Gal alpha 1----3(Fuc alpha 1----2)----GD1b) have been also identified. The DRG neurons contained only the neutral glycolipid, asialo form of IA. All these antigens reacted equivalently in the high performance thin layer chromatography-immuno overlay assay with the TC6, LD2, and LA4 mAbs. The molecular specificity of the three mAbs was determined by rection with a variety of possible antigens and appears to be the same. All three mAbs required terminal Gal alpha 1----3(Fuc alpha 1----2)Gal beta 1----3GalNAc (or 4GlcNAc) for full reactivity. Only partial reactivity was observed with compounds in which alpha-Fuc was removed. The observed restricted reactivity of mAbs TC6, LD2, and LA4 in subsets of DRG neurons and in ventral and dorsal areas of lamina IIB may be due to different topographical expression of the antigen in the neuronal membrane.

A novel sialylhexasaccharide from human milk: Purification by affinity chromatography on immobilized wheat germ agglutinin

A sialylhexasaccharide fraction (S-5) of human milk was obtained as described by A. Kobata and V. Ginsburg [(1972) Arch. Biochem. Biophys. 150, 273–281] and labeled by reduction with NaB[ 3H] 4. When subjected to affinity chromatography on immobilized wheat germ agglutinin (WGA), a single component representing 60% of the S-5 fraction was retarded by the column. The asialo derivative of the WGA-retarded oligosaccharide had a higher affinity for the WGA column than the native sialyloligosaccharide. The neutral hexaose was identified as lacto- N-neohexaose by sequential exoglycosidase digestions in combination with gel filtration analyses of digestion products. Enzymatic removal of the nonsialylated branch of the intact sialyloligosaccharide by jack bean β-galactosidase and β- N-acetylhexosaminidase resulted in a single sialyl[ 3H]tetraose which was identified as sialyltetrasaccharide c (NeuAcα2–6Galβ1–4GlcNAcβ1–3Galβ1–4GlcO[ 3H]) by cochromatography with authentic standard and specific antibody binding. Independent evidence for the structure of the sialylhexasaccharide was obtained by 500-MHz 1H NMR spectroscopy of the WGA-purified oligosaccharide before and after neuraminidase digestion. The structural data are consistent with the following, previously undescribed, sialylhexaose in human milk: ▪

Glycolipid-lectin interactions: Reactivity of lectins from Helix pomatia, Wisteria floribunda, and Dolichos biflorus with glycolipids containing N-acetylgalactosamine

The autoradiographic detection of 125I-labeled lectins binding to glycolipids on thinlayer chromatograms can be used to rapidly analyze total glycolipid extracts of cells or tissues for specific oligosaccharide structures. The Helix pomatia lectin which binds with high affinity to terminal α-linked GalNAc residues did not bind to globoside (terminal β1-3GalNAc) but did bind the ganglioside GM 2 and its asialo derivative which have terminal β1-4GalNAc residues. The lectin from Dolichos biflorus bound specifically to the Forssman glycolipid with relatively low affinity. The lectin from Wisteria floribunda was bound to Forssman glycolipid, globoside, and the asialo derivative of the ganglioside GM 2. The interactions of these lectins with the glycolipid-derived, 3-labeled oligosaccharides was also analyzed by affinity chromatography. The results indicated that the reactivity of multivalent carbohydrate-binding proteins with polyvalent surfaces of glycolipids is strong enough to permit detection of low-affinity interactions that may not be observed in binding assays that are based on carbohydrate-protein interactions in solution. The autoradiographic analysis of 125I- Helix pomatia lectin binding to thin-layer chromatograms of total lipid extracts from human erythrocyte membranes detected the quantitative differences in the A-active glycolipids from type A 1 and A 2 Cells.

Structure of tumor-associated carbohydrate antigen Ca 19-9 on human seminal-plasma glycoproteins from healthy donors

The monoclonal antibody-defined, tumor-associated antigen Ca 19-9, chemically identical with the sialylated Lewisa-carbohydrate determinant of a monoganglioside and a mucin, was demonstrated by radioimmunoassay to be present in large amounts as component of fucose-rich sialoglycoproteins, which had been extracted from human seminal plasma of healthy donors. The carbohydrate antigen of these glycoproteins (m greater than 205 kDa and m 115 kDa), which are presumably secreted by the prostatic gland, was absent in seminal plasma from blood-group-Lewis-negative men. The Ca 19-9 active sialyl-oligosaccharide was cleaved from the proteins by mild alkaline borohydride treatment and was shown to chromatograph on gradient elution from DEAE-Sephadex with the fraction of monosialylated saccharide alditols (MS-SP). The asialo derivative of the major saccharide alditol in this fraction was composed of L-fucose, D-galactose, N-acetyl-D-glucosamine and N-acetyl-D-galactosaminitol in the molar proportions 1:2:1:1 and chromatographed on Bio-Gel P2 according to approximately seven hexose units. A methylation analysis of the sialylated saccharide alditol in fraction MS-SP, which had been purified by high-pressure liquid chromatography, revealed the presence of terminal, non-reducing L-fucose, 3-O-substituted D-galactose, 3,4 di-O-substituted N-acetyl-D-glucosamine and 3-O-substituted N-acetyl-D-galactosaminitol. The presented data and the fragmentation pattern obtained on direct probe EI and FAB+ mass spectrometry of the permethylated asialo derivative are in accordance with the structure of a sialylated pentasaccharide alditol (formula; see text).

Fucosyl gangliosides of PC12 pheochromocytoma cells

Three monosialogangliosides are highly labeled when PC12 pheochromocytoma cells are cultured in the presence of L-[ 3H] fucose, and two additional monosialogangliosides are labeled to a lesser extent. In contrast, neither of the two disialogangliosides of PC12 cells contains fucose residues. Removal of sialic acid and fucose by formic acid hydrolysis demonstrated the presence of 3 major ‘core’ structures in the monosialogangliosides, and a single asialo derivative of the disialogangliosides which has the same chromatographic mobility as one of the monosialoganglioside hydrolysis products. None of the major formic acid hydrolysis products of the PC12 cell gangliosides corresponds to asialo-GM 1, supporting our previous conclusion that PC12 cells do not contain significant amounts of brain-type gangliosides.

Stalosylglobotetraosylceramide: A marker for amyotropic lateral sclerosis

Gangliosides of healthy and pathologic muscles (amyotropic lateral sclerosis and facio-scapulo-humeral muscular dystrophy) were studied. Total ganglioside content of the affected muscles was approximately 2 fold higher than the unaffected muscles. Our results showed that ALS muscle contained a ganglioside which was absent in the unaffected and FSH muscular dystrophic muscles. Based on the results of hydrolysis with Vibrio cholerae neuraminidase and subsequent reactivity of the asialo derivative towards anti-globotetraosylceramide, we propose that the ALS ganglioside is sialosylglobtetraosylceramide, NeuAc(α2–3)GalNAc(β1–3)Gal(α1–4)Gal(β1–4)Glc-Cer.

Tissue locations for the turnover of radioactively labeled rat orosomucoid in vivo

Tissues involved in the turnover of rat serum orosomucoid were identified by methods designed to cause lysosomal trapping of radiolabel at the sites of glycoprotein degradation. 125I-, [ 3H]Raffinose-, and [1- 14C]glucosamine-labeled orosomucoid exhibited serum half-lives of 20, 20, and 27 h when injected intravenously into rats. As expected, the asialo derivative of [ 3H]raffinose-labeled rat orosomucoid was lost very rapidly from the circulation and recovered quantitatively in the liver within 30 min. At 50 h after injection of [ 3H]raffinose-asialo-orosomucoid the liver retained 38% of the radioactivity while the remainder was found in the gastrointestinal tract and urine. Chromatography of the urine on Bio-Gel P-4 revealed a single radioactive product that eluted similar to raffinose-lysine. The same material was found in the liver. This ability of the [ 3H]raffinose label to resist metabolic disposal was used to evaluate tissue catabolism of native rat orosomucoid. Comparison of the tissue radioactivity in experiments using 125I- and [ 3H]raffinose-labeled derivatives of the nondesialylated glycoprotein showed kidney, liver, and muscle to be most active in 3H accumulation. However, the [ 3H]raffinose metabolites excreted in the urine was markedly different from those produced from asialo-orosomucoid and in contrast there was minimal loss of label to the gastrointestinal tract from the native substrate. Leupeptin, an inhibitor of lysosomal thiol cathespins, was administered continuously to rats by a subcutaneous osmotic pump. At 24 h after injection of 125I-orosomucoid, leupeptin-treated rats showed a net 16% increase in tissue radioactivity above sham-operated animals and a corresponding decrease occurred in the radioactivity associated with the gastrointestinal tract and urine. Tissues that exhibited increases in radioactivity were kidney, muscle, liver, and hide. The different behavior of labeled native and asialo-orosomucoids suggests that the hepatic galactose receptor system plays, at most, a limited role in maintaining homeostasis of the native glycoprotein.

Isolation and characterisation of glycopeptides from digests of human tamm-horsfall glycoprotein

Glycopeptides were isolated from pronase digests of human Tamm-Horsfall glycoprotein and its asialo derivative. The carbohydrate moiety of the major glycopeptide preparation isolated from the former digests had an apparent molecular weight of 4300, and those of two glycopeptides isolated from the latter digests had molecular weights of 3600 and 2300. These data, together with the compositions of the glycopeptides, indicate that the Tamm-Horsfall glycoprotein has at least five asparagine residues substituted by complex carbohydrate moieties, three being of one type, relatively rich in galactose, and two containing more sialic acid but less galactose. A small amount of a mannose-rich glycopeptide was also recovered from the digests of the Tamm-Horsfall glycoprotein.

Isolation and characterization of a novel monosialosylpentahexosyl ceramide from Tay-Sachs brain.

A novel monosialoganglioside was isolated from Tay-Sachs brains. It represented about 0.1% of the total ganglioside mixture. Compositional analysis by gas-liquid chromatography indicated that it contained glucose, galactose, N-acetylgalactosamine, N-acetylneuraminic acid, and long chain base in the molar ratio of 1:2:2:1:1. The ganglioside was found to be resistant to neuraminidase (Clostridium perfringens), beta-hexosaminidase (jack bean), and beta-galactosidase. However, it could be degraded by a human liver beta-hexosaminidase preparation in the presence of an activator to produce a glycolipid chromatographically identical with authentic IV3NeuAc-GgOse4-ceramide. This glycolipid product was resistant to beta-galactosidase (jack bean), but could be readily degraded to GgOse4-ceramide by neuraminidase. Mild formic acid hydrolysis degraded the intact ganglioside to an asialo derivative chromatographically identical with the pentahexosyl ceramide (GalNAc-Gal-GalNAc-Gal-Glc-ceramide) derived from GD1a-GalNAc. The asialo derivative could then be degraded to GgOse4-ceramide and GgOse3-ceramide by sequential treatment with jack bean beta-hexosaminidase and beta-galactosidase. These data suggest that the novel ganglioside is a monosialosylpentahexosyl ceramide with the sialosyl group attached to the penultimate galactose moiety of the pentahexosyl ceramide backbone, and it has the following structure: GalNAc(beta 1-4)Gal(beta 1-3)GalNAc(beta 1-4)Gal(beta 1-4)Glc-ceramide (formula see text).

Carbohydrate on human factor VIII/von Willebrand factor. Impairment of function by removal of specific galactose residues.

Human factor VIII/von Willebrand factor protein containing 120 +/- 12 nmol of sialic acid and 135 +/- 13 nmol of galactose/mg of protein was digested with neuraminidase. The affinity of native factor VIII/von Willebrand factor and its asialo form for the hepatic lectin that specifically binds asialoglycoproteins was assessed from in vitro binding experiments. Native factor VIII/von Willebrand factor exhibited negligible affinity while binding of the asialo derivative was comparable to that observed for asialo-alpha1-acid glycoprotein. Incubation of asialo-factor VIII/von Willebrand factor with Streptococcus pneumoniae beta-galactosidase removed only 62% of the galactose but abolished binding to the purified hepatic lectin. When the asialo derivative was incubated with purified beta-D-galactoside alpha2 leads to 6 sialyltransferase and CMP-[14C]NeuAc, only 61% of the galactose incorporated [14C]NeuAc. From the known specificites of these enzymes, it is concluded that galactose residues important in lectin binding are present in a terminal Gal/beta1 leads to 4GlcNAc sequence on asialo-factor VIII/von Willebrand factor. The relative ristocetin-induced platelet aggregating activity of native, asialo-, and agalacto-factor VIII/von Willebrand factor was 100:38:12, respectively, while procoagulant activity was 100:100:103.

Sandhoff disease: ganglioside GM and asialo‐GM2 accumulation in the cerebrospinal fluid

In an earlier investigation it was found that ganglioside GM2 the typical neuronal storage product in GMz‐gangliosidosis, was accumulated also in the cerebrospinal fluid (CSF) of a Tay‐Sachs patient. The asialo derivative of ganglioside GM2, GM2, which is a concomitant storage product in the brain of these patients, was not detected in the CSF of the patient investigated (BERNHEIMER, 1968). In the present account, a report is made on the accumulation of ganglioside GM2 and of GM2 in the CSF of a patient affected by Sandhoff disease.

THE CAT AS A MODEL FOB ENZYME THERAPY IN GM2 GANGLIOSIDOSIS

A genetic neurodegenerative disorder with lysosomal storage of Gm2 ganglioside, its asialo derivative, and globoside, and β-hexosaminidase deficiency has been described in domestic cats (Cork et al, Science, 1977, in press). To strengthen the apparent analogy with human Gm2 gangliosidosis type II, feline β-hexosaminidase has been studied. Two major forms of this lysosomal enzyme, Hex A and Hex B, are present in organs and body fluids from normal animals, with biochemical and kinetic properties remarkably similar to those of the corresponding human enzymes. These data, and the deficiency of both components in affected cats, support the analogy between feline and human diseases. Thus it is possible to utilize this animal model for enzyme therapy experiments. Structural differences between human and feline β-hexosaminidase, however, may result in different cellular uptake, endocellular activity and stability, limiting the relevance of replacement experiments with feline enzyme. Thus, plasma clearance and organ disposition of human placental Hex A and Hex B has been studied in normal cats, utilizing the apparent absence of interspecific immunologic cross-reactivity. The results so far indicate a rapid, preferential hepatic uptake, possibly via a saturable receptor mechanism, with recovery of the exogenous enzymes in a lysosomal fraction. Further studies using human enzymes should result in enzyme replacement strategies, to be tested in diseased cats, relevant to therapeutic attempts in human Gm2 gangliosidosis.

Oligosaccharide storage in brains from patients with fucosidosis, GM1-gangliosidosis and GM2-gangliosidosis (Sandhoff's disease).

Abstract— Analysis of whole autopsy brain from a patient with fucosidosis (α‐fucosidase deficiency) revealed minor storage of H‐antigen glycolipid [Fuc (α, 1→2) Gal‐GlcNAc‐Gal‐Glc‐Ceramide] and a slightly abnormal ganglioside composition in the form of a two‐fold elevation of GM1 and the presence of a fucose‐containing glycolipid (a minor component) which co‐migrated with GD1a. The major storage materials in fucosidosis brain were an oligosaccharide (Fuc‐Gal‐GlcNAc‐Man[Fuc‐Gal‐GlcNAc‐Man]‐ManGlcNAc) and a disaccharide [Fuc(α, 1→6)‐GlcNAc] in the approximate ratio of 5:1. Lesser amounts of a related oligosaccharide (Gal‐GlcNAc‐Man[Gal‐GlcNAc‐Man]‐Man‐GlcNAc) were isolated from the brain of patients with GM1‐gangliosidosis (Types I and II) where the major storage material is known to be GM1‐ganglioside (Gal (β, 1→3)GalNAc(β, 1→4) [NeuNAcf(α, 2→3) Gal(β, 1→4)Glc‐Ceramide). Similarly, a related oligosaccharide (GlcNAc‐Man [GlcNAc‐Man]‐Man‐GlcNAc) was isolated from the brain of a patient with a total deficiency of N‐acetyl‐β‐d‐hexosaminidase (Sandhoff variant of GM2‐gangliosidosis) where the major storage products are known to be GM2‐ganglioside (GalNAc (β 1→4) [NeuNAc (α, 2→3)Gal(β, 1→4)Glc‐Ceramine) and its asialo derivative. These studies indicate that glycoproteins containing at least 2 mol of l‐fucose per oligosaccharide unit are normally catabolized in human brain. Further, it appears that such glycoproteins are initially catabolized by an endo‐N‐acetylglucosaminidase to release an oligosaccharide which is then degraded by the sequential action of exo‐glycosidases.

Methyl 5(6)-phenylsulfinyl-2-benzimidazolecarbamate, a new, potent anthelmintic.

1. Human hepatic "acid" beta-galactosidase preparations, which had been purified approximately 250-fold, were examined for activities toward 4-methylumbelliferyl beta-galactosylceramide, lactosylceramide, galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosyl-glucosylceramide(GM1-ganglioside) and galactosyl-N-acetylgalactosaminyl-galactosyl-glucosylceramide (asialo GM1-ganglioside). 2. The enzyme was active toward the synthetic substrate, GM1-ganglioside and asialo GM1-ganglioside but was inactive toward galactosylceramide. Under our assay conditions, optimized for lactosylceramidase II, the preparations were as active toward lactosylceramide as toward GM1-ganglioside or its asialo derivative. The apparent Km values for the three natural substrates were similar. When determined by the assay system of Wehger, D.A., Sattler, M., Clark, C. and McKelvey, H. (1974) Clin. Chim Acta 56, 199-206, lactosylceramide-cleaving activity was 0.2% of that determined by our assay system. This confirmed our previous suggestion that the Wenger assay system determines exclusively the activity of lactosylceramidase I, which is probably identical with galactosylceramide beta-galactosidase. 3. Crude sodium taurocholate was far more effective than pure taurocholate in stimulating hydrolysis of the three glycosphingolipids by the beta-galactosidase. However, crude taurocholate could largely be replaced by smaller amounts of sodium taurodeoxycholate, suggesting that the unique activating capacity of the crude taurocholate might be due to taurodeoxycholate present as the major impurity. 4. Cl- was generally stimulatory for hydrolysis of the natural glycosphingolipids by our enzyme preparation. Effects of additional oleic acid and Triton X-100 were generally minor in either direction. 5. When the enzyme preparation was diluted with water, activity toward the synthetic substrate declined rapidly while those toward the natural substrates were essentially stable. Activity toward the synthetic substrate remained much more stable when the enzyme was diluted with 0.1 M sodium citrate/phosphate buffer, pH 5.0. 6. These observations provide insight into the complex relationship among the human hepatic beta-galactosidases.

Activity of human hepatic β-galactosidase toward natural glycosphingolipid substrates

1. 1. Human hepatic “acid” β-galactosidase preparations, which had been purified approximately 250-fold, were examined for activities toward 4-me-thylumbelliferyl β-galactoside, galactosylceramide, lactosylceramide, galactosyl- N-acetylgalactosaminyl- N- acetylneuraminyll-galactosyl-glucosylceramide (G M1-ganglioside) and galactosyl- n-acetylgalactosaminyl-galactosyl-glucosylceramide (asialo G M1-ganglioside). 2. 2. The enzyme was active toward the synthetic substrate, G M1-ganglioside and asialo G M1-ganglioside but was inactive toward galactosylceramide. Under our assay conditions, optimized for lactosylceramidase II, the preparations were as active toward lactosylceramide as toward G M1-ganglioside or its asialo derivative. The apparent K m values for the three natural substrates were similar. When determined by the assay system of Wenger, D.A., Sattler, M., Clark, C. and McKelvey, H. (1974) Clin. Chim. Acta 56, 199–206, lactosylceramidecleaving activity was 0.2% of that determined by our assay system. This confirmed our previous suggestion that the Wenger assay system determines exclusively the activity of lactosylceramidase I, which is probably identical with galactosylceramide β-galactosidase. 3. 3. Crude sodium taurocholate was far more effective than pure taurocholate in stimulating hydrolysis of the three glycosphingolipids by the β-galactosidase. However, crude taurocholate could largely be replaced by smaller amounts of sodium taurodeoxycholate, suggesting that the unique activating capacity of the crude taurocholate might be due to taurodeoxycholate present as the major impurity. 4. 4. Cl − was generally stimulatory for hydrolysis of the natural glycosphingolipids by our enzyme preparation. Effects of additional oleic acid and Triton X-100 were generally minor in either direction. 5. 5. When the enzyme preparation was diluted with water, activity toward the synthetic substrate declined rapidly while those toward the natural substrates were essentially stable. Activity toward the synthetic substrate remained much more stable when the enzyme was diluted with 0.1 M sodium citrate/ phosphate buffer, pH 5.O. 6. 6. These observations provide insight into the complex relationship among the human hepatic β-galactosidases.

Synthesis and turnover of lysosomal glycoproteins. Relation to the molecular heterogeneity of the lysosomal enzymes

Many, if not all, of the lysosomal acid hydrolases exist in several molecular forms. For example, using polyacrylamide gel electrophoresis [l] and isoelectric focusing [2,3] we found two to five distinct forms of seven different hydrolases in rat kidney lysosomes. That this molecular heterogeneity may have biological importance is strongly suggested by the recent discovery that the acidic (A) form of a lysosomal hydrolase is lacking in two inherited lipid storage diseases, namely aryl sulfatase A in metachromatic leukodystrophy [4,5] and hexosaminidase A in TaySachs disease [6,7] . Apparently the corresponding basic (B) form of these hydrolases which is present in normal or elevated amounts in these diseases is incapable of hydrolyzing the accumulated sphingolipid, cerebroside sulfate in metachromatic leukodystrophy [4,5] and GM, ganglioside (and its asialo derivative) in Tay-Sachs disease [6,7] . These observations imply that the different molecular forms may display divergent substrate specificities and hydrolytic activities in vivo. We have shown that the various acid hydrolases in purified lysosomal fractions from rat kidney and liver are glycoproteins (GPs) [ 1,8,9] . A study of the effects of bacterial neuraminidase on the electrophoretic mobility [8] , and pIs [3] of a number of lysosomal hydrolases indicated that N-acetylneuraminic acid (NANA) residues are largely responsible for the solubility and electronegative charge of these enzymes. On the basis of the solubility and electrophoretic mobility of five acid hydrolases in rat kidney subcellular fractions [9, lo] , and