Hydrolysis of galactosylceramide is catalyzed by two genetically distinct acid beta-galactosidases

Two genetically distinct acid beta-galactosidases are apparently involved in the hydrolysis of galactosylceramide in fibroblasts. These beta-galactosidases were activated by different bile salts. The classical galactosylceramidase (galactosylceramidase I, EC 3.2.1.46) was activated by sodium taurocholate, while the other galactosylceramidase (galactosylceramidase II) was activated by sodium cholate. The former was genetically lacking in globoid cell leukodystrophy (GLD) and the latter in GM1 gangliosidosis. Galactosylceramidase II cross-reacted with antibody raised against purified GM1 ganglioside beta-galactosidase (EC 3.2.1.23) from the human placenta. The purified beta-galactosidase had galactosylceramidase II activity, which was competitively inhibited by GM1 ganglioside. Thus, galactosylceramidase II seems to be identical to GM1 ganglioside beta-galactosidase and lactosylceramidase II. Galactosylceramidase II had a very low affinity for galactosylsphingosine. In the galactosylceramide-loading tests using fibroblasts from patients with GLD and GM1 gangliosidosis, both cell lines hydrolyzed the incorporated galactosylceramide, with lower rates than control fibroblasts but higher than the fibroblasts from patients with I-cell disease, in which both galactosylceramidase I and II were deficient. These results indicate that galactosylceramide is hydrolyzed by two genetically distinct beta-galactosidases and explain well that galactosylsphingosine but not galactosylceramide accumulates in the brain of patients with GLD.     

Photometric and fluorometric continuous kinetic assay of acid phosphatases with new substrates possessing longwave absorption and emission maxima

For the measurement of the enzymatic activity of GM1-ganglioside (II3 NeuAcGgOse4Cer, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminosyl) galactosyl-glucosylceramide) beta-galactosidase in crude enzyme samples, a microassay using nonradioisotopic GM1-ganglioside was devised. To reduce the volume of the reaction mixture and eliminate the interferences due to the fluorescent contaminants in the reaction mixture, NADH, a product after the oxidation of the released galactose with NAD and beta-galactose dehydrogenase, was fluorometrically estimated by use of high-performance liquid chromatography. By this method, as little as 10 pmol of galactose can be detected. Using rat brain homogenates as an enzyme sample, the several parameters were reexamined to define the optimal conditions for the assay. This assay method was also applied to human cultured skin fibroblast homogenates, and it was found that this method can be used for the diagnosis of GM1-gangliosidosis, instead of the usual method using the radioisotope-labeled natural substrate.     

Partial purification and properties of porcine thymus lactosylceramide beta-galactosidase.

Porcine thymus lactosylceramide beta-galactosidase was purified by a simple procedure. In the final step of isoelectric focusing the enzyme was separated into two peaks of pI 6.3 (peak I) and 7.0 (peak II), which showed 3,600- and 4,000-fold enhancement of lactosylceramide-hydrolysing activity, respectively. The two peaks had identical mobility on polyacrylamide gel electrophoresis. The apparent molecular weight was 34,000. Neither monosialoganglioside (GM1) nor galactosylceramide was hydrolysed by the purified enzyme fractions. The optimal pH was at 4.6, and sodium taurocholate was essential for the reaction. The apparent Km was 2.3 x 10-5 M. The reaction was stimulated by sodium chloride and linoleic acid, while it was strongly inhibited by Triton X-100 and bovine serum albumin. Galactosylceramide, p-nitrophenyl beta-galactoside, and p-nitrophenol were weak inhibitors. No effects of GM1 and galactose were observed on the hydrolysis of lactosylceramide.     

Isolation and characterization of jack bean beta-galactosidase.

A simple procedure has been devised to isolate beta-galactosidase from jack bean meal. The final preparation gives one major protein banc in disc gel electrophoresis. The substrate specificity of this enzyme toward some natural oligosaccharides, glycoproteins, and sphingoglycolipids has been examined in detail. Among three isomers of N-acetyllactosamine, Galbeta1leads to4GlcNAc; while Galbeta1leads to3GlcNAc was hydrolyzed very slowly. This property can be used to distinguish the galactose linkage in asialo-GM1 (Galbeta1leads to3GalNAcbeta1leads to4Galbeta1leads to4Glcleads toCer) and that in lacto-N-neotetraosylceramide (Galbeta1leads to4GlcNAcbeta1leads to 3Galbeta1leads to4Glcleads toCer). For hydrolyzing glycolipids, the effect of sodium taurodeoxycholate and sodium taurochenodeoxycholate on the rate of hydrolysis was carefully examined. This enzyme hydrolyzes lactosylceramide and asialo-GM1 faster than GM1. These results suggest that in addition to the type and linkage of the penultimate sugar unit, the sugar unit at the distal position of the saccharide chain also affects the hydrolysis rate. It also readily liberates 80% D-galactosyl units from asialo alpha1-acid glycoprotein. Escherichia coli beta-galactosidase on the other hand cannot hydrolyze asialo-alpha1-acid glycoprotein, lactosylceramide, GM1, asialo-GM1, and lacto-N-neotetraosylceramide. The molecular weight of this enzyme is about 75,000 and the isoelectric point is pH 8.0. With p-nitrophenyl beta-D-galactopyranoside as substrate, optimal activity occurs at pH 2.8 with glycine-HCl buffer and at pH 3.5 with citrate-phosphate buffer. With lactose as substrate, the pH optimum in these two buffers are 2.8 and 4.0, respectively. Km values for p-nitrophenyl beta-D-galactopyranoside, o-nitrophenyl beta-D-galactopyranoside and lactose are 0.51 mM, 0.63 mM, and 12.23 mM, respectively. Many inhibitors for this enzyme including inorganic ions, monosaccharides, and glycosides are investigated. In contrast to E. coli beta-galactosidase, jack bean beta-galactosidase is not inhibited by p-aminophenyl thio-beta-D-galactopyranoside.     

Galactosylceramide- and lactosylceramide-loading studies in cultured fibroblasts from normal individuals and patients with globoid cell leukodystrophy (Krabbe's disease) and GM1-gangliosidosis

The metabolism of galactosylceramide and lactosylceramide in cultured fibroblasts was studied using the lipid-loading test. These compounds were incorporated into the fibroblasts yet only small amounts of the incorporated lipids were hydrolyzed unless additional phospholipid was mixed with the glycolipid before loading. Among phospholipids, phosphatidylserine was the most effective for incorporation and hydrolysis of the glycolipids, while phosphatidylcholine inhibited the incorporation of the glycolipids. Using filtration techniques, light scattering analyses and subcellular fractionation, the particle size of glycolipid in the culture medium was found to be critically important for the incorporation of the lipids into the cells and their transportation to the lysosomes. The particle sizes of the glycolipids were decreased by mixing with phosphatidylserine. Furthermore, the negative charge in phosphatidylserine may be necessary for the glycolipid transportation into the lysosomes. In fibroblasts from patients with globoid cell leukodystrophy, 40-50% of galactosylceramide was hydrolyzed on the 4th day of culture, a time when the control fibroblasts had hydrolyzed it about 80%. This finding is in contrast with observations made on fibroblasts with other sphingolipidoses which showed near-zero degradation in corresponding substrate-loading tests. In fibroblasts from patients with either globoid cell leukodystrophy of GM1-gangliosidosis, hydrolysis of lactosylceramide was fairly normal yet somewhat lower than control values on any day of culture, thereby indicating that, in the loading tests, lactosylceramide seems to be hydrolyzed with similar levels of enzyme activities by two distinct beta-galactosidases, galactosylceramidase and GM1-ganglioside beta-galactosidase.     

Utilization of ganglioside-degrading Paenibacillus sp. strain TS12 for production of glucosylceramide

Gangliosides, sialic acid-containing glycosphingolipids, are membrane constituents of vertebrates and are known to have important roles in cellular differentiation, adhesion, and recognition. We report here the isolation of a bacterium capable of degrading gangliotetraose-series gangliosides and a new method for the production of glucosylceramide with this bacterium. GM1a ganglioside was found to be sequentially degraded by Paenibacillus sp. strain TS12, which was isolated from soil, as follows: GM1a --> asialo GM1 --> asialo GM2 --> lactosylceramide --> glucosylceramide. TS12 was found to produce a series of ganglioside-degrading enzymes, such as sialidases, beta-galactosidases, and beta-hexosaminidases. TS12 also produced beta-glucosidases, but glucosylceramide was somewhat resistant to the bacterial enzyme under the conditions used. Taking advantage of the specificity, we developed a new method for the production of glucosylceramide using TS12 as a biocatalyst. The method involves the conversion of crude bovine brain gangliosides to glucosylceramide by coculture with TS12 and purification of the product by chromatography with Wakogel C-300 HG.     

Metabolism of galactosylceramide in the twitcher mouse, an animal model of human globoid cell leukodystrophy

The metabolism of galactosylceramide was investigated in normal and twitcher mice, an animal model for human globoid cell leukodystrophy. The findings were compared with data obtained on human tissues. In vitro studies demonstrated that there were two genetically distinct enzymes that hydrolyze galactosylceramide: galactosylceramidase I and II. The former was deficient in the twitcher, while the latter was intact. beta-Galactosidase preparations purified from normal mouse liver possessed the activity to hydrolyze galactosylceramide when the assay conditions for galactosylceramidase II was used. Therefore, galactosylceramidase II was considered to be identical to GM1 ganglioside beta-galactosidase. In contrast to the human enzyme, the murine beta-galactosidase had a relatively high Km value toward galactosylceramide. The galactosylceramide-loading test demonstrated that the twitcher fibroblasts hydrolyzed the lipid at lower rates than seen in cases of human globoid cell leukodystrophy fibroblasts. These differences in galactosylceramidase II between murine and human tissues suggest that galactosylceramide accumulates in twitcher mice but not in humans with globoid cell leukodystrophy, even though galactosylceramidase I is genetically deficient in both human and this mouse model.     

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.     

Utilization of Ganglioside-Degrading Paenibacillus sp. Strain TS12 for Production of Glucosylceramide

Gangliosides, sialic acid-containing glycosphingolipids, are membrane constituents of vertebrates and are known to have important roles in cellular differentiation, adhesion, and recognition. We report here the isolation of a bacterium capable of degrading gangliotetraose-series gangliosides and a new method for the production of glucosylceramide with this bacterium. GM1a ganglioside was found to be sequentially degraded by Paenibacillus sp. strain TS12, which was isolated from soil, as follows: GM1a → asialo GM1 → asialo GM2 → lactosylceramide → glucosylceramide. TS12 was found to produce a series of ganglioside-degrading enzymes, such as sialidases, β-galactosidases, and β-hexosaminidases. TS12 also produced β-glucosidases, but glucosylceramide was somewhat resistant to the bacterial enzyme under the conditions used. Taking advantage of the specificity, we developed a new method for the production of glucosylceramide using TS12 as a biocatalyst. The method involves the conversion of crude bovine brain gangliosides to glucosylceramide by coculture with TS12 and purification of the product by chromatography with Wakogel C-300 HG.     

Hydrolysis of GM1-ganglioside by human liver β-galactosidase isoenzymes

1. GM(1)-ganglioside, specifically tritiated in the terminal galactose, was hydrolysed by two forms of ;acid' methylumbelliferyl beta-galactosidase isolated on gel filtration. 2. Identification of GM(1)-ganglioside beta-galactosidase activity with the ;acid' methyl-umbelliferyl beta-galactosidases was based on the following: coincident elution profiles on gel filtration; simultaneous inactivation by heat and other treatments; stabilization of both activities by chloride ions; mutual inhibition of hydrolysis by the two substrates. 3. The two isoenzymes (I) and (II) showed general requirements for a mixture of anionic and nonionic detergents in the hydrolysis of the natural substrate. 4. Isoenzyme (I) differed from (II) in molecular size, pH-activity profile, relative resistance to dilution and in sensitivity to various inhibitors. 5. The most significant difference between the isoenzymes is in substrate saturation kinetics: (I) was hyperbolic whereas (II) was sigmoid. The apparent Michaelis constants were 28mum for (I) and 77mum for (II). Isoenzyme (I) was insensitive to GM(2)-ganglioside whereas (II) was inhibited, consistent with the hypothesis that GM(1)-ganglioside (and its analogue) acts as modifier in isoenzyme (II) but not in (I). 6. Isoenzyme (I) was membrane-bound whereas (II) was soluble; the former probably represents isoenzyme (II) bound to membrane components, thereby becoming activated. 7. Membranes may serve a dual role in enzyme catalysis involving lipids: as a medium where both enzyme and substrate are effectively concentrated, and as actual activator of enzymes through binding of the latter to specific membrane components.     

Properties of a protein activator of glycosphingolipid hydrolysis isolated from the liver of a patient with GM1 gangliosidosis,Type 1

The heat stable protein activator of GM1 ganglioside hydrolysis was isolated from the liver of a patient with GM1 gangliosidosis, Type 1. It was found to be present at a level about 35 times that found in a liver sample from an age matched control. This activator protein was demonstrated to stimulate the hydrolysis of GM1 ganglioside and GA1 (asialo-GM1 ganglioside) in the presence of purified GM1 ganglioside β-galactosidase without the need for bile salt detergents. It could not stimulate the hydrolysis of two other galactosphingolipids, galactosylceramide and lactosylceramide, in the presence of the same enzyme. Lactosylceramide was a good substrate for this enzyme when sodium glycodeoxycholate was included in the assay. This activator protein had two isoelectric points pH 4.1 and 4.6, and it had an apparent molecular weight of 27,000 by gel filtration.     

A beta-galactosidase isoenzyme from Turbo cornutus with substrate specificity toward GM1-ganglioside and glycoproteins

beta-Galactosidase from T. cornutus was resolved into two activity peaks by gel filtration column chromatography. The pH optima of the two peaks designated P1 and P2, were 5.5 and 3.0, respectively, when p-nitrophenyl-beta-D-galactopyranoside was used as the substrate. The molecular weights of P1 and P2 were 700,000 +/- 70,000 and 78,000 +/- 7800, respectively, when estimated by gel filtration chromatography. The activities of both forms of the enzymes are stimulated by anions such as Cl-, Br- and NO-3. While the activity of P1 was stimulated by low anion concentrations, P2 requires 700 times higher anion concentration for similar enhancement of activity. P1, the high molecular weight form hydrolyzes mainly galactose from small molecular weight beta-galactosides, such as p-nitrophenyl-beta-D-galactopyranoside, 4-methylumbelliferyl-beta-D-galactopyranoside, lactose, lactosylceramide and 3-O-beta-D-galactopyranosyl-D-arabinose, whereas P2, the low molecular weight form cleaves, in addition, all the beta-galactosides tested, including 2-hexadecanoylamino-4-nitrophenyl-beta-D-galactopyranoside, GM1-ganglioside, asialo-GM1-ganglioside, asialo fetuin, alpha 1-acid glycoproteins and the tryptic peptides of the glycoproteins. The optimal conditions for the hydrolysis of the terminal galactose from GM1-ganglioside which does not occur in gastropods, such as T. cornutus, was found to require 40 mM NaCl and 1 mM sodium taurodeoxycholate at pH 3.0 in 50 mM sodium citrate buffer, conditions similar to those by mammalian beta-galactosidase.     

I-Cell disease: Activities of lysosomal enzymes toward natural and synthetic substrates

Cultured skin fibroblasts from a patient with I-Cell disease (mucolipidosis II) were assayed for a number of lysosomal enzymes using both natural and synthetic substrates. The cells from this patient were found to have very low activity for galactosylceramide β-galactosidase, lactosylceramide β-galactosidases (using two assay methods that measure different enzymes), G_M1 ganglioside β-galactosidase and sphingomyelinase. Glucosylceramide β-glucosidase activity was found to be normal. Acid hydrolase activities toward many synthetic substrate were measured and all except β-glucosidase and acid phosphatase were found to be extremely low (as has been reported by others). Acid phosphatase and β-glucosidase were in the low normal range. These studies expand on previously published reports on I-Cell disease that only present data from synthetic substrates, and also report the fibroblast culture deficiencies of galactosyl-ceramide β-galactosidase (the Krabbe disease enzyme) and sphingomyelinase (the Niemann-Pick disease enzyme) activities for the first time. Those two enzymes do not have a readily available synthetic analog to assay. Acid β-galactosidase activity measured with both the 4-methylumbelliferyl derivative and G_M1 ganglioside was partially deficient in leukocytes prepared from this patient. New methods for measuring 4-methylumbelliferyl-β-D-glucoside and glucosylceramide β-glucosidase activities are also presented.     

Galactosylceramide beta-galactosidase in Krabbe disease: partial purification and characterization of the mutant enzyme

Galactosylceramide β-galactosidase (EC 3.2.1.46) has been partially purified from liver of a patient who died of Krabbe disease. Approximately 700-fold purification was achieved by solubilization, adsorption with immobilized concanavalin A, gel filtration through Bio-Gel A-1.5m and chromatography on immobilized sphingosine. The relative increase in crossreacting material and residual galactosylceramidase and lactosylceramidase I activities of the mutant enzyme was essentially identical to that obtained for the enzyme partially purified by the same procedure from normal liver control. An apparent molecular weight of about 750,000 and similar electrophoretic mobilities were observed for both enzymes. In contrast, catalytic properties and stability of the enzyme protein were severely affected in the mutant as compared to the normal enzyme. The apparent K_m values of the mutant enzyme for β-galactosidase activities toward galactosylceramide and lactosylceramide in the presence of pure sodium taurocholate were 14 and 4 times, respectively, higher than the normal values. Incubation for 4 min at 52 °C or dialysis against 1.3 m urea caused a 50% loss of residual enzymatic activity of the mutant enzyme, whereas a 35-min incubation or dialysis against 5.6 m urea was required for 50% inactivation of the normal enzyme. These findings indicate that the mutation in Krabbe disease leads to synthesis of normal quantities of catalytically and structurally altered protein.     

Transglycosylation and reverse hydrolysis reactions of endoglycoceramidase from the jellyfish, Cyanea nozakii

Endoglycoceramidase (EGCase: EC 3.2.1.123) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids. We report here transglycosylation and reverse hydrolysis reactions of EGCase from the jellyfish Cynaea nozakii. Various alkyl-GM1 oligosaccharides (alkyl-II(3)NeuAcGgOse4) were synthesized when GM1 ganglioside was treated with the EGCase in the presence of 1-alkanols. Among various 1-alkanols tested, methanol was found to be the most preferential acceptor, followed by 1-hexanol and 1-pentanol. GM1 was the best donor, followed by GD1b and GT1b, when methanol was used as an acceptor. However, neither globoside nor glucosylceramide was utilized by the enzyme as a donor substrate. The enzyme transferred oligosaccharides from various glycosphingolipids to NBD-ceramide, a fluorescent ceramide, producing NBD-labeled glycosphingolipids. In addition to the transglycosylation reaction, the enzyme catalyzed the reverse hydrolysis reaction; lactose was condensed to ceramide to generate lactosylceramide in the presence of the enzyme. These results indicate that the jellyfish enzyme will facilitate the synthesis of various neoglycoconjugates and glycosphingolipids.     

Isolation and characterization of the major glycosphingolipids from the liver of the rainbow trout (Oncorhynchus mykiss): identification of an abundant source of 9-O-acetyl GD3

The carbohydrate structures of the major glycosphingolipids from the liver of the rainbow trout Oncorhynchus mykiss have been examined. We have isolated and identified four major neutral (glucosylceramide, galactosylceramide, lactosylceramide, and globoside) and five acidic (sulfatide, GM3, GM2, GD1a, and 9-O-Acetyl GD3) glycosphingolipids from trout liver. They have been characterized by 1H nuclear magnetic resonance spectroscopy, methylation analysis, fast atom bombardment mass spectrometry, and specific monoclonal antibodies. Significantly, the relatively scarce ganglioside 9-O-acetyl GD3 was found to comprise approximately 23% of the total ganglioside content of normal rainbow trout liver. 9-O-Acetyl GD3 is, however, abundant in human melanoma and as such, trout liver may be a suitable source of this antigen.     

Purification of G-M-1-ganglioside and ceramide lactoside beta-galactosidase from rabbit brain.

The major beta-galactosidase of rabbit brain has been purified over 400-fold. The enzyme converts G-M-1-ganglioside; Gal beta-1 yields 3 GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-1) into Tay Sachs ganglioside GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-2-ganglioside) and ceramide lactoside, Gal beta-1 yields 4 Glc yields ceramide (Gal-Glc-Cer) into glucocerebroside, Glc yields ceramide (Glc-Cer). The enzyme also hydrolyzes the synthetic substrates NPh-Gal and MeUmb-Gal. It is eluted as a single peak from Sephadex G-200 columns when natural and synthetic substrates were used and has an isoelectric point of 6.3. We were unable to resolve activity towards G-M-1-ganglioside and Gal-Glc-Cer by polyacrylamide electrophoresis in two buffer systems. With G-M-1 the pH optimum was 4.3 in acetate buffer and the K-m value 78 mu-M while with Gal-Glc-Cer, a pH optimum of 4.5 and a K-m of 17 mu-M were found. Hydrolysis of both natural and synthetic substrates was inhibited by gamma-D-galactonolactone, D-galactose and lactose. The data strongly suggest that a single beta-galactosidase hydrolyzes all the substrates tested.     

Purification and properties of neutral β-galactosidases from human liver

Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ?-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM₁-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar K_m and V values; the more acidic form (designated β-galactosidase N₁) was more heat stable than the other form (designated β-galactosidase N₂). Antibodies evoked against the N₁ and N₂ β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.     

Hypothesis. Cytochrome c oxidase as a proton pump. A transition-state mechanism

The thermotropic behavior of mixtures of dipalmitoylphosphatidylcholine (DPPC) with natural glycosphingolipids (galactosylceramide, phrenosine, kerasine, glucosylceramide, lactosylceramide, asialo-GM1, sulfatide, GM3, GM1, GD1a, GT1b) in dilute aqueous dispersions were studied by high sensitivity differential scanning calorimetry over the entire composition range. The pretransition of DPPC is abolished and the cooperativity of the main transition decreases sharply at mole fractions of glycosphingolipids below 0.2. All systems exhibit non-ideal temperature-composition phase diagrams. The mono- and di-hexosylceramides are easily miscible with DPPC when the proportion of glycosphingolipids in the system is high. A limited quantity (1-6 molecules of DPPC per molecule of glycosphingolipid (GSL) can be incorporated into a homogeneously mixed lipid phase. Domains of DPPC, immiscible with the rest of a mixed GSL-DPPC phase that shows no cooperative phase transition, are established as DPPC exceeds a certain proportion in the system. One negative charge (sulfatide) or four neutral carbohydrate residues (asialo-GM1) in the oligosaccharide chain of the glycosphingolipids results in phase diagrams exhibiting coexistence of gel and liquid phases over a broad temperature-composition range. Systems containing gangliosides show complex phase diagrams, with more than one phase transition. However, no evidence for phase-separated domains of pure ganglioside species is found. The thermotropic behavior of systems containing DPPC and glycosphingolipids correlates well with their interactions in mixed monolayers at the air/water interface.     

Purification and properties of GM1 ganglioside beta-galactosidases from bovine brain

Two GM1-beta-galactosidases, beta-galactosidases I, and II, have been highly purified from bovine brain by procedures including acetone and butanol treatments, and chromatographies on Con A-Sepharose, PATG-Sepharose, and Sephadex G-200. beta-Galactosidase I was purified 30,000-fold and beta-galactosidase II 19,000-fold. Both enzymes appeared to be homogeneous, as judged from the results of polyacrylamide disc gel electrophoresis. Enzyme I had a molecular weight of 600,000-700,000 and enzyme II one of 68,000, as determined on gel filtration. On sodium dodecyl sulfate polyacrylamide slab gel electrophoresis under denaturing conditions, enzyme II gave a single band with a molecular weight of 62,000, while enzyme I gave two minor bands with molecular weights of 32,000 and 20,000 in addition to the major band at 62,000. Both enzymes liberated the terminal galactose from GM1 ganglioside and lactosylceramide but not from galactosylceramide. Enzyme I showed a pH optimum of 4.0 and was heat stable, while enzyme II showed a pH optimum of 5.0 and lost 50% of its activity in 15 min at 45 degrees C. Enzyme I showed a pI of 4.2 and enzyme II one of 5.9.