PROPERTIES OF ACID β-d-GALACTOSIDASE ISOLATED FROM I-CELL DISEASE BRAIN AND SPLEEN

Abstract— Acid 4-methylumbelliferyl β- d -galactosidase activity from autopsied I-cell disease brain and spleen tissues was 28% and 35% respectively of normal activity. Acid β- d -gatactosidase (β- d -galactoside galactohydrolase, EC 3.2.1.23) from two I-cell disease brains demonstrated a 5-fold increase over normal for the proportion of enzyme activity which did not adsorb to Concanavalin A-Sepharose 4B, while acid β- d -galactosidase from two I-cell disease spleens demonstrated a 21–35-fold increase in the proportion of unadsorbed enzyme activity. Normal and I-cell disease acid β- d -galactosidase present in crude brain and spleen supernatant fluids and in preparations partially purified on Concanavalin A-Sepharose 4B had similar apparent K _m values with respect to 4-methylumbelliferyl β- d -galactopyranoside and G_M1-ganglioside. Isoelectric focusing profiles of normal and I-cell disease acid β- d -galactosidase from crude brain and spleen-supernatant fluids and partially purified preparations were similar. Neuraminidase treatment and subsequent isoelectric focusing of the partially purified normal and I-cell disease enzyme preparations from brain and spleen revealed increases in the proportion of I-cell β- d -galactosidases found at neutral pH values, suggesting that the electrophoretic variations observed for the I-cell enzymes may not be attributed solely to changes in sialic acid composition.     

β-Galactosidases of Lilium pollen

Lily (Lilium auratum) pollen contains very high levels of β-galactosidase. There are three forms: β-galactosidase I and II differ in M_r, while β-galactosidase III is firmly bound in the pollen wall. The two cytoplasmic forms were separated and partially purified using a combination of chromatography on DEAE-cellulose, Sephadex G-200 and Sepharose 6B. Forms I and II appear to be glycoprotein in nature as shown by binding to Con A-Sepharose. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. The wall-bound enzyme, β-galactosidase III effectively hydrolysed nitrophenyl β-galactosidase but not lactose, and could not be released from the wall polysaccharide matrix by high salt concentrations or detergents. The total β-galactosidase activity of lily pollen remained constant during in vitro germination. A possible role for this enzyme may be in degradation of stylar arabinogalactans providing a carbon source for pollen tube nutrition.     

Characterization of beta-D-galactosidase isolated from I-cell disease liver.

The residual beta-D-galactosidase activity (10% of normal) present in an autopsy sample of liver derived from an I-cell patient has been characterized. The pH optima for both I-cell and normal acid 4-methylumbelliferyl beta-D-galactoside activities were 4.35. The adsorption and elution profiles of the I-cell enzyme from Con A-Sepharose were similar to those of normal liver beta-D-galactosidase. Although starch gel electrophoresis revealed the presence of beta-D-galactosidase A and B in I-cell disease liver, the A band was more diffuse and migrated less anodally than the A band from normal liver. The electrophoretic mobilities of both I-cell and normal beta-D-galactosidase A appeared to decrease after treatment with neuraminidase. Kinetic studies of the I-cell and normal level beta-D-galactosidase demonstrated similar apparent Km values with respect to the 4-methylumbelliferyl beta-D-galactoside and Gm1 ganglioside, whereas the Vmax values obtained for the I-cell enzyme were 10- to 12-fold lower than those of the normal enzyme for both substrates.     

Fabry's disease as an -galactosidosis: evidence for an -configuration in trihexosyl ceramide

Pure α-galactosidases, devoid of β-galactosidase activity, were purified from coffee beans, ficin (a crude extract from figs), rat and rabbit small intestine. With the exception of the coffee bean enzyme, all α-galactosidase preparations released galactose from ³H- or ¹⁴C-labeled trihexosyl ceramide obtained from patients with Fabry's disease. Galactose liberation was specifically inhibited by α-galactosides, such as melibiose and stachyose, while lactose had no effect. Our results corroborate the α-galactosidase deficiency reported in Fabry's disease and establish that the terminal galactosyl residue of the trihexosyl ceramide stored in this condition has an α-configuration.     

Chemical compositions of brain and myelin in two patients with multiple sulphatase deficiency (a variant form of metachromatic leukodystrophy)

The lipid composition of the brain, including myelin, was studied in detail in two cases with a variant form of metachromatic leukodystrophy (multiple sulphatase deficiency type). In the white matter, the sulphatide concentration was 3-4 times higher than the normal level in both cases. There was a significant accumulation of cholesterol sulphate in the brain, liver and kidney of both cases. The ganglioside pattern in the grey and white matter was abnormal, with a higher proportion of GM3, GM2 and GD3-gangliosides. Non-lipid hexosamine contents were increased 1.5-2 times in brain, 8-10 times in liver and 2-3 times in kidney. Increased amounts of glucocerobroside, ceramide lactoside and ceramide trihexoside were present in grey and white matter of both cases. Recovery of purified myelin from two patients' brains was much less than from control (1-2% in case 1 and 20-30% in case 2). The lipid composition of myelin was almost normal except for a higher proportion of sulphatide, with a decreased amount of cerebroside. The fatty acid compositions of myelin sulphatide and sphingomyelin were almost normal, while non-hydroxy fatty acids of cerebroside contained less long-chain fatty acids, as characterized by a significant increase of C16:0 and C18:0 fatty acids. The myelin polypeptide pattern by SDS-disc gel electrophoresis showed a relative decrease of basic protein and of proteolipid protein. A possible mechanism of myelin loss in MSD is discussed.     

Deficiency of monogalactosyl diglycerid beta-B-galactosidase activity in krabbe's disease

Monogalactosyl diglyceride has previously been demonstrated to be intimately associated with brain white matter, especially myelin. Enzymes responsible for its biosynthesis and degradation have been reported to be present in rat and mouse brain. In the present study, the β-galactosidase responsible for the degradation of this brain specific compound was demonstrated to be extremely deficient in brain, liver and skin fibroblasts from patients who died of Krabbe's disease. This deficiency is the third enzymatic block demonstrated in this disorder. The β-galactosidase activity toward galactocerebroside and psychosine is also extremely deficient. This finding provides new information about the substrate recognition pattern of this enzyme and about the possible etiology of globoid cell leukodystrophy.     

I-Cell disease: isoelectric focusing, concanavalin A-Sepharose 4B binding and kinetic properties of human liver acid beta-D-galactosidases.

Isoelectric focusing of the acid beta-D-galactosidases (beta-D-galactoside galactohydrolase, EC 3.2.1.23) in normal crude liver supernatant fluids demonstrated multiple isoelectric forms in the pH range 4.58-5.15, while corresponding I-cell disease samples showed an absence of isoelectric forms in the pH range 4.99-5.15. Concanavalin A-Sepharose 4B chromatography of the I-cell disease mutant C.A. demonstrated a 31% and 37% decrease in the binding of 4-methyl-umbelliferyl-beta-D-galactosidase and GM1 beta-D-galactosidase activities, respectively, when compared to normal samples. Isoelectric focusing profiles of the concanavalin A-Sepharose 4B alpha-methyl-D-mannoside effluents containing normal and I-cell disease acid beta-D-galactosidase were generally similar, but the unadsorbed I-cell disease enzyme from concanavalin A-Sepharose 4B demonstrated more activity in the pH range 4.21-4.49 than normals. Normal and I-cell disease acid beta-D-galactosidase "A" and "B", separated by gel column chromatography were found to have similar properties with respect to apparent molecular weights pH vs. activity profiles and apparent Km values for the 4 methylumbelliferyl-beta-D-galactopyranoside, GM1-ganglioside and asialofetuin (ASF) substrates. However, the apparent V values for the ICD samples were consistently reduced when compared to the results obtained with the corresponding normal fractions. The greatest decreases in apparent V were obtained for acid beta-D-galactosidase activities in I-cell disease crude supernatant fluids, and for the separated I-cell disease "B" enzyme. The differences in the isoelectric focusing profiles, the altered binding to concanavalin A-Sepharose 4B, and the reduced V values with natural and synthetic substrates may be related to changes in carbohydrate composition of I-cell disease acid beta-D-galactosidase.     

Abnormal cortical lysosomal β-hexosaminidase and β-galactosidase activity at post-synaptic sites during Alzheimer's disease progression

A critical role of endosomal–lysosomal system alteration in neurodegeneration is supported by several studies. Dysfunction of the lysosomal compartment is a common feature also in Alzheimer's disease. Altered expression of lysosomal glycohydrolases has been demonstrated not only in the brain and peripheral tissues of Alzheimer's disease patients, but also in presymptomatic subjects before degenerative phenomenon becomes evident. Moreover, the presence of glycohydrolases associated to the plasma membrane have been widely demonstrated and their alteration in pathological conditions has been documented. In particular, lipid microdomains-associated glycohydrolases can be functional to the maintenance of the proper glycosphingolipids pattern, especially at cell surface level, where they are crucial for the function of cell types such as neurons. In this study we investigated the localization of β-hexosaminidase and β-galactosidase glycohydrolases, both involved in step by step degradation of the GM1 to GM3 gangliosides, in lipid microdomains from the cortex of both an early and advanced TgCRND8 mouse model of Alzheimer's disease. Throughout immunoprecipitation experiments of purified cortical lipid microdomains, we demonstrated for the first time that β-hexosaminidase and β-galactosidase are associated with post-synaptic vesicles and that their activities are increased at both the early and the advanced stage of Alzheimer's disease. The early increase of lipid microdomain-associated β-hexosaminidase and β-galactosidase activities could have relevant implications for the pathophysiology of the disease since their possible pharmacological manipulation could shed light on new reliable targets and biological markers of Alzheimer's disease.     

Binding of hydrolases from wheat aleurone to concanavalin A- and wheat-germ agglutinin-sepharose

We have studied the ability of hydrolases (acid phosphatase and glycosidases) from the aleurone layers of resting wheat grains to interact with Con A- and WGA-Sepharose as a way to examine their glycoprotein nature. Aliquots (6–85% depending on the enzyme) of all the enzymes interacted with Con A-Sepharose. The major part of α-mannosidase activity (85%) was present in this form. Aliquots (2–20% depending on the enzyme) of the following four enzymes, β-galactosidase, α-mannosidase, β-N-acetylglucosaminidase and acid phosphatase, interacted with WGA-Sepharose. All the enzymes were found in forms which were unable to interact with either lectin. No forms of hydrolases interacting with both lectins were found in the crude extract. The specific activities of most of the enzymes recovered from the lectin-Sepharose gels were greater than those measured in the crude extract. In particular, the highest specific activities were found for β-N-acetylglucosaminidase and β-galactosidase recovered from WGA-Sepharose. Different lectin-binding forms of hydrolases were compared with respect to pH optimum and stability under various conditions (heat and guanidine hydrochloride treatments). The lectin-binding pattern of the hydrolases released in the incubation medium by the aleurone layers was similar to that reported above for the enzymes extracted from these tissues, suggesting that none of the hydrolase forms found in the aleurone layers is selectively released during incubation of these tissues.     

Brain sphingoglycolipids in Krabbe's globoid cell leucodystrophy

Abstract— Seven sphingoglycolipids were isolated from the white matter of a patient with globoid cell leucodystrophy (Krabbe's disease). After purification by saponification and column and preparative thin-layer chromatography, these compounds were analysed for the carbohydrate composition and sequence and for fatty acid composition by paper and gas-liquid chromatography. The compounds were identified as gluco- and galactocerebrosides, lactosyl-ceramide, digalactosy I-glucosyl-ceramide, two types of tetrahexosyl-ceramides (asialo-ganglioside and globoside), and sulphatide. Glucocerebrosideconstituted 13 percent of total cerebroside in white matter, but sulphatide contained only galactose. Galactocere-broside and sulphatide exhibited compositions of fatty acids similar to those in normal white matter, with only minor abnormalities. Other sphingoglycolipids showed fatty acid patterns with relatively high proportions of longer-chain fatty acids, rather than the predominant C_18:0 acid usually found in ceramide hexosides of the brain. Hematoside, also found in the white matter in a significant amount, similarly contained a large proportion of longer-chain fatty acids, whereas other gangliosides contained predominantly C_18:0 acid. The abnormal ceramide hexoside pattern was restricted mostly to white matter except for glucocerebroside, which constituted 32 per cent of grey matter cerebroside. We postulate that the visceral type of sphingoglycolipids may be constituents of globoid cells, abundantly present in white matter and considered to be cells of mesenchymal origin.     

Occurrence of glycoprotein glycosidases in mature seeds of mung bean (vigna radiata)

The presence of nine different glycosidases was demonstrated in the crude extract of mature mung bean seeds. N-Acetyl β-D-glucosaminidase, α-D-galactosidase and β-D-glucosidase were each resolved into two respective active forms by gel filtration. The other glycosidases showed single forms only. The apparent MWs of the glycosidases were determined. The glycosidases were absorbed to Con A-Sepharose column, with the exception of a small percentage of α-galactosidase and α-mannosidase which were eluted unretarded. The bound enzymes displayed varying affinities for the immobilized lectin, indicating differences in glycosylation. With the exception of β-galactosidase and invertase, all the glycosidase activities were detected in the protein bodies isolated from the seeds.     

Hurler's syndrome, an -L-iduronidase deficiency

The activity of a-L-iduronidase was found to be deficient in extracts prepared from livers, cultured fibroblasts and urine of patients with Hurler's syndrome. Extracts from livers and fibroblasts of Hunter's and Sanfilippo's syndromes did not show such a deficiency. There was a diminution of activity in extracts of “I-cell” fibroblasts. a-L-Iduronidase is believed to be the underlying enzymic defect of Hurler's disease.     

LACTOSYLCERAMIDASES IN CANINE GLOBOID CELL LEUKODYSTROPHY1

Lactosylceramide-cleaving activities were examined in the canine form of globoid cell leuko-dystrophy. Lactosylceramidase I activities were deficient in the brains and livers of affected dogs, while they were intermediate in heterozygous carriers. In contrast, lactosylceramidase II activities were normal in affected dogs. Examination of the total tissue activities, solubilization characteristics, and the behavior in Sephadex G-200 gel filtration, of the activities of lactosylceramidase I and II, galactosylceramidase, and 4-methylumbelliferyl β-galactosidase, indicated that, as in human tissues, lactosylceramidase 1 is probably identical with galactosylceramidase, and that lactosylceramidase II is closely associated with 4-methylumbelliferyl β-galactosidase. The canine form of globoid cell leukodystrophy is analogous with the human disease with respect to lactosylceramidases, and the lactosylceramidase I assay system of Wenger et al. (1974) is directly applicable also for canine tissues.     

NEUROCHEMISTRY OF THE MUCOPOLYSACCHARIDOSES: BRAIN GLYCOSAMINOGLYCANS, LIPIDS AND LYSOSOMAL ENZYMES IN MUCOPOLYSACCHARIDOSIS TYPE III B (α-N-ACETYLGLUCOSAMINIDASE DEFICIENCY)

Glycosaminoglycans, lipids and lysosomal enzymes were measured in brain, liver and spleen of a patient with mucopolysaccharidosis Type III B (α-N-Acetylglucosaminidase deficiency). The glycosaminoglycan content of the brain gray and white matter, leptomeninges, spleen and liver of the patient was 4, 3, 10, and 100 times greater than that of the respective tissues of normal controls. Partially degraded heparan sulfate, the concentration of which increased 17 times in the brain, accounted for the increased glycosaminoglycan content of all tissues. The concentration of the gangliosides G_M2, G_M3 and G_D3 was markedly increased in the gray matter, and to a smaller degree in the white matter. Ceramide dihexoside was also increased in the gray matter of the patient with MPS III B. The activity of α-N-Acetylglucosaminidase was absent from the brain and the liver and greatly diminished in the spleen. β-Glucuronidase. β-glucosaminidase and α-l -iduronidase were more active than normally and the activity of α-galactosidase and β-galactosidase was markedly reduced.     

Human Brain Cerebroside β-Galactosidase: Deficiency of Transgalactosidic Activity in Krabbe's Disease

Abstract: Under experimental conditions optimal for the assay of D-galactosyl- N -acylsphingosine galactohydrolase (EC 3.2.1.46) activity, homog-enates of neurologically normal human brain tissue could transfer galactose from galactosyl ceramide (gal-cer), lactosyl ceramide (lac-cer), 4-methylumbelliferyl- β-galactoside (4-MU-gal), or p -nitrophenyl- β-galactoside (PNP-gal) to [1-¹⁴C]oleoyl sphingosine, but homogenates of brain tissue from patients with Krabbe's disease lacked this ability. The rate of hydrolysis of ganglioside G_M1 and to a lesser extent, of PNP-gal by homogenates of Krabbe's brain tissue was also decreased. Activity of PNP- β-galactosidase in normal brain tissue, like that of cerebroside β-galactosidase from the same source, was considerably more heat-stable than the activity of either 4-MU- β-galactosidase or the predominant G_M1β-D-galactosidase (EC 3.2.1.23). Lac-cer and G_M1, as well as 4-MU-gal and PNP-gal, were competitive inhibitors of human-brain cerebroside β-galactosidase. These findings confirm the ability of mammalian cerebroside β-galactosidase to catalyze a transgalactosylation reaction and provide additional information on the substrate specificity of human brain cerebroside β-galactosidase.     

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.     

Is there a mechanism for introducing acid hydrolases into liver lysosomes that is independent of mannose 6-phosphate recognition? evidence from I-cell disease

We have examined frozen liver tissue for N-acetylglucosamine-l-phosphotransferase, an enzyme required for the formation of the mannose 6-phosphate recognition marker of lysosomal enzymes. Using [β³²P]-UDPGlcNAc and placental β-hexosaminidase B as N-acetylglucosamine l-phosphate donor and acceptor, respectively, we were unable to find activity of the transferase in 100,000 × g membranes prepared from livers of patients with I-cell disease, whereas activity was readily observed in membranes from control livers stored under the same conditions. Yet the activity of several lysosomal enzymes (β-N-acetylglucosaminidase, β-glucuronidase, α-mannosidase and α-$\text{L}$-iduronidase) was comparable in liver tissue of I-cell patients and controls, and only β-galactosidase activity showed a marked reduction. These results suggest that in contrast to cultured skin fibroblasts, liver may be able to introduce into lysosomes acid hydrolases that lack the mannose 6-phosphate recognition marker.     

Identification of a breast tumor-associated orosomucoid by concanavalin A affinity chromatography.

Con A-Sepharose affinity chromatography was utilized to examine the glycoproteins in phosphosaline extracts of normal and breast tumor tissues and breast patient sera. In extracts of normal breast tissue, normal sera and patient sera, all glycoproteins were eluted from the Con A-Sepharose with a linear gradient of 0.0-0.5 M alpha-methylmannose. Using breast tumor extracts, a glycoprotein peak which could not be eluted as with normal tissue extracts was observed. This tightly-binding peak could be eluted from the Con A-Sepharose with acetate buffer containing 1.0 M KCl. Polyacrylamide electrophoresis of this tightly-binding glycoprotein peak revealed one major glycoprotein and four minor glycoproteins. The major glycoprotein obtained from electrophoresis represented about 60% of the Con A-Sepharose tightly-binding protein and reacted with antiserum to human orosomucoid (alpha 1-acid glycoprotein). All glycoproteins isolated from tumor tissue extracts appeared to represent normal serum constituents as they were retained on an immunoadsorbent containing antibodies to normal serum proteins. The possible significance of the isolated tumor-associated orosomucoid is discussed.     

DIFFERENTIAL DETERMINATION OF HEXOSAMINIDASES A AND B AND OF TWO FORMS OF β-GALACTOSIDASE,IN THE LAYERS OF THE HUMAN CEREBELLUM1

—In order to determine whether or not various histological elements of the nervous system may differ in their relative content of hexosaminidase A and B (O'Brien , Okada , Chen and Fillerup , 1970) and in the‘acid’and‘neutral’forms of β-galactosidase (Ho and O'Brien , 1971), these isoenzymes were determined separately in the layers of human cerebellum. The proportion of the heat-stable hexosaminidase B was greater in the granular layer than in the molecular layer or underlying white matter. The activity of the‘neutral’form of β-galactosidase was very low compared to the‘acid’type, but its distribution was similar.     

Brain ceramide hexosides in Tay-Sachs disease and generalized gangliosidosis (GM1-gangliosidosis)

The carbohydrate composition was determined for ceramide hexosides isolated from brains of patients with Tay-Sachs disease and generalized gangliosidosis (hereby named GM1-gangliosidosis). Gray matter of patients with each disease showed a characteristic abnormal ceramide hexoside pattern. In Tay-Sachs gray matter, ceramide trihexoside is the major component, whereas ceramide tetrahexoside is barely detectable. In GM1-gangliosidosis, ceramide tetrahexoside is the major ceramide hexoside, while ceramide trihexoside is present only in small amount. These two major components have been characterized as the asialo derivatives of, respectively, the "Tay-Sachs ganglioside" (GM2-ganglioside) and the normal major monosialoganglioside (GM1-ganglioside). In both diseases, more than half the ceramide monohexoside of gray matter was glucocerebroside. Gray matter ceramide dihexoside, present in both diseases at higher than normal levels, was mostly ceramide lactoside, with possibly a small amount of ceramide digalactoside. Sulfatide contained only galactose. The abnormal ceramide hexoside pattern is limited to gray matter: white matter showed normal ceramide hexosides, i.e. a preponderance of monohexosides and sulfatide, with no detectable glucocerebroside.