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PubMed1976

Raffinose metabolism in Escherichia coli K12. Purification and properties of a new alpha-galactosidase specified by a transmissible plasmid.

The utilization by Escherichia coli K12 of raffinose as sole carbon source depends on a new raffinose transport system, an invertase and an alpha-galactosidase specified by the Raf-plasmid D1021. The alpha-galactosidase was purified to homogeneity from a mutant strain with constitutive synthesis of the enzyme. alpha-Galactosidase hydrolyzes p-nitrophenyl-alpha-D-galactoside (Km 0.14 mM), methyl-alpha-D-galactoside (Km 30mM), melibiose (Km 3.2 mM) and raffinose (Km 60 mM). The enzymatic activity is strongly inhibited by Ag+, p-chloromercuriphenyl sulfonic acid and, to a lesser extent, by iodoacetamide. Isoelectric focusing indicates the existence of one form of alpha-galactosidase with an isoelectric point of 5.1. The purified enzyme has an sw,20 value of 11.7 +/- 0.3S and a molecular weight of 329000 +/- 4000; this value is not reduced at high dilutions. When examined by dodecylsulphate gel electrophoresis, purified alpha-galactosidase yields a single subunit band of molecular weight 82000 suggesting that the intact enzyme consists of four subunits. Amino acid analysis indicates the presence of approximately 712 amino acid residues per quarter molecule including 8 half-cystine residues. No carbohydrate moiety has been detected. High resolution electron micrographs and Markham rotation of alpha-galactosidase show enzyme molecules of approximately 11 x 11 nm containing four globular subunits in a tetragonal arrangement. The plasmid-coded alpha-galactosidase differs from the homologous E. coli enzyme by substrate affinities, cofactor requirements, stability and toluene resistance. It can, therefore, be used as a marker enzyme suitable for the detection in vivo of Raf-plasmids.

Amino Acids↗

PubMed1993

Comparative study on the production of guar alpha-galactosidase by Saccharomyces cerevisiae SU50B and Hansenula polymorpha 8/2 in continuous cultures.

Saccharomyces cerevisiae SU50B and Hansenula polymorpha 8/2, both carrying a multicopy integrated guar alpha-galactosidase, have been cultivated in continuous cultures, using various mixtures of carbon sources and cultivation conditions. Both S. cerevisiae SU50B and H. polymorpha 8/2 are stable and produce high levels of extracellular alpha-galactosidase in continuous cultures for more than 500 h. For these expression systems the strong inducible promoter systems GAL7 and methanol oxidase, respectively, were used. The induction of alpha-galactosidase synthesis by galactose in SU50B is limited by the low galactose uptake. Apart from that, at high dilution rates, the glucose repression is substantial, and a maximal expression level of 28.6 mg of extracellular alpha-galactosidase.g (dry weight) of biomass-1 could be obtained. In H. polymorpha, the induction of alpha-galactosidase synthesis, in addition to methanol oxidase synthesis using formaldehyde, is very effective up to 42 mg of extracellular alpha-galactosidase.g (dry weight) of biomass-1. Productivities in terms of specific production rate enable a good comparison with those of other heterologous expression systems in the literature. The productivities found with S. cerevisiae SU50B and H. polymorpha, 3.25 and 5.5 mg of alpha-galactosidase.g of biomass-1.liter-1.h-1, respectively, rank among the highest reported in the literature. Enzyme production and secretion in H. polymorpha are more complex. A two-peaked optimum is found in enzyme production. No clear explanation of this phenomenon can be given.

Alcohol Oxidoreductases↗

PubMed2005

Role of the bga1-encoded extracellular {beta}-galactosidase of Hypocrea jecorina in cellulase induction by lactose.

Lactose is the only soluble and economically feasible carbon source for the production of cellulases or heterologous proteins regulated by cellulase expression signals by Hypocrea jecorina (Trichoderma reesei). We investigated the role of the major beta-galactosidase of H. jecorina in lactose metabolism and cellulase induction. A genomic copy of the bga1 gene was cloned, and this copy encodes a 1,023-amino-acid protein with a 20-amino-acid signal sequence. This protein has a molecular mass of 109.3 kDa, belongs to glycosyl hydrolase family 35, and is the major extracellular beta-galactosidase during growth on lactose. Its transcript was abundant during growth on l-arabinose and l-arabinitol but was much less common when the organism was grown on lactose, d-galactose, galactitol, d-xylose, and xylitol. Deltabga1 strains grow more slowly and accumulate less biomass on lactose, but the cellobiohydrolase I and II gene expression and the final cellulase yields were comparable to those of the parental strain. Overexpression of bga1 under the control of the pyruvate kinase promoter reduced the lag phase, increased growth on lactose, and limited transcription of cellobiohydrolases. We detected an additional extracellular beta-galactosidase activity that was not encoded by bga1 but no intracellular beta-galactosidase activity. In conclusion, cellulase production on lactose occurs when beta-galactosidase activity levels are low but decreases as the beta-galactosidase activities increase. The data indicate that bga1-encoded beta-galactosidase activity is a critical factor for cellulase production on lactose.

Cellulase↗

PubMed1970

Beta-galactosidase: isolation of and antibodies to incomplete chains.

A prematurely terminated polypeptide chain was purified to homogeneity from an Escherichia coli amber mutant strain containing the site of the mutation in the beta-galactosidase structural gene. The polypeptide was highly active against anti-beta-galactosidase, and had an amino acid composition similar to but not identical to that of beta-galactosidase. The molecular weight of the reduced, carboxymethylated chain in 6 m guanidine hydrochloride was found to be 89,000, in excellent agreement with the size predicted from the position of the mutation. This result adds further support to the conclusion that the gene specifies the structure of a single polypeptide chain. Antisera were prepared against partially purified preparations of this polypeptide and a similar one, of molecular weight about 100,000, produced by another amber mutant. These sera had lower titers towards beta-galactosidase than anti-beta-galactosidase. In the double-diffusion test, they reacted towards extracts of nonsense and deletion mutant strains in a pattern similar to that previously observed with anti-beta-galactosidase. A sensitive immunological test for cross-reacting protein was devised based on the inhibition by beta-galactosidase of the reaction between such protein and antibodies prepared against incomplete chains.

Amino Acids↗

PubMed1975

Kinectics of beta-galactosidase synthesis in Escherichia coli at 5 C.

The defect in protein synthesis that is observed in Escherichia coli after transfer to low temperature was studied. For the enzyme beta-galactosidase, the elongation reactions of transcription and translation can take place slowly but normally at 5 C. The time necessary to complete the coupled synthesis of the beta-galactosidase messenger ribonucleic acid and polypeptide chain was found to be about 80 min at 5 C. From this result and from the known length of the beta-galactosidase monomer, it is possible to calculate that at 5 C one amino acid is added to the growing polypeptide chain every 4 s. The initiation of transcription of the beta-galactosidase messenger is inhibited after transfer to 5 C. This fact alone, however, cannot account for all of the phenomena observed at 5 C, because a given amount of messenger yields less enzyme at 5 C than it does at 37 C. Furthermore, in cells induced for short periods at 37 C, the capacity to synthesize beta-galactosidase after transfer to 5 C was found to accumulate linearily with the square of the time of induction. Two alternative models could account for these data. If all ribosomes that initiate translation at 37 C yield complete beta-galactosidase polypeptide chains at 5 C, then an inhibition of translation initiation after transfer to 5 C must be invoked to explain the results. If, on the other hand, a substantial portion of the ribosomes that initiate translation at 37 C do not yield complete beta-galactosidase polypeptides at 5 C, then intracistronic polarity could account for the data, and there is no need to invoke an inhibition of translation initiation at 5 C.

Bacterial Proteins↗

PubMedDate not supplied

Evaluation of beta-galactosidase activity in tissue in the presence of blood.

The reporter gene for beta-galactosidase is frequently used to determine the efficiency of gene transfer in arteries. However, blood is often present in arterial explants and may compromise the results by the presence of hemoglobin. The light absorption of hemoglobin is similar to the absorption of several colorimetric products of the commonly used beta-galactosidase substrates, including o-nitrophenyl-beta-D-galactopyranoside (ONPG) and chlorophenol red galactopyranoside (CPRG). This may result in false-positive measurements of beta-galactosidase enzyme activity. The aim of this investigation was to determine the most appropriate method for quantification of beta-galactosidase activity in the presence of blood. Colorimetric substrates (ONPG, CPRG) or the chemiluminescent Galacton-Plus substrate were used, and light absorption was measured at different concentrations of erythrocyte extract. Among the beta-galactosidase substrates tested, CPRG was the most appropriate, allowing detection of enzyme activity at concentrations as low as 0.05 mU, independent of blood contamination. Addition of reducer stabilized enzyme activity for at least 5 h. Endogenous beta-galactosidase activity was evaluated and used to correct results. CPRG substrate, in combination with the reducer agent mercaptoethanol, was found to be the optimal reagent for quantifying beta-galactosidase activity in the presence of blood after nonviral in vivo reporter gene transfection, even with a relatively low transfer efficiency.

Animals↗

PubMed1997

Visualization of beta-galactosidase by enzyme and immunohistochemistry in the olfactory bulb of transgenic mice carrying the LacZ transgene.

In the olfactory bulb (OB) of a transgenic mouse line that carries the bacterial LacZ gene under the control of the 5'-regulatory region of the GAD67 gene, expression of the beta-galactosidase was confined almost exclusively to the non-GABAergic mitral and tufted cells. By light microscopy, enzyme histochemistry showed strong staining in the cell bodies and faint diffuse staining in the axons and dendrites. With immunohistochemistry for beta-galactosidase the entire cytoplasm, including the axons and dendrites, was strongly stained. By electron microscopy, beta-galactosidase enzyme histochemistry resulted in a submicroscopic reaction product that was diffusely distributed in the cytoplasm of neurons. In addition, large deposits of the reaction product were also seen attached to the cytoplasmic side of the membranes. In contrast, when the intracellular localization of beta-galactosidase was determined by immunohistochemistry, homogeneous cytoplasmic staining was obtained that filled the entire cytoplasm including the terminal dendrites and fine axons. Therefore, synaptic contacts of the beta-galactosidase-positive output neurons with other beta-galactosidase-negative neuronal cells were readily recognized in the OB. As we demonstrated, transgenic mouse lines expressing the LacZ reporter gene in a well-defined neuronal subpopulation can be used to follow beta-galactosidase-positive neurons and to directly identify their synaptic connections.

Animals↗

PubMed1994

Acid beta-galactosidase: a developmentally regulated marker of endocrine cell precursors in the human fetal pancreas.

Isolation of endocrine cell precursors from the human fetal pancreas will be important to the study of islet cytodifferentiation and eventually for islet transplantation in insulin-dependent diabetes. These precursor cells, from which all four islet endocrine cell types arise, are present within fetal pancreatic ductal epithelium. After enzymatic digestion and culture of the fetal pancreas, we obtained cell clusters resembling islets, but with a high content of undifferentiated cells. Histochemical staining revealed very high acid beta-galactosidase activity in over 70% of cells within the clusters. After transplantation into athymic nude mice, the islet-like cell clusters gave rise to tissue rich in differentiated endocrine cells, but low in beta-galactosidase activity. The histochemical finding of high acid beta-galactosidase activity in endocrine precursor cells was confirmed by direct measurement of lysosomal enzyme activities. In addition, we found that the expression of acid beta-galactosidase was developmentally regulated, peaking at 18-24 weeks gestation and declining to low levels in adult islets. Using a fluorogenic beta-galactosidase substrate, we were able to isolate a subpopulation of cells high in acid beta-galactosidase activity using fluorescence-activated flow cytometry. Evidence identifying these cells as potential islet cell precursors includes, besides the transplantation experiments, the colocalization in vitro of tyrosine hydroxylase, a marker of embryonic islet cells. Thus, our results indicate that high acid beta-galactosidase activity serves as a marker for a population of fetal pancreatic cells with the potential to differentiate and grow into mature pancreatic endocrine cells.

Biomarkers↗

PubMed1999

Comparative histochemical and biochemical studies on acid beta-galactosidase activity in the experimentally injured rabbit cornea and tear fluid using the sensitive substrate beta-galactoside-4-trifluoromethylumbelliferyl (HFC).

Comparative histochemical and biochemical studies on acid beta-galactosidase activity in the rabbit eye after various experimental injuries were performed using the same sensitive fluorogenic substrate beta-galactoside-4-trifluoromethylumbelliferyl (HFC). The aim of the study was to examine whether the severity of corneal damage corresponds with the level of the enzyme activity in the tear fluid. As until recently the substrate beta-galactoside-4-HFC had not been used for the histochemical detection of acid beta-galactosidase in the cornea, results obtained with this substrate in a fluorescent method were compared in parallel cryostat sections with results obtained using the substrate 5-bromo-4-chloro-3-indoxyl beta-galactoside in the indigogenic method (previously shown to be very sensitive for the detection of acid beta-galactosidase activity in the cornea). Both methods revealed similar localization and changes in enzyme activity; using beta-galactoside-4-HFC an acceptable cellular localization was achieved. For the measurement of acid beta-galactosidase activity in the tear fluid a semiquantitative biochemical method was elaborated using filter paper punches with the substrate (beta-galactoside-4-HFC) soaked with tears and incubated at 37 degrees C. The time of the first appearance of a greenish-yellow fluorescence (enzyme positivity) was recorded by UV lamp and compared with the appearance of fluorescence in calibrated punches containing known acid beta-galactosidase activities. The results show that beta-galactoside-4-HFC is useful for the biochemical assessment of acid beta-galactosidase activity in the tear fluid. Comparing histochemical and biochemical results, it can be concluded that increased enzymatic activity in tears parallels the severity of corneal damage. Further studies are necessary to evaluate whether the detection of acid beta-galactosidase activity in tears might be useful for diagnostic purposes in humans.

Animals↗

PubMed1994

Factors affecting the ability of a high beta-galactosidase yogurt to enhance lactose absorption.

Lactose in yogurt is better absorbed by lactase-deficient subjects than is an equivalent quantity of lactose in milk, presumably because of the microbial activity of the beta-galactosidase present in yogurt. In this study, we describe a process that increases the beta-galactosidase of yogurt 5- to 6-fold and the ability of this high lactase yogurt to enhance lactose absorption in lactase-deficient subjects. These subjects ingested the yogurt meals after a 12-h fast, and lactose malabsorption was determined by measuring breath hydrogen. Breath hydrogen was reduced 39% following ingestion of high lactase yogurt from that after consumption of conventional yogurt, indicating that the high lactase yogurt enhanced lactose absorption. However, the reduction after high lactase yogurt was less than expected, given the 5- to 6-fold increment in beta-galactosidase measured in vitro. In vivo activity of beta-galactosidase requires that the enzyme resist acid denaturation in the stomach. The beta-galactosidase in high lactase yogurt was much less acid resistant than was the beta-galactosidase in conventional yogurt, and the relative inability of high lactase yogurt to enhance lactose absorption was likely due to the destruction of the beta-galactosidase in the stomach.

Absorption↗

PubMed1975

Biochemical and immunological studies of purified mouse beta-galactosidase.

Beta-Galactosidase (EC 3.2.1.23) has been purified from the livers of C57BL/6J mice. The enzyme migrated as a single band of protein on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the denatured and reduced enzyme was 63,000. The native form of beta-galactosidase appeared to be a tetramer of 240,000 at pH 5.0, which was reversibly dissociated at alkaline pH to a dimer with apparent molecular weight of 113,000. Multiple charge isomers of beta-galactosidase were resolved by polyacrylamide gel electrophoresis and ion exchange chromatography. Treatment of beta-galactosidase with neuraminidase markedly reduced its electrophoretic mobility. Purified enzyme as well as crude liver extract hydrolyzed p-nitrophenyl-beta-D-fucoside at one-tenth the rate of hydrolysis of the beta-galactoside. Antiserum to the purified enzyme precipitated the major portion of beta-galactosidase activity of mouse liver, brain, and kidney. This antiserum cross-reacts with beta-galactosidases from rat and Chinese hamster, but not with human, porcine, or bovine beta-galactosidase.

Animals↗

PubMed1964

EFFECT OF THE TEMPERATURE OF GROWTH OF ESCHERICHIA COLI ON THE FORMATION OF BETA-GALACTOSIDASE.

Marr, Allen G. (University of California, Davis), John L. Ingraham, and Craig L. Squires. Effect of the temperature of growth of Escherichia coli on the formation of beta-galactosidase. J. Bacteriol. 87:356-362. 1964.-The synthesis of beta-galactosidase was measured during exponential growth of Escherichia coli in a succinate-minimal medium over a temperature range of 10 to 43 C for the following: (i) a constitutive strain, and (ii) an inducible cryptic strain, induced maximally with isopropyl-thio-beta-d-galactopyranoside (IPTG), or induced submaximally with IPTG. The differential rates of synthesis of beta-galactosidase were identical for the constitutive strain and for the fully induced strain; the rates were constant from 20 to 43 C, and decreased progressively with a decrease in temperature below 20 C. Thus, in the absence of specific repression, the ability of E. coli to produce beta-galactosidase decreases at low temperature. The differential rate of the submaximally induced culture was minimal between 20 and 30 C, and increased progressively with temperature both above 30 C and below 20 C. That the repressor concentration is maximal at 20 C was established by measuring the rate of induced synthesis of beta-galactosidase as a function of the concentration of IPTG; the relative concentrations of repressor were 1.00:3.28:0.25 at 40, 20, and 10 C, respectively. After an abrupt change in temperature, the differential rate of a submaximally induced culture changed gradually to the rate of the steady state, which is in agreement with the proposal that the effect of temperature is on the concentration of repressor and not on the equilibrium between repressor and its site of action. The effect of temperature on catabolic repression was determined by comparing the differential rate of synthesis of beta-galactosidase by a constitutive strain grown in succinate-minimal medium with the rate in glucose-minimal medium at various temperatures; the ratio of the rates in the two media decreased progressively and approached 2.0 as the temperature of growth was increased.

Cold Temperature↗

PubMed1978

Lactosyl ceramidosis: deficient activity of neutral beta-galactosidase in liver and cultivated fibroblasts?

Neutral beta-galactosidase was partially purified from liver of normal controls, a patient with Niemann-Pick disease type A and the previously described patient with lactosyl ceramidosis using Concanavalin A-Sepharose adsorption and Sephadex G-100 gel filtration. The partially purified fractions were essentially free of galactosyl ceramide beta-galactosidase and GM1 beta-galactosidase activities. The normal and Niemann-Pick fractions were found to hydrolyze lactosyl ceramide, in the presence of sodium taurodeoxycholate, at a pH optimum of 5.6 as well as aryl beta-galactosides and aryl beta-glucosides at pH 6.2. The corresponding fraction from the lactosyl ceramidosis liver contained only 1--4% of the normal activity towards artificial substrates and lactosyl ceramide. Cross-reacting material identical to the normal was demonstrated in this fraction with antiserum raised against purified neutral beta-galactosidase, but no activity was observed in the precipitin line when stained with naphthol AS-LC-beta-galactoside or naphthol AS-LC-beta-glucoside. A similar deficiency of neutral beta-galactosidase activity was demonstrated in cultivated fibroblasts of the patient with lactosyl ceramidosis. Following adsorption on Concanavalin A-Sepharose and anti-GM1 beta-galactosidase antibody-Sepharose conjugates and chromatography on DEAE cellulose, fibroblast lysates from the patient exhibited 3% of normal activity towards 4-methyl-umbelliferyl beta-glucoside at pH 6.2 and 12% of normal activity towards lactosyl ceramide at pH 5.6. These data suggest that neutral beta-galactosidase may have an in vivo role in the cleavage of lactosyl ceramide and that a deficiency of this activity may be related to the lactosyl ceramide accumulation observed in the patient with lactosyl ceramidosis.

Cells, Cultured↗

PubMed2002

Pectin-bound beta-galactosidase present in cell walls of carrot cells under the different calcium status.

Glycosyl-hydrolytic enzymes from suspension-cultured carrot (Daucus carota L. cv. Kintoki) cells grown in calcium (Ca2+)-deficient and normal liquid media were studied after extraction successively by K-phosphate (pH 7.0) and Na-acetate (pH 5.2) containing 3 M LiCl. The same activities were detected in two protein fractions from control and Ca2+-deprived cells. The specific activities of alpha-galactosidase and polygalacturonase decreased under Ca2+ deprivation, but beta-galactosidase activity in the buffer-soluble protein from Ca2+-deprived cells increased 1.7-fold compared to control cells. Upon ion exchange and size-exclusion chromatography the fraction (Ca-Ia-I) in the buffer-soluble protein from Ca2+-deprived cells represented beta-galactosidase activity associated with a galacturonic acid-rich polysaccharide peak, whereas the corresponding fraction could hardly be detected in the buffer-soluble protein from control cells. Several of the same glycosidase activities were detected in the extract solubilized with cyclohexane-trans-1,2-diaminetetra-acetate (CDTA) from active cell walls of Ca2+-deprived cells as in the extract of control cells, but the beta-galactosidase activity was considerably reduced under Ca2+ deprivation. Following the same chromatography the fraction (CDTA-Ca-1) of beta-galactosidase activity in the extract solubilized with CDTA from active cell walls of Ca2+-deprived cells was also completely overlapping with the peak of galacturonic acid-rich polysaccharide. The molecular mass of fractions Ca-Ia-I and CDTA-Ca-1 was 300 kDa, and the polysaccharides in these two fractions were composed of approximately equal amounts of rhamnosyl and galacturonosyl residues. These results suggest that the increase of beta-galactosidase in the buffer-soluble protein fraction from Ca2+-deprived cells is the result of solubilization of a part of the acidic pectic polymer-bound beta-galactosidase due to the structural changes in the cell walls that occur during Ca2+ deprivation.

Journal Article↗

PubMed1980

Legume: alpha-GALACTOSIDASE FORMS DEVOID OF HEMAGGLUTININ ACTIVITY.

Twenty different legume species (20 genera) were examined for alpha-galactosidase and hemagglutinin activities. Although all of the species contained enzyme activity, only 13 of 20 contained hemagglutinin activities and none displayed a hemagglutinin activity comparable to the previously described alpha-galactosidase-hemagglutinins.The alpha-galactosidase activities in the 20 species possessed remarkably similar kinetic behavior and carbohydrate specificities. All were inhibited by galactose, xylose, and inositol (very similar K(i) values from plant to plant) and had very similar K(m) values for the substrate, p-nitrophenyl alpha-galactoside.Gel filtration analysis of extracts from nine species suggests that legume alpha-galactosidase activities may frequently reside in two molecular weight forms. However, all these species contained a large molecular weight enzyme activity with a size comparable to the alpha-galactosidase-hemagglutinins.Immunochemical studies reveal that the alpha-galactosidases in these plants are immunologically related to an alpha-galactosidase-hemagglutinin and, therefore, are related to one another.These studies suggest that each of the legume species studied (and perhaps all members of this plant family) contain a homologue from a specific class of alpha-galactosidase. Although the previously described alpha-galactosidase-hemagglutinins appear to be members from this enzyme class, these proteins most frequently occur as forms devoid of hemagglutinin activity.

Journal Article↗

PubMed1984

Substrate specificity and distribution of acid beta-galactosidase activities in seizure-susceptible and non-susceptible strains of mice.

The properties and distribution of beta-galactosidase were studied in the mouse brain using the artificial substrate methylumbelliferyl-beta-galactoside. Enzyme activities were compared between an audiogenic seizure-susceptible mouse strain (DBA/2) and three non-susceptible strains of mice (BALB/c, C3H/He and Swiss A2G). At all ages, DBA/2 mice have significantly lower beta-galactosidase activity compared with the three other mouse strains: this is attributed to the different alleles present at the Bgs locus. The low activity of beta-galactosidase is also evident when the natural substrate GMl-ganglioside is hydrolyzed. In contrast to this low GMl-ganglioside-beta-galactosidase activity, there is no difference in the activity of the second form of acid beta-galactosidase, galactosylceramidase, in DBA/2 mice at 7 and 14 days. However, at 21 and 28 days the activity is significantly lower in DBA/2 mice compared with the other strains of mice. These results on beta-galactosidase activity in the brain of seizure-susceptible and non-susceptible mice are discussed in relation to published levels of GMl-ganglioside and galactosylceramide present in the developing mouse brain.

Acoustic Stimulation↗

PubMed1980

Fibroblast alpha-galactosidase A activity for identification of Fabry's disease heterozygotes.

The identification of female carriers of Fabry's disease is important for genetic counselling since prenatal diagnosis of affected fetuses is possible. The activities of either total alpha-galactosidase or alpha-galactosidase A in cultured fibroblasts were similar in Fabry carriers and controls and cannot therefore be used for carrier detection. Better discrimination between carriers and controls was found when total alpha-galactosidase activity was expressed as a ratio to beta-galactosidase activity, but overlap still occurred. However, there was complete discrimination between the ratio of alpha-galactosidase A to beta-galactosidase in cultured fibroblasts from five carriers of Fabry's disease and either 11 controls, seven hemizygote affected males or two of their female relatives.

Adult↗

PubMed1987

Hydrolysis of galactosylsphingosine and lactosylsphingosine by beta-galactosidases in human brain and cultured fibroblasts.

Enzymatic properties of beta-galactosidases with galactosylsphingosine (psychosine) and lactosylsphingosine as the substrates were examined. Although bile salts were stimulatory on the hydrolysis of the glycolipids in normal brain and cultured fibroblasts, the hydrolytic activities could be readily assayed, without detergents. The in vitro hydrolysis of lactosylsphingosine in cultured fibroblast homogenates was catalyzed by two enzymes, as is the case with the hydrolysis of galactosylceramide and lactosylceramide. Lactosylsphingosine beta-galactosidase activities assayed in the absence and the presence of taurocholate (probably lactosylceramidase I) were deficient in fibroblasts from patients with globoid cell leukodystrophy, while the activity assayed with sodium cholate (probably lactosylceramidase II) was deficient in GM1 gangliosidosis fibroblasts. In contrast, galactosylsphingosine beta-galactosidase was not activated by cholate and the enzyme activities assayed with the no-additive and taurocholate systems were deficient in brain and fibroblasts from patients with globoid cell leukodystrophy, thereby indicating that the hydrolysis of galactosylsphingosine is catalyzed by one enzyme, galactosylceramidase I. Exogenous lipids and an activator protein purified from normal spleen activated galactosylsphingosine beta-galactosidase but they were inhibitory to lactosylsphingosine beta-galactosidase. Because the Km values of lactosylsphingosine beta-galactosidase assayed with cholate were several magnitude higher than those obtained with the no-additive system and because lactosylsphingosine is readily hydrolyzed with the no-additive system in vitro, it is likely that the in vivo hydrolysis of the lipid is catalyzed by only one enzyme, lactosylceramidase I.

Bile Acids and Salts↗