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PubMed1987

Efficacy testing of beta-galactosidase with H2 breath test in patients with carbohydrate malabsorption.

The clinical efficacy and the potential side-effects of beta-galactosidase were studied in adult lactose intolerance. Various randomized oral tolerance tests were performed using lactose solution (35 g), glucose + galactose solution (17.5 + 17.5 g), native, skimmed milk and milk pretreated with beta-galactosidase. In each case, simultaneous examinations were made of the glucose concentration of capillary blood by an instrument constructed by the authors, of the H2 content of expired air as also of the subjective complaints and of the number of stools and their pH. It was established that pretreatment of milk with beta-galactosidase has a beneficial effect in adult lactose maldigestion, since it stops dyspeptic complaints and diarrhoea due to milk, it reduces the H2 content of expired air increases blood glucose concentration. Measuring the H2 content of the breath by using and instrument constructed by the authors, exact data can be obtained noninvasively, and rapidly on the degree of carbohydrate malabsorption in patients with lactose-intolerance.

Adult↗
PubMed1988

Effect of swainsonine on the processing and turnover of lysosomal beta-galactosidase and beta-glucuronidase from mouse peritoneal macrophages.

The effect of swainsonine, an inhibitor of Golgi alpha-mannosidase II and lysosomal alpha-mannosidase, on the synthesis, processing, and turnover of two glycoproteins, lysosomal beta-galactosidase and lysosomal beta-glucuronidase, has been studied in cultured mouse peritoneal macrophages. No effect of the inhibitor on the relative rates of synthesis of the precursor form of either enzyme was observed. On the other hand, carbohydrate processing of beta-galactosidase and beta-glucuronidase was markedly altered by swainsonine, consistent with a blockage by the inhibitor of the removal of the alpha-1,3- and alpha-1,6-linked mannose residues which occurs in normal processing. In homogenates of both normal and swainsonine-treated cells, the precursor forms of the enzymes were found exclusively in the light membrane fraction on Percoll gradients and the mature forms exclusively in the lysosomal fractions indicating that translocation from Golgi to lysosomes and proteolytic processing in the lysosome were not impaired by the presence of abnormal oligosaccharide side chains. There was no detectable effect of swainsonine during a 4-day chase period on the total cellular turnover of these enzymes which involves two processes, secretion and degradation. In the absence of swainsonine, secretion represented about 40% of the total turnover of beta-galactosidase and about 50% with beta-glucuronidase. The presence of swainsonine increased these proportions to about 60 and 70%, respectively.

Alkaloids↗
PubMed1987

[Expression of beta-galactosidase in recombinant nonintegrated plasmids in evaluating the functional activity of vaccinia virus promoters].

The recombinant plasmids pVL1 and pVL2 were constructed for insertion and expression of alien genetic information in HindIII-F fragment of vaccinia virus DNA under the control of the strong early-late promoter of the protein 7.5. The late promoter of the main late protein 11K of vaccinia virus was cloned. These as well as other vector plasmids have been used to express the procaryotic beta-galactosidase gene. Functional activity of the genetic engineering constructions was estimated by transitory expression of beta-galactosidase after plasmid DNA transfection into the chicken fibroblasts embryo culture infected with vaccinia virus. The promoters of the genes for 7.5K and 11K proteins permitted the high level of beta-galactosidase expression. Using of the early promoter of the central part of HindIII-F fragment DNA from vaccinia virus was less efficient for expression of the enzyme.

Galactosidases↗
PubMed1988

[Expression in Escherichia coli of the human leukocyte interferon alpha2 gene fused with N-terminal fragment of beta-galactosidase].

Genes for leucocyte interferon and alpha-donor of galactosidase were fused by deletion mutagenesis or by site-directed mutagenesis. In both cases the fused protein was expressed. The protein having an antiviral activity of leucocyte interferon was easily detected in bacteria and solutions by the reaction of beta-galactosidase alpha-complementation and retained the antigenic determinants of interferon and beta-galactosidase. The use of fused proteins for optimization of gene expression and for the analysis of interferon structure-function relationship is discussed.

Cloning, Molecular↗
PubMed1988

GM1 ganglioside beta-galactosidases from bovine liver.

Two GM1 ganglioside beta-galactosidases, multimeric form (enzyme I) and monomeric form (enzyme IV), have been purified from bovine liver by the procedures comprising Sephadex G-100 gel filtration, affinity chromatographies on Concanavalin A (Con A)-Sepharose and p-aminophenyl thio-beta-galactoside-CH-Sepharose (PATG-Sepharose) and Sephadex G-200 gel filtration. The multimeric form of the enzyme was purified 13,000-fold and monomeric form was 68,700-fold. On sodium dodecyl sulfate poly acrylamide gel electrophoresis, the monomeric form of the enzyme gave a single protein band with a molecular weight of 65,000, while the multimeric form gave two minor protein bands with molecular weights of 32,000 and 20,000 in addition to the major band at 65,000. Both enzymes liberated the terminal galactose from GM1 ganglioside and lactosylceramide. Enzyme I showed a broad pH optimum between pH 4.3 and 5.0, while enzyme IV was most active at pH 4.75. The pI values of beta-galactosidases I and IV were 4.6 and 5.8, respectively. Both enzymes were quite stable upon preincubation at 45 degrees C under acidic condition (pH 4.5), but rapidly lost their activities under neutral condition (pH 7.0). The apparent Km values for GM1 ganglioside of beta-galactosidases I and IV were calculated to be 2.0 x 10(-4) M and 3.3 x 10(-4) M, respectively.

Animals↗
PubMed1977

The presence and distribution of beta-galactosidases in the small intestinal mucosa of the neonatal dog.

The beta-galactosidase activity of the small intestinal mucosa of the neonatal dog has been studied over the pH range 3-0 - 5-8 using different substrates and in the presence and absence of p-chloromercuribenzoate. Partial separation of two beta-galactosidases has been achieved by gel filtration. The results suggest that three beta-galactosidases, comparable with those in other mammals, exist in canine small intestinal mucosa.

Animals↗
PubMed1978

Conformational adaptability of the active site of beta-galactosidase. Interaction of the enzyme with some substrate analogous effectors.

The action of different effectors, glycosides, and alcohols on the reactions catalyzed by beta-galactosidase is analyzed in this paper. Effectors as large as tri- and tetrasaccharides have no effect on the enzyme activity, suggesting that the binding site has rather small size. Most of the beta-galactosides produce a competitive inhibition. The other compounds assayed behave either as noncompetitive inhibitors, and they are deadened inhibitors, or as uncompetitive inhibitors which exhibit a better affinity for the chemical intermediate than for free enzyme; nearly all of them give transfer products. The analysis of the data indicates that the active center of beta-galactosidase is made up of two subsites: a galactose and a glucose subsite. This latter site is in a more favorable conformation in the galactosylenzyme than in free enzyme; possibly it might even by generated by the galactose binding. Conformational rearrangements of the active center deduced from the inhibition data have been directly observed by differential spectroscopy. The conformational adaptability of the enzyme and its consequence for the functional properties of beta-galactosidase are discussed.

Binding Sites↗
PubMed1981

Limited proteolysis. Early steps in the processing of large premature termination fragments of beta-galactosidase in Escherichia coli.

The mechanism by which large premature termination fragments of beta-galactosidase were degraded in Escherichia coli was studied using quantitative immunoprecipitation techniques. Two different lacZ nonsense mutants which produced apparent primary translation products of 96,000 and 109,000 daltons, respectively, were both shown to produce a second beta-galactosidase-related polypeptide of Mr = 90,000. These 90,000-dalton polypeptides appeared to be the same in both strains since they co-migrated when analyzed as a mixture on sodium dodecyl sulfate-polyacrylamide gels and were indistinguishable when analyzed by one-dimensional peptide mapping. Pulse-chase experiments established a stoichiometric precursor-product relationship between the primary mutant gene products (called the A polypeptides) and the common 90,000-dalton polypeptide (called the B polypeptide). No intermediates were detected between the A and B polypeptides. We propose that there is a common pathway for the degradation of these different large fragments of beta-galactosidase. According to this model, the first step would be a specific endoproteolytic cleavage of the primary translation product which produces the 90,000-dalton polypeptide as a common intermediate. The kinetic analysis demonstrated a first order decay of both A and B polypeptides but, surprisingly, the first order rate constant for the decay of A appeared dependent upon the induction regimen. This result suggested that degradation may possibly be autoregulated either by the intracellular level of A or by other intermediates in the degradation pathway.

Antigen-Antibody Complex↗
PubMedDate not supplied

[Alpha-galactosidase producers among yeast cultures].

The capacity for biosynthesis of alpha-galactosidase was examined in 89 yeast cultures belonging to 21 genera during submerged cultivation on a medium containing dry whey. This capacity was found only in one genus, namely Schwanniomyces. Optimal cultivation conditions were selected for the strain Schw. alluvius 1167 that showed the highest alpha-galactosidase activity. As a result, activity of alpha-galactosidase was increased 4.8 times as compared with the initial level.

Galactosidases↗
PubMed1980

Enzyme therapy XVII: metabolic and immunologic evaluation of alpha- galactosidase A replacement in Fabry disease.

A pilot trial of enzyme replacement using splenic and plasma forms of alpha-galactosidase A was undertaken in 2 brothers with Fabry disease, an X-linked glycosphingolipid storage disease. Partially purified preparations of alpha-galactosidase A from human spleen and plasma Cohn fraction IV-1 were prepared aseptically for in vivo administration. The disappearance of enzymatic activity from plasma, levels of circulating substrate, and potential immune response were evaluated following IV administration of 6 unentrapped doses (2,000 U/kg) of each enzyme form to the respective recipient during a 117-day period. Repeated injections were well tolerated. The circulating half-life of the splenic form was about 10 min whereas that for the plasma form was approximately 70 min. No immune response was detected by skin and immunodiffusion tests or by alterations in the maximal activity or clearance kinetics for either enzyme following successive administrations. After each dose of the splenic form, the concentration of the accumulated circulating substrate globotriaosylceramide, decreased maximally (approximately 50% of initial values) in 15 min and returned to preinfusion levels by 2-3 hr. In marked contrast, injection of the plasma form decreased the circulating substrate levels 50-70% by 2-6 hr; the concentrations of globotriaosylceramide gradually returned to preinfusion values by 36-72 hr. Two consecutive doses of the plasma form, administered on days 1 and 3, reduced the circulating substrate concentration to normal levels. Prior to the 6th enzyme administration, circulating substrate was stable-isotope labeled by the infusion of dideutero-glucose, and the effects of each enzyme form on circulating substrate degradation and reaccumulation were determined. The results of this study indicated that labeled (newly synthesized) substrate reaccumulated following injection of the splenic enzyme whereas both unlabeled (previously stored?) and labeled substrate reaccumulated in the circulation after administration of the plasma form. These studies demonstrated the differential disappearance kinetics of the splenic and plasma forms of alpha-galactosidase A, their differential effects on circulating substrate degradation and reaccumulation, as well as the lack of an immune response to repeated administrations of these homologous, unentrapped enzymes.

Adult↗
PubMed1981

Affinity purification of alpha-galactosidase A from human spleen, placenta, and plasma with elimination of pyrogen contamination. Properties of the purified splenic enzyme compared to other forms.

The substrate analog alpha-D-galactosylamine was synthesized, linked to 6-aminohexanoic acid, and coupled to carboxyhexyl-Sepharose. This affinity support permitted the purification of human alpha-galactosidase A (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) from spleen, placenta, and plasma. When used in conjunction with conventional procedures, affinity chromatography enabled the rapid and specific purification of alpha-galactosidase A from each source. Significantly, pyrogenic endotoxins were eliminated from enzyme preparations by the use of the affinity column. Splenic alpha-galactosidase A was purified in high yield (38%) with a specific activity of 1.9 X 10(6) units/mg. The purified enzyme was a homodimer with a native molecular weight of 101,000 and a subunit weight of 49,800. The UV absorption coefficient was E280 1% = 18 and the lambda max was 282 nm. The plasma form was purified with a markedly improved yield to a specific activity (229,000 units/mg) which was 3 times greater than that achieved previously. The enzymes from plasma, spleen, and placenta were immunologically identical. The physical and kinetic properties of the purified enzymes were consistent with and confirmed previous findings.

Carbohydrates↗
PubMed1982

Pseudodeficiency of alpha-galactosidase A.

Apparent deficiency of alpha-galactosidase A was observed in a 51-year-old, clinically healthy male, with no clinical symptoms of Fabry disease, and without excess urinary excretion of ceramide trihexoside. The deficiency, which was similar to that found in Fabry disease patients, could be demonstrated using both synthetic and natural substrates. This pseudodeficiency was transmitted in his family by classical X-linked inheritance. His wife showed enzyme activity in the normal range, two daughters were heterozygotes for this mutation as demonstrated by hair root assay, and three sons showed normal alpha-galactosidase activity. Kinetic studies in cultured skin fibroblasts indicated a five-fold increase in the apparent Km and a greater heat stability of the residual alpha-galactosidase activity when compared to controls. These data indicate that the residual enzyme activity in this mutation behaves similarly to that observed in Fabry disease patients but does not cause any clinical abnormalities.

Adolescent↗
PubMedDate not supplied

[Microbial alpha-galactosidase (a review)].

The review discusses properties, distribution and potential use of microbial alpha-galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22), the enzyme catalyzing degradation of alpha-D-galactoside bonds. Recent years have witnessed many publications describing microbial alpha-galactosidase which, in contrast to the similar enzyme from higher plants, has been poorly studied. The microbial enzyme has certain specificities: a smaller substrate specificity, existence in one molecular form, etc. The present communication is an attempt to systematize the data about microbial alpha-galactosidase and to outline the most important investigations for the future.

Animals↗
PubMed1983

Characterization of a glycoprotein alpha-galactosidase from lentil seeds (Lens culinaris).

alpha-Galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22), an enzyme responsible for mobilizing the raffinose family of oligosaccharides in legume seeds, has been isolated from lentils (Lens culinaris) and purified about 4,000-fold. The Sephadex gel filtration profile showed the presence of two forms of the enzyme, alpha-galactosidase I with an apparent Mr = 160,000 and alpha-galactosidase II of Mr = 40,000. Enzyme II readily aggregates to form I when any attempt is made to concentrate the solution. Thus, only enzyme I was purified and its properties studied. The multistep purification procedure included affinity binding of the enzyme to concanavalin A-Sepharose, indicating its glycoprotein nature with glucose/mannose termini of the carbohydrate moieties. The amino acid and carbohydrate compositions of the native enzyme and that of the glycopeptide obtained from pronase-digested, denatured enzyme have been determined. Asparagine seems to be involved in forming the linkage with the carbohydrate moiety. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of enzyme I shows a single protein band with Mr = 40,000 which also stains with periodic acid-Schiff reagent. Thus, the enzyme consists of four identical glycoprotein subunits. The isoelectric point of the enzyme is 8.0. The pH optima of enzyme I and II are 6.1 and 4.7, respectively. The substrate specificity and the mode of substrate inhibition of enzyme I is discussed. The effect of temperature on Vmax and Km of the enzyme is presented; at pH 6.1 the energy of activation is 62.1 kJ/mol and the delta H value is -34.3 kJ/mol in the temperature range 20-50 degrees C. Preliminary studies show that enzyme I possesses hemagglutinating properties with glucose/mannose specificity.

Fabaceae↗
PubMed1984

Endo-beta-D-galactosidases of Bacteroides fragilis and Escherichia freundii hydrolyze linear but not branched oligosaccharide domains of glycolipids of the neolacto series.

The specificities of the endo-beta-galactosidases of Bacteroides fragilis and Escherichia freundii towards linear and branched oligosaccharides of the lacto-N-glycosyl series were investigated using as substrates glycolipids containing (a) linear neolactotetra - or hexaosyl sequences, (b) branched biantennary neolactooctaosyl sequences, and (c) triantennary neolactononaor dodecaglycosyl sequences. Glycolipid and oligosaccharide hydrolysis products were identified by tlc and/or paper chromatography. The rate of hydrolysis was assessed in time course experiments in which the oligosaccharides released were quantified as 3H-labeled alditols. The salient observations were as follows. (i) With the substrates thus far tested in the present and a previous study ( Scudder , P., Uemura , K., Dolby , J., Fukuda, M.N., and Feizi , T. (1983) Biochem. J. 213, 485-494), the endo-beta-galactosidases from B. fragilis and E. freudii have indistinguishable specificities. (ii) The beta-galactosidic linkage of the branch point sequence (Formula: see text) is completely resistant to hydrolysis by these enzymes, although the unbranched sequence GlcNAc beta 1-3Gal beta 1-4GlcNAc/Glc is readily cleaved. (iii) At an optimal concentration of detergent, the endo-beta-galactosidase susceptibility of the GlcNAc beta 1-3Gal beta 1-4Glc sequence near the ceramide moiety of branched glycolipids is similar to that of the corresponding sequence in linear glycolipids.

Bacteroides fragilis↗
PubMed1981

Purification and characterization of endo-beta-galactosidase from Escherichia freundii induced by hog gastric mucin.

A new procedure for inducing and purifying endo-beta-galactosidase from Escherichia freundii was described. The enzyme was found to be induced with high efficiency in culture medium containing Smith-degraded hog gastric mucin, which was prepared from a commercially available starting material. Endo-beta-galactosidase was then purified by ammonium sulfate fractionation, DEAE-Sephadex chromatography, and affinity chromatography on Sepharose conjugated with the Smith-degraded mucin. The enzyme thus purified by only three steps showed no other glycosidase or protease activities and had higher specific activity compared to the previous method. This new method has a great advantage since the gastric mucin is abundantly available and the efficiency of enzyme production was high without significant induction of exoglycosidase. The hydrolysis of oligosaccharides, glycosphingolipid, and keratansulfate was studied by using this newly purified enzyme. Kinetic data indicate that hydrolyzability of these substrates is largely affected by substrate concentration, enzyme concentration and the structure of substrates. Based on these results, the specificity of E. freundii endo-beta-galactosidase was discussed.

Animals↗
PubMed1981

Cell-free expression of the beta-galactosidase gene: a model system to study the effects of nucleotide analogs on transcription-translation.

A cell-free system for the expression of the beta-galactosidase gene was employed to study the effects of the UTP and CTP analogs: s2UTP, s2CTP, f5UTP and rTTP on transcription-translation. From the analogs investigated, only rTTP turned out to be able to substitute UTP in the cell-free synthesis of beta-galactosidase. In case of the other analogs listed above, the incorporation of even a small fraction of analog into rRNA resulted in drastic inhibition of beta-galactosidase synthesis.

Escherichia coli↗
PubMed1982

Mapping of aminoacylase-1 and beta-galactosidase-A to homologous regions of human chromosome 3 and mouse chromosome 9 suggests location of additional genes.

Conserved linkage groups have been found on the X and autosomal chromosomes in several mammalian species. The identification of conserved chromosomal regions has potential for predicting gene location in mammals, particularly in humans. The genes for human aminoacylase-1 (ACY1, N-acylamino acid aminohydrolase, E.C.3.5.1.14), an enzyme in amino acid metabolism, and beta-galactosidase-A (GLB1, E.C.3.2.1.23), deficient in GM1-gangliosidosis, have been assigned to human chromosome 3. Using human-mouse somatic cell hybrids segregating translocations of human chromosome 3, expression of both ACY1 and GLB1 correlated with the presence of the p21 leads to q21 region of chromosome 3. In a previous study, assignment of these genes to mouse chromosome 9 used mouse-Chinese hamster somatic cell hybrids, eliminating mouse chromosomes. To approximate the size of the conserved region in the mouse, experiments were performed with recombinant inbred mouse strains. An electrophoretic variant of ACY-1 in mouse strains was used to map the Acy-1 gene 10.7 map U from the beta-galactosidase locus. These data suggest that there is a region of homology within the p21 leads to q21 region of human chromosome 3 and a segment of mouse chromosome 9. Since the mouse transferrin gene (Trf) is closely linked to the aminoacylase and beta-galactosidase loci, we predict that the human transferrin (TF) gene is on chromosome 3.

Amidohydrolases↗