Activities of brush border lactase, acid -galactosidase, and hetero- -galactosidase in the jejunum of the Zambian African.
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Adolescent↗
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Beta-galactosidase activity of the jejunum and ileum of suckling rats. Comparison of activities of beta-galactosidase at different concentration of substrates (o-nitrophenyl-beta-D-galactoside and lactose) at different pH.
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Animals↗
Enzymological properties and immunological characterization of alpha-galactosidase isoenzymes from normal and Fabry human liver.
1. A method is described for the rapid isolation of alpha-galactosidases A and B (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) from normal human liver. 2. When the same method is applied to Fabry liver, most of the alpha-galactosidase activity is recovered in the fraction corresponding to normal alpha-galactosidase B. In agreement with Romeo, G., D'Urso, M., Pisacane, A., Blum, E., De Falco, A. and Ruffilli, A. (1975) Biochem. Genet. 13, 615-628) [18], a small amount of alpha-galactosidase activity is found in the fraction corresponding to normal alpha-galactosidase A. 3. The kinetic properties of the B-like activity from Fabry liver are similar to those of normal alpha-galactosidase B. In agreement with Romeo et al. [18], it was found that the kinetic properties of the A-like activity from Fabry liver are similar to those of normal alpha-galactosidase A. 4. Using antisera raised against normal alpha-galactosidase A and normal alpha-galactosidase B, it is shown that the normal alpha-galactosidase isoenzymes are immunologically distinct and that the B-like activity from Fabry liver is immunologically related to normal alpha-galactosidase B. Furthermore, the A-like activity from Fabry liver is immunologically related to normal alpha-galactosidase B and not to normal alpha-galactosidase A. 5. Normal alpha-galactosidase B is converted into an A-like form during storage. 6. It is concluded that the B-like alpha-galactosidase in Fabry tissues is identical to normal alpha-galactosidase B, and that the small amount of A-like activity found in Fabry material is due to a modified form of alpha-galactosidase B.
Animals↗
Properties of the residual alpha-galactosidase activity in the tissues of a Fabry hemizygote.
The properties of the residual alpha-galactosidase activity in kidney, liver, spleen, fibroblasts and urine of a Fabry hemizygote have been studied using p-nitrophenyl-alpha-galactoside and 4-methylumbelliferyl-alpha-galactoside as substrates. In addition, alpha-galactosidase activity in urine has been determined with ceramidetrihexoside as substrate. The residual alpha-galactosidase activity of Fabry, measured with artificial substrate, is stimulated (6-35%) by myo-inositol and only slightly inhibited by melibiose (7-17%) in all the materials used. In contrast, the alpha-galactosidase of normal tissues and urine is inhibited (36-48%) by myo-inositol and inhibited to a much greater extent (40-50%) by melibiose. The KM for artificial substrate of the residual activity of Fabry is higher than that of the alpha-galactosidase in normal kidney, liver, spleen, fibroblasts and urine. The residual activity of Fabry is generally more stable to heating than the activity in the normal materials, although exceptions were noted. When these properties are compared with those of the alpha-galactosidase isoenzymes in normal tissues and body fluids, the residual activity of Fabry material seems to be very similar to the minor component of normal tissue (alpha-galactosidase B). Moreover, the pH optimum curve of this minor component and of the Fabry alpha-galactosidase in urine are similar, whereas the major isoenzyme (alpha-galactosidase A) shows a curve much more like that of normal urine. The findings with ceramidetrihexoside as substrate indicate a possible discrepancy. Alpha-Galactosidase A hydrolyses ceramidetrihexoside, Fabry urine preparation does not. However, alpha-galactosidase B of normal urine shows a slight but definite ceramidetrihexosidase activity. No contamination of the B preparation with alpha-galactosidase A could be detected. The minimum hypothesis, supported by most of the experimental evidence, is that the residual activity of Fabry and normal alpha-galactosidase B are identical.
Disaccharides↗
Improvement of the nutritive value of soybean meal by protease and alpha-galactosidase treatment in broiler cockerels and broiler chicks.
1. Tube-fed broiler cockerels were used in three experiments to measure the effect of different enzyme treatments on true metabolisable energy (TME) and true nitrogen digestibility (TND) of commercial solvent-extracted, heat-treated soybean meal (SBM). 2. In experiment 1, proteases P2 and P3 (from Aspergillus niger) and alpha-galactosidase (from A. oryzae) improved TME and TND while protease P1 (from Bacillus subtilis) had little effect. The effects of enzyme treatment were similar whether treatment was applied by pre-incubation of enzymes (proteases P1, P2 and P3, 1.0 g/kg; alpha-galactosidase, 0.1 g/kg) with SBM for 2 h at 50 degrees C or by simple mixing of enzymes (proteases P1, P2 and P3, 0.25 g/kg; alpha-galactosidase, 0.1 g/kg) with SBM prior to feeding. 3. In experiment 2, the effects of mixing SBM with each of protease P1 (0 or 0.25 g/kg), protease P3 (0 or 0.25 g/kg) or alpha-galactosidase (0 or 0.1 g/kg) alone or in all possible combinations were studied. Effects of protease P1 were limited, but protease P3 and alpha-galactosidase improved TME and TND. There were significant interactions between protease P3 and alpha-galactosidase for both TME and TND; the response when enzymes were applied together was less than that expected from application of the enzyme preparations individually. 4. In experiment 3, the effect of varying the concentration of protease P3 (0, 0.1, 0.25 and 1.0 g/kg SBM dry matter) and alpha-galactosidase (0, 0.025, 0.0625 and 0.25 g/kg SBM dry matter) mixed with SBM alone or in all possible combinations of protease P3 and alpha-galactosidase were assessed. Increases in TME and TND for both enzymes were non-linear with the greatest response occurring at the lowest concentration of each enzyme. There were also interactions between the two enzyme preparations. 5. Finally, either protease P3 (0, 0.1 and 1.0 g/kg SBM dry matter) or alpha-galactosidase (0, 0.025 and 0.25 g/kg SBM dry matter) were mixed with SBM alone or in all possible combinations and treated SBMs incorporated into semi-purified diets containing 450 g SBM/kg as the sole source of dietary N and offered to growing broiler chicks for 21 d. Similar patterns of increases in chick growth rate and diet digestibility to those recorded in experiment 3 were obtained when protease P3 and alpha-galactosidase where included in the diets. 6. It is concluded that responses measured by tube-feeding SBM treated with protease P3 and alpha-galactosidase were similar to responses obtained with growing broiler chicks. Further, these responses were obtained by simple addition of enzymes to diets and nutritive value of SBM was improved by both protease and alpha-galactosidase treatment.
Animal Feed↗
Beta-D-galactosidase of rat spermatozoa: subcellular distribution, substrate specificity, and molecular changes during epididymal maturation.
In previous studies, we reported that rat epididymal fluid acid beta-D-galactosidase, which optimally cleaves a synthetic substrate (PNP beta-D-galactoside) at pH 3.5, shows maximum activity at pH 6.8 when a glycoprotein is used as a substrate [Skudlarek MD, Tulsiani DRP, Orgebin-Crist M-C. Biochem J 1992; 286: 907-914]. We now describe a similar pH-dependent substrate preference for rat sperm beta-D-galactosidase. We found that only 10-14% of total beta-D-galactosidase (and other glycosidase) activity was associated with spermatozoa. The remaining enzyme activities were present in soluble form in the luminal fluid. When the glycosidase levels were expressed per 10(6) sperm, all enzymes showed a progressive increase in spermatozoa from the caput to the corpus or proximal cauda followed by a sharp decline in spermatozoa from the distal cauda epididymidis. The observed decrease in beta-D-galactosidase activity could not be explained by the loss of cytoplasmic droplets (which have a low enzyme activity relative to spermatozoa) or the presence of inhibitors/activators of the enzyme activity in spermatozoa from the proximal or distal epididymis. However, we found that the changes in beta-D-galactosidase activity during sperm maturation in the epididymis were accompanied by changes in the molecular form(s) of the enzyme. Western blot analysis using an antibody to beta-D-galactosidase showed a progressive processing of the 82-kDa immunoreactive band in caput spermatozoa to an 80-kDa diffuse band in cauda spermatozoa. The sperm-associated beta-D-galactosidase form(s) does not appear to be due to adsorption and/or binding of the luminal fluid beta-D-galactosidase, which contained a 97-kDa form in fluid from the caput and two forms, of 97 kDa and 84 kDa, in corpus and cauda fluids. The observed difference in the molecular forms of the sperm and luminal fluid was found to be due to differential glycosylation, since de-N-glycosylation of various forms of beta-D-galactosidase generated a single immunoreactive form of 70 kDa. Subcellular localization studies and assay for the beta-D-galactosidase activity in the enriched plasma membrane and acrosomal membrane fractions suggested the likelihood that the activity of beta-D-galactosidase and other glycosidases is present in the acrosome and is readily released during sperm disruption. The evidence suggests that sperm beta-D-galactosidase may be functional within the acidic environment of the acrosome during sperm maturation as well as in the neutral environment of the oviduct after the zona-induced acrosome reaction.
Animals↗
A Bacteroides ovatus chromosomal locus which contains an alpha-galactosidase gene may be important for colonization of the gastrointestinal tract.
An alpha-galactosidase gene has been cloned from the human colonic Bacteroides species Bacteroides ovatus 0038. This alpha-galactosidase appears to be distinct from two previously characterized alpha-galactosidases, I and II, from the same strain and has been designated alpha-galactosidase III. Partially purified alpha-galactosidase III from Escherichia coli EM24 containing pFG61 delta SE had a pI of 7.6, as compared with the reported pI values for the known alpha-galactosidases of 5.6 for I and 6.9 for II. Its molecular weight as estimated on sodium dodecyl sulfate-polyacrylamide gels was 78,000, whereas the molecular weights of alpha-galactosidases I and II were 85,000 and 80,500, respectively. The only substrate hydrolyzed by alpha-galactosidase III was melibiose, whereas the other two alpha-galactosidases were able to degrade melibiose, raffinose, and stachyose and partially degraded guar gum. alpha-Galactosidase III had a pH optimum of 6.7 to 7.2. Finally, a single crossover insertion which disrupted the gene in the B. ovatus chromosome had no effect on expression of alpha-galactosidases I and II. Although this insertion had no effect on the ability of B. ovatus to grow in laboratory medium on any of the galactoside-containing carbohydrates tested, the insertion mutant was outcompeted by wild type when a combination of mutant and wild type was used to colonize germfree mice. Insertions on either side of the gene had the same effect. Thus, the locus which contains alpha-galactosidase III may be important for colonization in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Two gene products for beta-galactosidase are differentially expressed in the mouse salivary glands.
The specific activity of GM1 ganglioside beta-galactosidase, also known as lysosomal or acidic beta-galactosidase, and the neutral beta-galactosidase were determined in the mouse three major salivary glands and compared to other tissues. Our data indicate that at pH 4.4, lysosomal beta-galactosidase activity in the submandibular gland and the sublingual gland of the mature male is the higher than in the parotid gland, kidney, and skeletal muscle. At pH 7.3, neutral beta-galactosidase activity is overall much lower and is higher in the submandibular gland compared to the sublingual and the parotid glands, kidney, and muscle. En bloc histochemical staining of tissues using x-gal as a substrate at pH 4.4 demonstrates high beta-galactosidase activity in all three salivary glands in comparison to skeletal muscle. At pH 7.3, the submandibular gland demonstrates higher activity, whereas the parotid appears negative and the sublingual gland demonstrates intermediate activity levels. En bloc staining using x-fucose (another substrate of lysosomal beta-galactosidase) demonstrates high activity in all three glands at pH 4.4, and no activity in any of the glands at pH 7.3. Microscopic histochemistry indicates that beta-galactosidase activity is localized to parenchymal cells. Thus, the two gene products for beta-galactosidase are differentially expressed in the salivary glands. These novel findings question the previous use of the bacterial beta-galactosidase (lacZ) as a reporter gene in the salivary glands. Endogenous beta-galactosidase activity in the salivary glands is probably related to glycoprotein metabolism, processing glycoconjugates containing a terminal beta-galactosidic linkage. Further studies of beta-galactosidase function and differential regulation in these tissues are needed.
Animals↗
Isolation and characterization of a new endo-beta-galactosidase from Diplococcus pneumoniae.
An endo-beta-galactosidase, which hydrolyzes the internal beta-galactosidic linkages of R----GlcNAc (or GalNAc) beta 1----3Gal beta 1----4GlcNAc (or Glc), was isolated from the culture supernatant of Diplococcus pneumoniae. The enzyme, named endo-beta-galactosidase DII, hydrolyzed linear N-acetyllactosamine repeating structures in glycolipids and glycopeptides to release oligosaccharides. The specificity of endo-beta-galactosidase DII is the same as that of Escherichia freundii endo-beta-galactosidase as far as described above, but the following differences between these two enzymes were found: Branched lactosaminyl glycolipids and H-antigenic glycolipids were resistant to endo-beta-galactosidase DII, even when linear structure was present at the inner part. Throughout the enzymic hydrolysis, endo-beta-galactosidase DII released mostly small oligosaccharides (tetra-, tri-, and disaccharides) from substrates, suggesting that the enzyme split off the oligosaccharides stepwise from the nonreducing terminal. Lactosaminoglycans were partially hydrolyzed by endo-beta-galactosidase DII to produce small oligosaccharides as the major product and residual glycopeptides. The residual glycopeptides were readily hydrolyzed by E. freundii endo-beta-galactosidase to produce various sizes of oligosaccharides. Keratan sulfate was not degraded by endo-beta-galactosidase DII. These properties of endo-beta-galactosidase DII characterize it as a new endo-beta-galactosidase with a unique specificity.
Anion Exchange Protein 1, Erythrocyte↗
Studies of the M15 beta-galactosidase complementation process.
M15 beta-Galactosidase was activated by heat-denatured wild-type beta-galactosidase, urea, and heat-denatured wild-type beta-galactosidase, a peptide made up of residues 6-44 of beta-galactosidase and CB2, the peptide that is normally used for complementation (residues 3-92 of beta-galactosidase). In each case roughly equal activation levels were attained. Heat-denatured wild-type beta-galactosidase was present as a finely divided visible white precipitate both before and after complementation. The heat-denatured protein by itself did not migrate on native PAGE and both the protein and the activity that occurred as a result of the complementation also remained at the point of application. The N-terminal ends of the heat-denatured wild-type beta-galactosidase must have been available for complementation and must have been mobile enough to allow tetramer to form despite being aggregated. Beta-galactosidase denatured by both urea and heat resulted in a streak of interacting protein on the native PAGE. Upon activation, a streak (indicating that interaction was still occurring) was still present, but it moves more slowly. Complementation using a peptide called XP (made up of residues 6-44 plus an additional nine C-terminal amino acids) resulted in three discrete forms of active enzyme at ratios of peptide to M15 beta-galactosidase monomer of less than 1:1. The fastest migrating of the three bands predominated at ratios near 1:1. A single active tetrameric form of M15 beta-galactosidase was formed with CB2. In both of these last two cases an active slow-moving diffuse band also formed (possibly a dimer of the tetramer). A quantitation of the amount of peptide bound to M15 beta-galactosidase by titration with XP and with CB2 and by using gel filtration after an excess of fluorescent-labeled XP was added showed that peptide bound in a 1:1 ratio (peptide/monomer) when full activity was achieved. These fluorescent studies also showed that peptide initially bound to dimer and that the tetramer was then formed.
Electrophoresis, Polyacrylamide Gel↗
Cloning, sequence, and expression of a blood group B active recombinant alpha-D-galactosidase from pinto bean (Phaseolus vulgaris).
A cDNA encoding pinto bean alpha-D-galactosidase [E.C. 3.2.1.22] was obtained by amplification of cDNA using highly conserved sequences found in eucaryotic alpha-D-galactosidases. Subsequently a full length Phaseolus cDNA clone was obtained that is 1537 nt long and contains untranslated 5' and 3' sequences. The nucleotide sequence of the cDNA has a high degree of homology with other eucaryotic alpha-D-galactosidase genes. The recombinant alpha-D-galactosidase (rGal) was expressed in Escherichia coli and purified by ion exchange and affinity chromatography. Purified rGal was homogeneous by SDS-PAGE and had relative masses of 40.1 and 45.4 kDa under nonreducing and reducing conditions, respectively. The N-terminal sequence of the expressed protein contained the sequence GNGLGQTPPMG corresponding to that deduced from the cDNA sequence. The native molecular weight for rGal was determined to be 32.18 kDa by Sephacryl S-200 chromatography. The specific activity of the rGal was 349 mu moles of PNP-alpha-D-galactopyranoside hydrolyzed per mg of pure rGal per min. rGal was highly specific for alpha-D-galactosyl residues and degraded B oligosaccharide. No detectable hemagglutinin or protease activity was present in the preparations. Furthermore, rGal was active against the blood group B antigen on native human erythrocytes in cell suspension assays. The only detectable RBC phenotypic change was loss of the B and P1 epitopes. Recombinant Phaseolus vulgaris alpha-D-galactosidase may have useful biotechnical applications in the potential mass production of enzymatically converted, universally transfusable type O RBCs. alpha-D-galactosidase [E.C. 3.2.1.22] has been purified from a variety of procaryotic and eucaryotic species. Most alpha-D-galactosidases have similar low molecular weight substrate specificities, but activity against high molecular weight substrates is variable. Terminal alpha-D-galactoside residues are present in glycoproteins and glycolipids. Some alpha-D-galactosidases have activity against alpha-D-galactosyl residues on cell membrane glycoconjugates. Glycosidases with this property are useful for carbohydrate structural studies and biotechnical applications. Enzymes free of other glycosidase activities with activity near neutral pH are particularly useful for membrane modification studies on native cells. Complex sugar chains in glycolipids and glycoproteins have often been implicated in the growth and development of eucaryotes. In particular, complex sugar chains play an important role in the recognition of self in the immune system. Some alpha-D-galactosidases can modify certain carbohydrate membrane epitopes, thereby modulating the immune response. For example, the blood group B epitope expressed on erythrocytes contains a terminal alpha-D-galactosyl residue. Individuals lacking this antigen produce naturally occurring complement fixing antibodies to the B epitope. Hydrolysis of this terminal saccharide destroys the antigenic activity of the B determinant producing H antigen (blood type O) on erythrocytes. Only rare individuals produce clinically significant antibodies to the H antigen, and therefore, type O red blood cells are "universally" compatible and in great demand. Dhar purified alpha-D-galactosidase isozymes from Phaseolus vulgaris and characterized their activity. To our knowledge, our laboratory, in a brief report, is the first to describe the cloning of the gene and the use of recombinant enzyme for seroconverting blood type B to O cells. This paper describes the cloning, sequence, expression, purification, and characterization of recombinant alpha-D-galactosidase. Activity of the recombinant enzyme on the native human erythrocyte blood group B epitope is shown.
ABO Blood-Group System↗
A beta-galactosidase gene is expressed during mature fruit abscission of 'Valencia' orange (Citrus sinensis).
beta-galactosidases have been detected in a wide range of plants and are characterized by their ability to hydrolyse terminal non-reducing beta-D-galactosyl residues from beta-D-galactosides. These enzymes have been detected in a wide range of plant organs and tissues. In a search for differentially expressed genes during the abscission process in citrus, sequences encoding beta-galactosidase were identified. Three cDNA fragments of a beta-galactosidase gene were isolated from a cDNA subtraction library constructed from mature fruit abscission zones 48 h after the application of a mature fruit-specific abscission agent, 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMN-pyrazole). Based on sequence information derived from these fragments, a full-length cDNA of 2847 nucleotides (GenBank accession number AY029198) encoding beta-galactosidase was isolated from mature fruit abscission zones by 5'- and 3'-RACE approaches. The beta-galactosidase cDNA encoded a protein of 737 amino acid residues with a calculated molecular weight of 82 kDa. The deduced protein was highly homologous to plant beta-galactosidases expressed in fruit ripening. Southern blot analysis demonstrated that at least two closely related beta-galactosidase genes were present in 'Valencia' orange. Temporal expression patterns in mature fruit abscission zones indicated beta-galactosidase mRNA was detected 48 h after treatment of CMN-pyrazole and ethephon in mature fruit abscission zones. beta-galactosidase transcripts were detected in leaf abscission zones only after ethephon application. The citrus beta-galactosidase was expressed in stamens and petals of fully opened flowers and young fruitlets. The results suggest that this beta-galactosidase may play a role during abscission as well as early growth and development processes in flowers and fruitlets.
Amino Acid Sequence↗
Intestinal lactase (beta-galactosidase) and other disaccharidase activities of suckling and adult common brushtail possums, Trichosurus vulpecula (Marsupialia:Phalangeridae).
Small-intestinal disaccharidase activities of eight suckling T. vulpecula, aged from 34 to 150 days, and of two adult animals were investigated. Intestinal maltase, isomaltase and sucrase activities increased with age, whereas lactase activities decreased. Trehalase activities were relatively high in all animals and showed no obvious age-related changes. Three separate beta-galactosidase activities, one neutral and two acid, acted on lactose. The neutral beta-galactosidase activity appeared to be due to a brush border enzyme similar to that of eutherian mammals, whereas the acid beta-galactosidases were soluble and probably of lysosomal origin. One of these, acid beta-galactosidase-1, had similar properties to the sole intestinal beta-galactosidase of macropodid marsupials, whereas the other, acid beta-galactosidase-2, has not previously been described. Galactosyl oligosaccharides isolated from macropodid milk were readily hydrolysed by both acid beta-galactosidases but not by the neutral beta-galactosidase. The total intestinal lactase activity in animals aged up to 125 days was due mainly to acid beta-galactosidase-1, whereas in older animals it was due mostly to the neutral beta-galactosidase; this suggests that late in lactation the young T. vulpecula change from a macropodid mode of digestion of galactosyl oligosaccharides to a eutherian mechanism for the digestion of lactose. These findings may have implications for the hand-rearing of orphaned T. vulpecula.
Animals↗
Purification and characterization of two alpha-galactosidases associated with catabolism of guar gum and other alpha-galactosides by Bacteroides ovatus.
When Bacteroides ovatus is grown on guar gum, a galactomannan, it produces alpha-galactosidase I which is different from alpha-galactosidase II which it produces when grown on galactose, melibiose, raffinose, or stachyose. We have purified both of these enzymes to apparent homogeneity. Both enzymes appear to be trimers and have similar pH optima (5.9 to 6.4 for alpha-galactosidase I, 6.3 to 6.5 for alpha-galactosidase II). However, alpha-galactosidase I has a pI of 5.6 and a monomeric molecular weight of 85,000, whereas alpha-galactosidase II has a pI of 6.9 and a monomeric molecular weight of 80,500. alpha-Galactosidase I has a lower affinity for melibiose, raffinose, and stachyose (Km values of 20.8, 98.1, and 8.5 mM, respectively) than does alpha-galactosidase II (Km values of 2.3, 5.9, and 0.3 mM, respectively). Neither enzyme was able to remove galactose residues from intact guar gum, but both were capable of removing galactose residues from guar gum which had been degraded into large fragments by mannanase. The increase in specific activity of alpha-galactosidase which was associated with growth on guar gum was due to an increase in the specific activity of enzyme I. Low, constitutive levels of enzyme II also were produced. By contrast, enzyme II was the only alpha-galactosidase that was detectable in bacteria which had been grown on galactose, melibiose, raffinose, or stachyose.
Bacteroides↗
Expression and nucleotide sequence of the Lactobacillus bulgaricus beta-galactosidase gene cloned in Escherichia coli.
The Lactobacillus bulgaricus beta-galactosidase gene was cloned on a ca. 7-kilobase-pair HindIII fragment in the vector pKK223-3 and expressed in Escherichia coli by using its own promoter. The nucleotide sequence of the gene and approximately 400 bases of 3'- and 5'-flanking sequences was determined. The amino acid sequence of the beta-galactosidase, deduced from the nucleotide sequence of the gene, yielded a monomeric molecular mass of ca. 114 kilodaltons, slightly smaller than the E. coli lacZ and Klebsiella pneumoniae lacZ enzymes but larger than the E. coli evolved (ebgA) beta-galactosidase. The cloned beta-galactosidase was found to be indistinguishable from the native enzyme by several criteria. From amino acid sequence alignments, the L. bulgaricus beta-galactosidase has a 30 to 34% similarity to the E. coli lacZ, E. coli ebgA, and K. pneumoniae lacZ enzymes. There are seven regions of high similarity common to all four of these beta-galactosidases. Also, the putative active-site residues (Glu-461 and Tyr-503 in the E. coli lacZ beta-galactosidase) are conserved in the L. bulgaricus enzyme as well as in the other two beta-galactosidases mentioned above. The conservation of active-site amino acids and the large regions of similarity suggest that all four of these beta-galactosidases evolved from a common ancestral gene. However, these enzymes are quite different from the thermophilic beta-galactosidase encoded by the Bacillus stearothermophilus bgaB gene.
Amino Acid Sequence↗
The relation between human lysosomal beta-galactosidase and its protective protein.
Cultured skin fibroblasts from patients with the lysosomal storage disease galactosialidosis lack a 54-kDa protein which is a precursor of 32-kDa and 20-kDa proteins, which immunoprecipitate with human anti-beta-galactosidase antiserum. The lack of a 32-kDa "protective protein" results in a combined deficiency of beta-galactosidase and sialidase. The mechanism of protection of lysosomal beta-galactosidase against proteolytic degradation is elucidated by sucrose density gradient centrifugation and immunoprecipitation studies. In normal fibroblasts at the low intralysosomal pH, more than 85% of beta-galactosidase exists as a high molecular weight (600-700 kDa) multimer and about 10% as a monomer of 64-kDa. In mutant cells from galactosialidosis patients, the residual enzyme activity, about 10%, is present as a monomer and no multimer exists. After addition of the 54-kDa precursor form of the protective protein, the density pattern of beta-galactosidase in galactosialidosis cells is normalized. Immunoprecipitation studies after sucrose density gradient centrifugation on homogenate and on purified beta-galactosidase from normal fibroblasts show that the protective protein is associated only with the multimeric form of beta-galactosidase. We propose that intralysosomal protection against proteolysis of beta-galactosidase and sialidase is accomplished by aggregation into a high molecular weight complex consisting of multimeric beta-galactosidase, sialidase, and protective protein. The genetic deficiency of the latter, as in galactosialidosis, results in a rapid degradation of monomeric beta-galactosidase and a loss of sialidase activity.
Electrophoresis, Polyacrylamide Gel↗
Characterization of alpha-galactosidase isoenzymes in normal and Fabry human-Chinese Hamster somatic cell hybrids.
The alpha-galactosidases in normal man-Chinese hamster somatic cell hybrids were investigation with antibodies specific for human alpha-galactosidase A and antibodies specific for Chinese hamster alpha-galactosidase. It was found that an isoenzyme in hybrid cells, which has an electrophoretic mobility between that of human alpha-galactosidase A and Chinese hamster alpha-galactosidase, contains immunologic determinants of both human and Chinese hamster origin, suggesting that it is a heteropolymeric molecule. Moreover, the locus for human alpha-galactosidase, which was found to be X-linked, is the locus coding for alpha-galactosidase A. Hybrids isolated after fusion of Chinese hamster cells with cells of a patient with Fabry's disease did not express human alpha-galactosidase A or the heteropolymeric molecule even in the presence of the active human X chromosome, indicating that the deficiency of alpha-galactosidase A in Fabry's disease is probably due to a mutation in a structural gene resulting in the inability to form immunologically detectable and functionally active molecules of alpha-galactosidase A.
Animals↗
Quaternary structure, Mg2+ interactions, and some kinetic properties of the beta-galactosidase from Thermoanaerobacterium thermosulfurigenes EM1.
The beta-galactosidase from Thermoanaerobacterium thermosulfurigenes EM1 was found to be a dimer with a monomer molecular weight of about 85,000. It lacks the alpha-peptide and an important alpha-helix that are both needed for dimer-dimer interaction and there is no homology in other important dimer-dimer interaction areas. These differences in structure probably account for the dimeric (rather than tetrameric) structure. Only 0.19 Mg2+ bound per monomer and Mg2+ had only small effects on the activity and heat stability. The absence of residues equivalent to Glu-416 and His-418 (two of the three ligands to Mg2+ in the beta-galactosidase from Escherichia coli) probably accounts for the low level of Mg2+ binding and the consequent lack of response to Mg2+. Both Na+ and K+ also had no effect on the activity. The enzyme activity with o-nitrophenyl-beta-D-galactopyanoside (ONPG) was very similar to that with p-nitrophenyl-beta-D-beta-D-galactopyranoside (PNPG) and the ONPG pH profile was very similar to the PNPG pH profile. These differences are in contrast to the E.coli beta-galactosidase, which dramatically discriminates between these two substrates. The lack of discrimination by the T. thermosulfurigenes beta-galactosidase could be due to the absence of the sequence equivalent to residues 910-1023 of the E. coli beta-galactosidase. Trp-999 is probably of the most importance. Trp-999 of the E. coli beta-galactosidase is important for aglycone binding and ONPG and PNPG differ only in their aglycones. The suggestion that the aglycone site of the T. thermosulfurigenes beta-galactosidase is different was strengthened by competitive inhibition studies. Compared to E. coli beta-galactosidase, D-galactonolactone was a very good inhibitor of the T. thermosulfurigenes enzyme, while L-ribose inhibited poorly. These are transition-state analogs and the results indicate that T. thermosulfurigenes beta-galactosidase binds the transition state differently than does E. coli beta-galactosidase. Methanol and glucose were good acceptors of galactose, and allolactose was formed when glucose was the acceptor. Allolactose could not, however, be detected by TLC when lactose was the substrate. The differences noted may be due to the thermophilic nature of T. thermosulfurigenes.
Bacteria, Anaerobic↗