Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “GALACTOSIDASE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,045 records · Page 58Linked to original sources
PubMed1989

Alpha-factor leader sequence-directed transport of Escherichia coli beta-galactosidase in the secretory pathway of Saccharomyces cerevisiae.

The construction of two fused genes is described. One involves the in-frame fusion of the yeast prepro-alpha-factor coding sequence, and the Escherichia coli lac Z gene. The second gene fusion utilizes a 103 bp yeast invertase NH2-terminal coding sequence at the fusion junction of the hybrid gene described above. The gene fusions, under the control of the alpha-factor promoter, expressed active beta-galactosidase in alpha haploid yeast cells. The activity could be regulated in a temperature-sensitive sir3 mutant. The incorporation of the invertase coding sequence at the MF alpha 1-lacZ fusion junction provided significantly higher levels of beta-galactosidase activity. A substantial quantity of the hybrid proteins generated from the gene fusions was primarily localized in the intracellular membranes of yeast cells, while a processed form could be secreted into the periplasm.

Blotting, Northern↗
PubMed1980

The phage promoter responsible for the expression of the inserted beta-galactosidase gene in bacteriophage lambda plac5.

The lac transducing phage, lambda plac5, carries a segment of the E. coli lac operon on the left side of the b2 region of the lambda phage. In the absence of additional cyclic AMP, beta-galactosidase can only be expressed from the phage promoter, and the expression of the inserted lac promoter is suppressed. This phage promoter responsible for beta-galactosidase synthesis is shown to be under the control of the cI and N gene products; however, the repressive action of the cro gene product at high multiplicity of infection is not observed although some turn off at very late time is detected. To pin down this phage promoter, results described in this communication and those described elsewhere can rule out the promoter PI, PR, P'R, and the promoter PL also looks rather unlikely. No firm identification of this phage promoter has been made, but the promoter(s) in the b2 region (the b2 promoter) is proposed. The phage promoter responsible for beta-galacrosidase synthesis is shown to be a weak promoter, requires the Q gene product or one (or more) of the late gene products for activation, and the time of expression is very late.

Bacteriophage lambda↗
PubMed1989

Functional relationship among TATA sequences, gene induction and transcription initiation in the beta-galactosidase, LAC4, gene from Kluyveromyces lactis.

In the 5' non-coding region of the beta-galactosidase, LAC4, gene of Kluyveromyces lactis, three TATA-like sequences are present at -230, -170 and -142 from the ATG translation start site. By means of deletion mutations in the TATA region, at least two of these TATA sequences, those at -230 and -142, were shown to be required for normal gene expression. Evidence is presented for a functional hierarchy and cooperation between these TATA sequences. The deletion or a change in the position of the TATA sequences affects both beta-galactosidase induction and the location of RNA initiation sites. The TATA sequence at -230 alone is sufficient for correct gene induction when it is moved to a position 41 bp from the major RNA initiation sites located around -110; the -142 TATA alone contributes only partly to gene induction. We suggest a functional distinction between these two related regulatory sequences. This functional distinction might be established by sequence differences and/or targets of unlike specific DNA binding protein(s). A conformational analysis of the LAC4 promoter showed that under torsional stress the functional elements UAS, TATA boxes RNA initiation sites and ATG can be detected as P1-sensitive sites. Possible functions of DNA structural alterations on gene expression are discussed.

Chromosome Deletion↗
PubMed1978

Relationship between genetic variation in thermal stability and electrophoretic mobility of mouse beta-galactosidase.

We have examined the relationships between the genetic determinants for mouse beta-galactosidase heat stability and electrophoretic mobility, in order to clarify previous reports indicating that a variation for enzyme heat stability is restricted to kidney while that for electrophoretic mobility is expressed in all tissues. We find that the two phenotypes show concordant strain distributions and cosegregate in genetic crosses. In contrast to a previous report, the thermal stability variation is expressed in all tissues, although the absolute rate of enzyme inactivation is tissue specific. The evidence supports the notion that a single beta-galactosidase structural locus is expressed in all tissues and that the differences in enzyme stability between tissues results from posttranslational enzyme modification.

Animals↗
PubMed1984

Quantitative cytochemistry of beta-galactosidase in normal and enzyme deficient (gal) pollen of Brassica campestris: application of the indigogenic method.

The available cytochemical methods for localization of beta-galactosidase have been evaluated using pollen grains of Brassica campestris. beta-Galactosidase-deficient pollen (gal), served as a control. Azo dye methods involving naphthyl substrates showed high and nonspecific background staining to the exine. The indigogenic method, employing 5-bromo-4-chloro-3-indoxyl beta-D-galactoside (X-gal) as the enzyme substrate, gave specific opaque-blue final reaction product, while mutant pollen grains remained colourless. Final reaction product formation was blocked by D-galactono-1,4-lactone, thus demonstrating the specificity of the enzyme reaction. Using microspectrophotometry, the absorbance of the final reaction product was found to be a linear function of incubation time and section thickness in cryostat sections up to 8 micron thick and was only slightly reduced by glutaraldehyde prefixation. The validity of the indigogenic method for quantitative analysis was confirmed by using an enzyme-containing polyacrylamide gel model system and enzyme-coupled Sepharose 4B beads. Cellular sites of enzymic activity have been determined using plastic sections: final reaction product occurred in the intine wall layer and peripheral cytoplasm.

Galactosidases↗
PubMedDate not supplied

Effect of environmental toxicants on enzyme biosynthesis: a comparison of beta-galactosidase, alpha-glucosidase and tryptophanase.

Except for beta-galactosidase, little is known about the effect of environmental toxicants on enzyme induction. The information could be potentially useful for the development of low-cost and rapid ecotoxicity assays. The effect of toxicants on the de novo biosynthesis of three inducible enzymes, beta-galactosidase and tryptophanase in E. coli and alpha-glucosidase in B. subtilis was investigated. Biosynthesis of alpha-glucosidase was the most sensitive to environmental toxicants, particularly pentachlorophenol and sodium dodecyl sulfate. The sensitivity of B. subtilis to toxicants was further increased when Tween 80 was incorporated in the growth medium.

Bacteria↗
PubMed1987

A simple competitive enzyme-linked immunosorbent assay using antigen-beta-galactosidase fusions.

The fusion of the N-terminal 461 bp of the human interferon-alpha 2 (INF) in frame to the beta-galactosidase gene from Escherichia coli is described. The presence of the expected DNA sequence was shown by restriction mapping and DNA sequencing. A fusion protein was demonstrated in crude extracts of E. coli by Western blots using polyclonal anti-beta-galactosidase and monoclonal anti-IFN antibodies. Using monoclonal antibodies specific for the N-terminal region of IFN-alpha and cell-free extracts from an E. coli strain containing the fusion protein, we set up a simple competitive enzyme-linked immunosorbent assay for human interferon. The test described here was linear down to a lower detection limit of at least 1000 Units, or 5 ng human IFN.

Antibodies, Monoclonal↗
PubMed1984

Binding and reactivity at the "glucose" site of galactosyl-beta-galactosidase (Escherichia coli).

A large number of sugars and alcohols were tested to see how well they bound and how readily they reacted at the "glucose" site of the galactosyl form of beta-galactosidase. Two classes of compounds were found to bind well to the galactosyl form of the enzyme. One class contained sugars and alcohols similar in structure to D-glucose in its pyranose ring form, and the other class was composed of relatively hydrophobic sugars and alcohols. On the other hand, several factors seemed to control k4. Large k4 values were found for straight-chain alcohols as compared to the values for the corresponding ring sugars. Also, if the acceptors had hydroxyl groups at the end of the molecule, the reactivity (k4) was greater than if hydroxyl groups were only in the middle of the molecule. In addition, if there was a hydroxyl at an asymmetric carbon next to a terminal hydroxymethyl group, it was necessary that it be in the same orientation as the D configuration of glucose; otherwise, the k4 was low. Overall, the results showed that it is the binding effect, more than the reactivity, which is responsible for the specificity at the "glucose" site. More specifically, these studies showed that the reason glucose is such an ideal molecule for transgalactosylation is that it leaves the galactosyl form of the enzyme very slowly, that is, k-a is relatively small. Thus, glucose remains attached to the galactosyl form of beta-galactosidase for a sufficient time to allow transgalactosylation to occur, while other acceptors, despite being as reactive (or more reactive) in terms of their k4 values, dissociate from the "glucose" site of the galactosyl form of the enzyme very readily and thus are poor acceptors.

Binding Sites↗
PubMed1990

Anomerization and hydrolysis of lactose by beta-galactosidase from Saccharomyces lactis.

Beta-Galactosidase from Saccharomyces lactis was found to be able to catalyze both the anomerization of alpha-lactose and the hydrolysis of beta-lactose; the rate of hydrolysis appeared to be four times higher with a 1:1 mixture of alpha and beta lactose than with a freshly prepared solution of alpha-lactose. The enzyme was also found to be unable to hydrolyze alpha-lactose. Thus, it appears that beta-galactosidase from S. lactis has its hydrolytic activity on lactose adapted only to the naturally more abundant beta-lactose.

Galactosidases↗
PubMed1978

Pilot scale purification of alpha-galactosidase A from Cohn fraction IV-1 of human plasma.

Human plasma alpha-galactosidase A (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was purified 7000-fold over plasma levels from Cohn Fraction IV-1. The yield per kg starting material averaged 11 000 units (nmol galactose liberated per h) and the specific activity was about 600 units per mg protein with 4-methylumbelliferyl-alpha-D-galactoside. The ratio of 4-methylumbelliferyl-alpha-galactosidase to ceramide trihexosidase activities was 6.2. Both activities were heat labile and exhibited the same relative mobilities on polyacrylamide gel electrophoresis. Enzymatic activity was stable for at least 4 months at 4 and -20 degrees C. The endotoxin concentration of this preparation averaged 0.26 mg per mg protein.

Galactosidases↗
PubMed1978

Properties of immobilized fig alpha-galactosidase and effect on ceramide-3 content of plasma from patients with Fabry's disease.

The possibility of lowering the level of ceramide-3 (galactosyl-alpha(1 leads to 4)-galactosyl-beta(1 leads to 4)-glucosyl-beta(1 leads to 1)-ceramide) in the plasma of patients with Fabry's disease was investigated. An immobilized alpha-galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was prepared by coupling purified fig alpha-galactosidase to Sepharose 4B. The pH optimum for the hydrolysis of the artificial substrate p-nitro-phenyl-alpha-D-galactopyranoside was shifted by approx. 0.5--1.0 pH unit to higher pH values upon coupling of the enzyme to Sepharose 4B. The immobilized enzyme was more stable than the native enzyme to incubation at 60 degrees C. The immobilized enzyme was able to hydrolyse ceramide-3 either at pH 4.5 or at pH 7.4 in an artificial system in which sodium taurocholate was used to solubilize the substrate. In contrast, when the immobilized enzyme was incubated with normal plasma or plasma from a patient with Fabry's disease, in which elevated levels of ceramide-3 occur, no hydrolysis of the glycosphingo-lipid could be detected. The results suggest that lowering of level of ceramide-3 in plasma from patients with Fabry's disease by enzymic means is not feasible.

Drug Stability↗
PubMed1979

Factors affecting the hydrolysis of ceramide-3 by alpha-galactosidase A from human liver.

1. The effect of detergents on the catalytic properties of alpha-galactosidase from human liver was studied using p-nitrophenyl-alpha-galactoside and galactosyl-alpha(1 leads to 4)-galactosyl-beta(1 leads to 4)-glucosylceramide (ceramide-3) as substrates. 2. The hydrolysis of p-nitrophenyl-alpha-galactoside by alpha-galactosidase was inhibited by commercial preparations of sodium taurocholate and by taurocholate purified from these preparations by thin-layer chromatography. The extent of inhibition was dependent on the concentration of the detergent and on the amount of protein present. The impurities present in the preparation also inhibited the hydrolysis. 3. The inhibition of taurocholate preparations of p-nitrophenyl-alpha-galactoside hydrolysis was pH-dependent. 4. The inhibition by taurocholate of p-nitrophenyl-alpha-galactoside hydrolysis can be partly overcome by adding glycosphingolipids. 5. No significant hydrolysis of ceramide-3 occurs in the absence of detergent. Upon adding increasing concentrations of taurocholate, the rate of hydrolysis increases to a maximum value. At still higher taurocholate concentrations the activity decreases. 6. The concentrations of taurocholate giving a maximal rate of hydrolysis of ceramide-3 is dependent on the amount of protein present and independent of the ceramide-3 concentration. 7. When the pH dependence of the rate of hydrolysis of ceramide-3 was measured in the presence of a commercially available preparation of pure taurocholate or of crude taurocholate, curves with different shapes were obtained.

Ceramides↗
PubMed1985

m-Fluorotyrosine substitution in beta-galactosidase; evidence for the existence of a catalytically active tyrosine.

The pH profiles of beta-galactosidase, having tyr replaced by m-fluorotyrosine, were compared to those of normal enzyme. The inflection point on the alkaline side was lowered about 1.5 pH units in the fluoro-enzyme, corresponding to the difference in the phenolic pKa values of m-fluorotyrosine and tyr. When glycosidic bond breakage was rate-limiting, the Vm at pH 7.0 was higher for the fluoro-enzyme. When hydrolysis was rate-limiting or when acceptors which made transgalactosylis rate-limiting were used, the Vm was lower for the fluoro-enzyme. This shows that a tyr in beta-galactosidase is a general-acid catalyst in the glycosidic bond breaking reaction and a tyr (probably the same one) is a general-base catalyst in the hydrolytic reaction.

Drug Stability↗
PubMed1987

beta-Galactosidase from rat epididymal fluid is bound by a recognition site attached to membranes of the epididymis different from the phosphomannosyl receptor.

In order to know if the beta-galactosidase of the rat epididymal fluid, as other secreted acid hydrolases, carries a marker in its molecule, we studied the binding of this enzyme to cellular membranes of the epididymal tissue. The binding, like that mediated by the phosphomannosyl receptor, was saturable, did not require calcium, had a Kd in the nM range and was inhibited by phosphatase or metaperiodate treatment of the enzyme. However fructose 6-phosphate derivates were more effective competitive inhibitors than mannose 6-phosphate. The binding capacity of the membranes were extractable with Triton X-100 and incorporable into liposomes. Trypsin inhibited the binding capacity of Triton extracts but it did not affect the affinity of intact cellular membranes for beta-galactosidase. The results suggest that a phosphorylated carbohydrate of the enzyme is bound by a recognizing site of the cellular membranes different from the phosphomannosyl receptor.

Animals↗
PubMed1988

Purification and analysis of the structure of alpha-galactosidase from Escherichia coli.

Alpha-Galactosidase, the product of the melA gene, was purified from a strain of Escherichia coli harboring a plasmid carrying melA, which over-produced the alpha-galactosidase. An apparent molecular weight was determined to be 50 kDa. The amino acid composition of this enzyme was determined. The result indicates that this enzyme is a hydrophilic and acidic protein. We have subjected the purified enzyme to 20 cycles of N-terminal sequence analysis. This verified the translation start site of the melA gene and the predicted N-terminal sequence.

Amino Acid Sequence↗
PubMed1989

Secretion of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae using the signal sequence from the glucoamylase-encoding STA2 gene.

The budding yeast Saccharomyces cerevisiae is a safe and widely used host for the production of recombinant DNA-derived proteins. We have used the signal sequence from the S. diastaticus STA2 gene, encoding glucoamylase II, to secrete Escherichia coli beta-galactosidase, encoded by the lacZ gene. In frame STA2/lacZ gene fusions have been constructed and expressed in S. cerevisiae under the control of either the STA2 or the galactose inducible GAL1-10 upstream promoters. Fairly high amounts of the enzyme (up to 76% of total activity, depending on the growth conditions) are secreted in the periplasmic space. Adding yeast extract and peptone to the growth medium results in a dramatic increase in both synthesis and secretion of beta-galactosidase.

Amino Acid Sequence↗
PubMed1990

Site specific mutants of beta-galactosidase show that Tyr-503 is unimportant in Mg2+ binding but that Glu-461 is very important and may be a ligand to Mg2+.

The Mg2+ concentrations required for half maximal activity, the dissociation constants, and the free energies of binding for Mg2+ bound to wild type beta-galactosidase and several site specific mutants are reported. The mutants have one of the following substitutions: Glu-461 substituted with Asp, Gln, Gly, His, or Lys; or Tyr-503 substituted with Phe, His or Cys. Substitutions for Tyr-503 had little effect on the affinity of the enzyme for Mg2+, implying that Tyr-503 is not involved in Mg2+ binding. Neutrally charged amino acids substituted for the negatively charged Glu-461 significantly decreased the affinity of the enzyme for Mg2+ and substitution of positively charged amino acids at this position further decreased the affinity. On the other hand, substitution by Asp (negative charge) at position 461 had no effect on the binding. Thus, the negatively charged side chain of Glu-461 is important for divalent cation binding to beta-galactosidase.

Amino Acid Sequence↗
PubMed1984

Purification of a beta-D-galactosidase from bovine liver by affinity chromatography.

A beta-D-galactosidase from bovine liver was purified to apparent homogeneity. The major purification step was affinity chromatography on a column of D-galactose attached to a Sepharose support activated with divinyl sulfone. Affinity media prepared by binding ligands to Sepharose activated with cyanogen bromide were unsuitable for purification of the enzyme, even though such media have been used to purify beta-D-galactosidases from other sources. The molecular weight of the denatured enzyme was 67,000. The molecular weight of the native enzyme at pH 7.0 was 68,000, and at pH 4.5 or 5.0, was 141,000. These data suggest that the enzyme has a single, fundamental subunit with a molecular weight of 67,000, and that the enzyme exists as a monomer at pH 7.0, and a dimer at pH 4.5 or 5.0. The Vmax values of the enzyme with p-nitrophenyl beta-D-galactoside, p-nitrophenyl beta-D-fucoside, lactose, and beta-Gal-(1----4)-beta-GlcNAc-1---- OC6H4NO2 -p were 10,204, 11,550, 9,479, and 8,859 nmol/min/mg of protein, respectively, and the Km values for these substrates were 0.08, 14.9, 14.2, and 1.6mM, respectively. D-Galactose, beta-D- galactosylamine , p-aminophenyl 1-thio-beta-D-galactoside, and D- galactono -1,4-lactone were competitive inhibitors of the enzyme, with Ki values of 0.9, 0.6, 0.6, and 0.8mM, respectively. The enzyme catalyzed the transfer of the D-galactosyl group from p-nitrophenyl beta-D-galactoside to D-glucose. The pH optimum of the enzyme was 4.5, and the pI was 4.7.

Animals↗