SEARCH · PubMed Health

Results for “GALACTOSIDASE”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

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

At least 307 records · Page 17Linked to original sources

PubMed2000

The structure of the alpha-galactosidase gene loci in Thermus brockianus ITI360 and Thermus thermophilus TH125.

The Thermus thermophilus TH125 alpha-galactosidase gene, agaT, and flanking sequences were cloned in Escherichia coli and sequenced as well as flanking sequences of the previously cloned agaT from Thermus brockianus ITI360. Different structures of putative alpha-galactosidase operons in the two Thermus strains were revealed. Downstream of and overlapping with the alpha-galactosidase genes of both strains, a gene was identified that is similar to the galactose-1-phosphate uridylyltransferase gene (galT) of E. coli and Streptomyces lividans. Upstream of the agaT of T. brockianus ITI360, four open reading frames were observed. The deduced translation products displayed similarity to components of bacterial binding protein-dependent transport systems and a beta-galactosidase. No galactoside utilization genes were identified upstream of agaT in T. thermophilus TH125. The inactivation of the alpha-galactosidase genes of both strains by insertional mutagenesis led to an inability to use melibiose or galactose as a single carbohydrate source. An attempt was made to isolate a gene encoding the enzyme responsible for para-nitrophenyl-(pNP-) beta-galactoside hydrolyzing activity in T. thermophilus TH125. A gene designated bglT was cloned and expressed in E. coli. The inactivation of the bglT gene led to 55% reduction of the pNP-beta-galactoside hydrolyzing activity in the mutant strain in comparison to the wild type.

Bacterial Proteins↗

PubMed1990

Increase in the catalytic rate of beta-galactosidase by selection in chemostats at changing dilution rates.

The experiments presented in this paper explore whether mutants with catalytically more active beta-galactosidase (E.C. 3.2.1.23) can be selected in lactose-limited chemostats. This experimental system has been chosen because lactose metabolism in Escherichia coli is well understood both from a biochemical and genetic point of view. In a lactose-limited chemostat with constant dilution rates, both beta-galactosidase and the lactose permease increased in quantity. The catalytic rate (kcat) of beta-galactosidase in populations showed no change. On the other hand, in chemostats with periodically changing dilution rates, the catalytic rate of beta-galactosidase increased dramatically. Therefore, the selection for beta-galactosidase with improved catalytic rate occurs in chemostats with fluctuating dilution rates but not in ones with constant dilution. These observations may be of value in the selection of other enzymes with enhanced catalytic rates.

Bacteriological Techniques↗

PubMed1978

Characterization of the acid beta-D-galactosidases from human urine.

Acid beta-D-galactosidases (EC 3.2.1.23) from human urine samples have been characterized using GM1-ganglioside, asialofetuin, and 4-MU-beta-D galactopyranoside. Sepharose 6-B column chromatography of crude urine supernatant fluids resolved three forms of acid beta-D-galactosidase activity with apparent molecular weights of 500 X 10(3)--700 X 10(3) (I), 90 X 10(3)--120 X 10(3) (II), and 20 X 10(3)--27 X 10(3) (III), which hydrolyzed 4-MU-beta-D-galactopyranoside, GM1-ganglioside and asialofetuin. The crude urine supernatant fluids and the separated forms of acid beta-D-galactosidase exhibited similar apparent KM values for the respective substrates. Starch gel electrophoresis of urine samples at pH 7.0 revealed a slow anodally migrating form of acid beta-D-galactosidase which electrophoretically corresponded to form I and a faster anodally migrating form corresponding to form II. Form III migrated as a composite of forms I and II suggesting that aggregation to the larger molecular weight activity forms occurred during starch gel electrophoresis. This report represents the first characterization of urinary acid beta-D-galactosidase with respect to naturally occurring glycolipid and glycoprotein substrates. In addition, data is presented to indicate that the enzyme may be composed of an enzymatically active form with an apparent molecular weight of 20 X 10(3)--27 X10(3), which is also capable of hydrolyzing the glycolipid and glycoprotein substrates.

Chromatography, Gel↗

PubMed1985

A study of the low beta-galactosidase activity in cystinotic fibroblasts: effects of cysteamine.

beta-Galactosidase activity but not beta-glucuronidase, N-acetyl-beta-D-galactosaminidase or arylsulphatase A activity, is known to be significantly lower in cultured human skin fibroblasts from patients with cystinosis than in cells from control subjects. Incubation of cell homogenates with disulphide or thiol compounds did not affect beta-galactosidase activity, suggesting that decreased beta-galactosidase activity in affected cells was not caused by the presence of inhibiting substances or absence of activating substances. Incubating cells with 0.5 or 1.0 mmol/l cysteamine, a substance used in the clinical treatment of cystinosis because it depletes cells of excess cystine, greatly decreased beta-galactosidase activity in both cystinotic and normal cells. This effect is shown to result from enzyme instability in lysosomes with raised pH and increased thiol concentration. Thus, cysteamine, although effective in depleting cystinotic cells of excess cystine, may have the undesired side-effect of severely decreasing lysosomal beta-galactosidase.

Cells, Cultured↗

PubMed1985

A microassay for acid beta-galactosidase activity toward asialofetuin.

To study the enzymatic properties of beta-galactosidase from the patients with a beta-galactosidase deficiency such as GM1 gangliosidosis, determination of enzymatic activity with naturally occurring substrates, asialofetuin in addition to another natural substrate, GM1 ganglioside, is essentially required. With a previously reported, simple and sensitive fluorometric assay for GM1 ganglioside beta-galactosidase using high performance liquid chromatography (HPLC), optimal reaction conditions were determined for the assay of acid beta-galactosidase activity toward asialofetuin in skin fibroblast homogenates. Under these conditions, reduced enzymatic activities could be detected in cultured skin fibroblasts from patients with type 1 and 3 GM1 gangliosidoses and mucopolysaccharidosis IV-B (Morquio B syndrome). This method was applicable to study of the enzymatic properties of the mutant beta-galactosidase and provided an alternative to assays employing radioactive or artificial substrates.

Asialoglycoproteins↗

PubMed1989

Defined monoclonal antibodies to Escherichia coli beta-galactosidase as a tool for characterisation of recombinant expression products.

Mouse monoclonal antibodies were prepared against beta-galactosidase (EC 3.2.1.23) of Escherichia coli. The binding sites of these monoclonal antibodies within the beta-galactosidase molecule were estimated by immunoblot analyses to various defined peptide regions of beta-galactosidase, encoded by expression plasmids. Monoclonal antibodies were characterised, which either bind to the amino-terminal or to the carboxy-terminal region or to an internal section of beta-galactosidase. These defined monoclonal antibodies were shown to be a useful tool for characterisation of beta-galactosidase fusion proteins expressed in Escherichia coli.

Animals↗

PubMed1986

Beta-galactosidase in the seminal plasma and reproductive organs of the bull.

The distribution of beta-galactosidase activity was studied in different reproductive organs, seminal plasma and spermatozoa of the bull. The highest specific activity of beta-galactosidase was found in testis and in different parts of the epididymis, where the activity seemed to be partly in secretory (cauda secretion) and partly in non-secretory, bound form (caput to cauda epididymidis). Gel filtration on Sepharose 6B at pH 7.0 revealed two beta-galactosidase forms (GF-1, Mr approximately 500,000-600,000 and GF-2, Mr approximately 190,000-220,000) in reproductive organs and seminal plasma. The pH-optimum of both beta-galactosidase forms was about 3.75-4.75. Hg2+ and p-chloromercuribenzoate inhibited strongly these activities. Further, form GF-2 seemed to be slightly more sensitive to the thermal inactivation at 50-70 degrees C than form GF-1. In chromatofocusing beta-galactosidase activities in bull seminal plasma coeluted with those of the cauda epididymidis (pI-values 7.5-6.4). On the contrary, prostate, Cowper's gland, testis, ampulla and seminal vesicles had enzyme activities eluting at lower pI-values (6.3-4.2). Thus, the seminal plasma activity is mainly an indicator for the function of the epididymal cauda.

Animals↗

PubMed1978

Molecular cloning and expression in E. coli of a yeast gene coding for beta-galactosidase.

The yeast Kluyveromyces lactis synthesizes a beta-galactosidase (EC 3.2.1.32) which is inducible by lactose. We have isolated the gene that codes for this enzyme using recombinant DNA techniques. K. lactis DNA was partially digested with the restriction endonuclease Eco R1 and joined to Eco R1-digested pBR322 plasmid DNA using DNA ligase. ligase. A lac-mutant of Escherichia coli lacking the structural gene for beta-galactosidase was transformed with ligated DNA. Three lac+ transformants containing recombinant plasmids were selected. Two of the plasmids (pK15 and pK17) contain four Eco R1-K. lactis DNA fragments having molecular weights of 2.2, 1.4, 0.55 and 0.5 x 10(6) daltons. The other plasmid (pK16) lacks the smallest fragment. E. coli carrying any of these plasmids produce beta-galactosidase activity that has a sedimentation coefficient and immunological determinants that are nearly identical to K. lactis beta-galactosidase and distinctly different from E. coli beta-galactosidase. DNA-DNA hybridization studies show that the four Eco R1 fragments in pK15 hybridize to K. lactis but not to E. coli DNA.

DNA, Recombinant↗

PubMed1986

A quantitative assay for the detection of hepatitis B virus DNA employing a biotin-labeled DNA probe and the avidin-beta-galactosidase complex.

We have developed a procedure for the quantitation of specific DNA which employs nonradioisotopic probes and beta-galactosidase as a detector. The sample DNA was immobilized on a nitrocellulose filter paper. After the filter paper had been processed to hybridization with a biotinylated probe DNA, the paper was incubated with avidin-beta-galactosidase complex. The optimum ratio of avidin to biotinylated beta-galactosidase for preparation of a complex between the two was determined. The filter paper was punched. Each punched piece was put into a microtiter well and beta-galactosidase activity was measured using 4-methylumbelliferyl beta-D-galactosidase as a substrate. By this method, we were able to quantify as little as a few picograms of specific DNA. The application of this method for the quantitative assay of hepatitis B virus DNA in serum sample is also described. The sensitivity for the detection of the DNA by our method was practically comparable to that of the conventional radioisotopic method. The validity of our method for detection of the virus DNA was further supported by comparison with the serological data.

Avidin↗

PubMed1982

Aggregation properties of beta-galactosidase of human urine and degradation of its natural substrates by a purified preparation of the enzyme.

Acid beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) was purified to near homogeneity from normal human urine by two affinity chromatography steps. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate the major protein band had an apparent molecular weight of 59000, thus being 5000 daltons smaller than the protein purified from human liver. Upon gel filtration on Sephadex G-150 column the purified enzyme had an apparent molecular weight of 70000 of pH 7.0. At pH 4.0 partial aggregation to a dimer of an apparent molecular weight of 150000 was found. Addition of 0.1 M galactose caused at pH 3.5, but not at pH 4.0 and 7.0, an increased formation of multimeric beta-galactosidase which eluted with the void volume of the column. Crude beta-galactosidase from human urine showed a higher aggregation tendency than the purified enzyme. None of the conditions produced an enzyme species of an apparent molecular weight of less than 40000. pH-activity profiles were measured against p-nitrophenyl-beta-D-galactoside, 3H-labelled GM1-ganglioside, [3H]keratan sulfate and the pentasaccharide O-beta-(1 leads to 4)-[6-3H]galactopyranosyl-O-beta-(1 leads to 2)-2-deoxy-2-acetamidoglycopyranosyl-O-alpha-(1 leads to 6)-mannopyranosyl-O-beta-(1 leads to 4)-mannopyranosyl-2-deoxy-2-acetamidoglucopyranoside. While p-nitrophenyl-beta-D-galactopyranoside and GM1-ganglioside were optimally hydrolyzed at pH 4.0, keratan sulfate and the pentasaccharide were optimally degraded at pH 4.3 and pH 5.0, respectively. With the chromogenic substrate and with GM1-ganglioside Km values of 0.33 mM were calculated. At pH 3.5 the hydrolysis of the synthetic substrate did not follow Michaelis-Menten kinetics. Two enzyme species appeared with Km values of 0.006 mM and 3.2 mM, respectively. The affinity of beta-galactosidase for [3H]keratan sulfate and the 3H-labelled pentasaccharide was at least one order of magnitude lower than for the amphiphilic substrates. Keratan sulfate and GM1-ganglioside did not act as competitive inhibitors of p-nitrophenyl-beta-galactosidase at the concentration tested. These findings could be explained by the existence of different binding sites for the substrates used.

Galactosidases↗

PubMed1982

Regulation of alpha-galactosidase synthesis in Saccharomyces cerevisiae and effect of cerulenin on the secretion of this enzyme.

Saccharomyces cerevisiae -136ts MEL10 (a thermosensitive mutant whose RNA synthesis is inhibited at 37 degrees C but is normal at 23 degrees C), when grown at 23 degrees C in the presence of galactose, melibiose or L-arabinose, these cells synthesize alpha-galactosidase mRNA. In the simultaneous presence of both galactose and glucose the transcription of alpha-galactosidase mRNA is blocked. Glucose also interferes with mRNA translation, but the degree of inhibition depends on concentration and time of addition of the hexose to induced cells. It has been found that the final concentration of alpha-galactosidase produced by induced cells when transferred at the non-permissive temperature (37 degrees C) is inversely proportional to the incubation time in glucose. Cerulenin inhibits lipid formation on growing yeasts, but protein synthesis and selective permeability are not affected. The antibiotic partially inhibits secretion of alpha-galactosidase with a parallel accumulation of this enzyme in membranous structures, specially at the level of the plasma membrane. Induction of alpha-galactosidase or cerulenin addition to growing cells, results in changes in the polypeptide composition of the plasma membrane.

Antifungal Agents↗

PubMed1983

Enhanced stability of beta-galactosidase in parenchymal and nonparenchymal liver cells by conjugation with dextran.

In order to enhance the stability of beta-galactosidase, we conjugated the enzyme with dextran T-10 (Mr approx. 10 000). The conjugate contained 9-10 mol dextran/mol protein (beta-galactosidase, Mr 68 000), and the specific activity retained after conjugation was 90 +/- 4% (n = 3) of the initial activity. Uptake and degradation of native and conjugated beta-galactosidase in isolated hepatocytes and nonparenchymal liver cells was studied. There was a marked increase in stability against degradation in both cell types when beta-galactosidase was conjugated with Dextran. The degradation of dextran-conjugated enzyme was reduced by 35% in hepatocytes and by 43% in nonparenchymal cells, after 80 and 40 min, respectively, as compared with the free enzyme. However, there was insignificant difference between the uptake of native and conjugated enzyme into the liver cells. Upon intravenous infusion into rats, native and conjugated enzyme were cleared from plasma with only a slight difference in the clearance rate. The observed stability of dextran-conjugated beta-galactosidase towards cellular degradation was in accordance with the in vitro experiments. The conjugate showed marked thermal stability at 50 degrees C and enhanced resistance towards proteolysis by the broad specific protease subtilopeptidase A. This demonstrates that dextran conjugation may be used as a means of stabilizing lysosomal enzymes for therapeutic purposes.

Animals↗

PubMed1987

Strong inhibitory effect of furanoses and sugar lactones on beta-galactosidase Escherichia coli.

Various sugars and their lactones were tested for their inhibition of beta-galactosidase (Escherichia coli). L-Ribose, which in the furanose form has a hydroxyl configuration similar to that of D-galactose at positions equivalent to the 3- and 4-positions of D-galactose, was a very strong inhibitor, and D-lyxose, which in the furanose form also resembles D-galactose, was a much better inhibitor than expected. Structural comparisons prelude the pyranose forms of these sugars from being significant contributors to the inhibition, and inhibition at different temperatures (at which there are different furanose concentrations) strongly supported the conclusion that the furanose form is inhibitory. Studies with sugar derivatives that can only be in the furanose form also supported the conclusion. This is the first report of the inhibitory effect of furanose on beta-galactosidase. Lactones were also inhibitory. Every lactone tested was much more inhibitory than was its parent sugar. D-Galactonolactone was especially good. Experiments indicated that it was D-galactono-1,5-lactone rather than D-galactono-1,4-lactone which was inhibitory. Inhibition of beta-galactosidases from mammalian sources by lactones has been reported previously, but this is the first report of the effect of beta-galactosidase from E. coli. Since furanoses in the envelope form are analogous (in some ways) to half-chair or sofa conformations and since lactones with six-membered rings probably have half-chair or sofa conformations, the results indicate that beta-galactosidase probably destabilizes its substrate into a planar conformation of some type and that the galactose in the transition state may, therefore, also be quite planar.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate Conformation↗

PubMed1982

Activation of beta-galactosidase by monoclonal antibodies.

Four monoclonal cell lines secreting antibodies that activate the beta-galactosidase protein from lac-aba strains of Escherichia coli have been isolated. One of the antibodies, BG 79, inhibits the normal beta-galactosidase from E. coli in addition to its activation of the protein from mutants. Moreover, when in combination with any of the other activating antibodies, BG 79 exhibits synergistic activation of the beta-galactosidase protein, and the synergistically activated enzyme is stimulated by methanol, although most of the proteins activated by single antibodies are inhibited by methanol. The equilibrium of binding of BG 79 to the beta-galactosidase protein is not affected by the presence of a second antibody, and the half-time for activation by BG 79 is only slightly, though significantly, increased by preincubation of the protein with the second antibody. Our results imply that activation of beta-galactosidase proteins is not a simple correction of a conformational defect, and that many distinct active conformations are available to the enzyme.

Antibodies, Monoclonal↗

PubMed1989

Cytochemical observation of regulated bacterial beta-galactosidase gene expression in mammalian cells.

Bacterial beta-galactosidase, encoded by the lacZ gene, serves as a sensitive cytochemical marker in eukaryotic cells and tissues. In transient expression experiments, human and simian cells stain blue 48 hr after transfection with a plasmid containing a lacZ gene, whose expression is directed by a simian virus 40 promoter containing a synthetic lactose operator sequence. Transfection efficiency was about 0.6%. Incorporation of an operator sequence within the promoter permits regulation of beta-galactosidase gene expression by the lacI gene product, the lac repressor. When cells were cotransfected with the lacZ plasmid and a second plasmid containing the lacI gene, beta-galactosidase activity was extinguished. Its activity could be reestablished to original levels upon application of isopropyl beta-D-thiogalactoside to transfected cells. A cell line that stably carries both the lacI and lacZ genes was efficiently induced to synthesize beta-galactosidase after isopropyl beta-D-thiogalactoside administration. In transient expression experiments and in stably transfected lines, repression and induction of beta-galactosidase activity were predominantly at the transcriptional level.

Animals↗

PubMed1984

Preparation of a monomeric 2,4-dinitrophenyl Fab'-beta-D-galactosidase conjugate for immunoenzymometric assay.

A method is described for the preparation of a monomeric Fab'-beta-D-galactosidase conjugate, which is required for the development of a sensitive immunoenzymometric assay. Anti-human IgG F(ab')2 was labeled with 2,4-dinitrophenyl groups, split into Fab' by reduction and reacted with excess maleimide groups which had been introduced into beta-D-galactosidase through thiol groups using N,N'-o-phenylenedimaleimide. The monomeric 2,4-dinitrophenyl Fab'-beta-D-galactosidase conjugate was subsequently separated from unconjugated beta-D-galactosidase by affinity chromatography on a column of (anti-2,4-dinitrophenyl) IgG-Sepharose 4B. In the monomeric conjugate preparation, 98% of beta-D-galactosidase activity was associated with Fab' and 90% was associated with specific (anti-human IgG) Fab'. This conjugate allowed the measurement of 0.1 fmol of human IgG by an immunoenzymometric assay technique.

2,4-Dinitrophenol↗

PubMed1989

Structure and expression of the Lactococcus lactis gene for phospho-beta-galactosidase (lacG) in Escherichia coli and L. lactis.

The Lactococcus lactis subsp. lactis 712 lacG gene encoding phospho-beta-galactosidase was isolated from the lactose mini-plasmid pMG820 and cloned and expressed in Escherichia coli and L. lactis. The low phospho-beta-galactosidase activity in L. lactis transformed with high-copy-number plasmids containing the lacG gene contrasted with the high activity found in L. lactis containing the original, low-copy-number lactose plasmid pMG820, and indicated that the original lactose promoter was absent from the cloned DNA. In E. coli the phospho-beta-galactosidase could be overproduced using the strong inducible lambda PL promoter, which allowed a rapid purification of the active enzyme. The complete nucleotide sequence of the L. lactis lacG gene and its surrounding regions was determined. The deduced amino acid sequence was confirmed by comparison with the amino acid composition of the purified phospho-beta-galactosidase and its amino-terminal sequence. This also allowed the exact positioning of the lacG gene and identification of its characteristic Gram-positive translation initiation signals. The homologous expression data and the sequence organization of the L. lactis lacG gene indicate that the gene is organized into a large lactose operon which contains an intergenic promoter located in an inverted repeat immediately preceding the lacG gene. The organization and sequence of the L. lactis lacG gene were compared with those of the highly homologous lacG gene from Staphylococcus aureus. A remarkable bias for leucine codons was observed in the lacG genes of these two species. Heterogramic homology was observed between the deduced amino acid sequence of the L. lactis phospho-beta-galactosidase, that of the functionally analogous E. coli phospho-beta-glucosidase, and that of an Agrobacterium beta-glucosidase (cellobiase).

Amino Acid Sequence↗

PubMed1987

Signal sequence and DNA-mediated expression of human lysosomal alpha-galactosidase A.

Twelve complementary DNA clones for human lysosomal alpha-galactosidase A were isolated from an Okayama-Berg library constructed from SV40-transformed human fibroblasts. The identity of these clones was confirmed by complete colinearity of the nucleotide-deduced amino acid sequence with that determined by direct chemical sequencing of human placental alpha-galactosidase A. Hybridization of the alpha-galactosidase A cDNA to genomic DNA from individuals with varying numbers of X chromosomes as well as from interspecies somatic-cell hybrids showed only a single locus in the genome at Xq 13.1-Xq 22. One cDNA clone (pcD-AG210) contained the complete coding sequence for both the signal peptide and mature alpha-galactosidase A. The signal peptide of 31 amino acids contains the expected hydrophobic domains consisting of Leu-Gly-Cys-Ala-Leu-Ala-Leu and Phe-Leu-Ala-Leu-Val and has Ala at the signal peptidase cleavage site. Twelve out of fifteen G residues flanking the 5' end of the cDNA in pcD-AG210 were removed and the truncated fragment was ligated into the original vector. This construct, pcD-AG502, encoded enzymatically active human alpha-galactosidase A in monkey COS cells.

Amino Acid Sequence↗