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PubMed1978

On the evolution of beta-galactosidase.

The amino acid sequence of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) has been compared to itself and to other proteins. Two segments, each of about 380 amino acids, comprising the first three-fourths of the polypeptide chain, were found to be very similar to each other. It is concluded that they are homologous. The carboxyl-terminal fourth has a high percentage of amino acid identities with dihydrofolate reductase of Escherichia coli, suggesting these sequences also are homologous. A model for the origin of beta-galactosidase is presented. The overall similarity of beta-galactosidase to lac repressor does not appear to be significant.

Amino Acid Sequence↗
PubMed1981

Yeast genes fused to beta-galactosidase in Escherichia coli can be expressed normally in yeast.

A plasmid was constructed that allows the selection in vivo of gene fusions between the Escherichia coli beta-galactosidase gene and the yeast (Saccharomyces cerevisiae) URA3 gene. A large yeast DNA fragment containing the URA3 gene was placed upstream of an amino-terminally deleted version of the lacZ gene. The plasmid vehicle contains sequences that allow selection and maintenance of the plasmid in both yeast and E. coli. Selection for Lac+ in E. coli yielded numerous deletions that fused the lacZ gene to the URA3 gene and flanking yeast sequences, to the bacterial tetracycline-resistance gene from the parent plasmid pBR322, and to the yeast 2-micrometer plasmid DNA. Some of these fusion plasmids produced beta-galactosidase activity when introduced into yeast. One of the fusions to the URA3 gene itself has been shown to place the expression of beta-galactosidase activity under uracil regulation in yeasts.

Escherichia coli↗
PubMed1988

Structural organization of the human alpha-galactosidase A gene: further evidence for the absence of a 3' untranslated region.

Human alpha-galactosidase A (alpha-D-galactoside galactohydrolase; EC 3.2.1.22) is a lysosomal hydrolase encoded by a gene localized to the chromosomal region Xq22. The deficient activity of this enzyme results in Fabry disease, an X chromosome-linked recessive disorder that leads to premature death in affected males. For studies of the structure and function of alpha-galactosidase A and for characterization of the genetic lesions in families with Fabry disease, the full-length cDNA was isolated, sequenced, and used to screen human genomic libraries. The 1393-base-pair full-length cDNA had a 60-nucleotide 5' untranslated region and encoded a precursor peptide of 429 amino acids including a signal peptide of 31 residues. Three overlapping lambda clones spanning 32 kilobases were identified that contained the entire approximately equal to 12-kilobase chromosomal gene as well as approximately equal to 9 and approximately equal to 11 kilobases of 5' and 3' flanking sequence, respectively. The gene had seven exons. The genomic exonic and full-length cDNA sequences were identical. All intron-exon splice junctions conformed to the GT/AT consensus sequence. The 5' flanking region of this lysosomal housekeeping gene contained Sp1 and CCAAT box promoter elements as well as sequences corresponding to the activator protein 1 (AP1), octanucleotide ("OCTA"), and "core" enhancer elements. There was an upstream "HTF" island (Hpa II tiny fragments) followed by four direct repeats of the "chorion box" enhancer. The unique lack of a 3' untranslated sequence in the alpha-galactosidase A cDNA was confirmed by sequencing additional cDNA clones and the genomic 3' region.

Amino Acid Sequence↗
PubMed1988

Fluorescence-activated cell analysis and sorting of viable mammalian cells based on beta-D-galactosidase activity after transduction of Escherichia coli lacZ.

We demonstrate that individual cells infected with and expressing a recombinant retrovirus carrying the Escherichia coli beta-galactosidase gene (lacZ) can be viably stained, analyzed, sorted, and cloned by fluorescence-activated cell sorting based on the levels of lacZ expressed. To accomplish this we have devised a method to enzymatically generate and maintain fluorescence in live mammalian cells. Accumulation of fluorescent products in cells is linear with time, with a direct correlation of fluorescence to enzymatic activity. This technology for beta-galactosidase detection is more sensitive than other available cytochemical or biochemical methods. We have used this procedure to show that the expression of psi-2-MMuLVSVnlsLacZ in the T-cell lymphoma BW5147 and the B-cell hybridoma SP2/0 is not completely stable and that subclones selected by the fluorescence-activated cell sorter for low lacZ activity demonstrate distinctly lower average expression of LacZ. These findings indicate the utility of beta-galactosidase as a reporter molecule at the single-cell level for studies of gene regulation, including studies of promoter efficacy, enhancer activity, trans-acting factors, and other regulatory elements.

Animals↗
PubMed1988

Properties of acid beta-galactosidase from fowl spermatozoa.

1. Two forms, I and II, of beta-galactosidase [EC 3.2.1.23] from fowl spermatozoa were separated by Blue-Sepharose CL-6B chromatography. 2. The two forms of the enzyme yielded different pI values (4.1 and 4.7 for form I and 5.3 for form II). 3. Their gel filtration patterns were also different: form I resolved into two peaks with Mr about 65,000 and 80,000, whereas form II resolved into a single peak with an Mr of about 65,000. 4. Both forms had similar optimum pH, pH stability, thermal stability and Km values. 5. Differences between the characteristics of beta-galactosidase forms from spermatozoa and from seminal plasma suggest that the beta-galactosidases investigated in this study are spermatozoa-specific.

Animals↗
PubMed1990

Isolation, characterization, and mapping of a human acid beta-galactosidase cDNA.

A lambda gt11 human testicular cDNA library was screened with degenerate oligonucleotide probe mixtures based on amino acid sequence data generated from cyanogen bromide fragments and tryptic fragments of purified human beta-galactosidase. Six positive clones were identified after screening 2 x 10(6) plaques. The sequences of these six clones were determined and found to be derived from two different cDNAs. The sequence of the longest of these cDNAs is nearly identical to that recently determined by Oshima et al. (1988). It codes for a 76-kD protein and all 11 peptides that were generated from the purified enzyme. The second clone is shorter by 393 bp in the central portion of the coding region. Analysis by Northern blotting revealed the presence of a single mRNA species of 2.45 kb in lymphoblasts and testicular tissue. It is deduced from the amino acid sequence data that proteolytic processing of the precursor form of beta-galactosidase must occur by cleavage in the carboxy-terminal portion of the polypeptide perhaps around amino acid 530 at a uniquely hydrophilic sequence. Using a probe generated from the 3' region of the cDNA, we have mapped the locus coding for human beta-galactosidase to chromosome 3p21-3pter.

Amino Acid Sequence↗
PubMed1985

Dietary manipulation of postprandial colonic lactose fermentation: II. Addition of exogenous, microbial beta-galactosidases at mealtime.

The feasibility and efficacy of adding microbial beta-galactosidase enzymes directly to milk at the time of consumption was explored in adult lactose-malabsorbers. The hydrogen breath test, and on one occasion, the rise in blood glucose, were used as indices of the completeness of intraintestinal hydrolysis and absorption of milk lactose. When added to 360 ml of cow milk containing 18 g of lactose, empirical dosages of three beta-galactosidases--one from Kluyveromyces (yeast) and two from Aspergillus (fungal)--had some effectiveness in reducing postprandial H2 excretion, although no in vivo treatment at the dosages chosen was as effective as pre-incubation of the milk in vitro. The yeast enzyme also reduced symptom frequency as compared to intact milk and enhanced postprandial rises in blood glucose. The replacement therapy with exogenous, food-grade beta-galactosidases may provide a useful intervention to reduce lactose malabsorption and milk intolerance in individuals with primary lactase deficiency.

Adult↗
PubMed1980

Mutations affecting synthesis of beta-galactosidase activity in the yeast Kluyveromyces lactis.

Fifty-one mutants of Kluyveromyces lactis that cannot grow on lactose (Lac-) were isolated and characterized. All the mutations are in nuclear genes, are recessive in their wild-type allele and define seven complementation groups, which we designate lac3 through lac9. Strains bearing mutations in lac3, lac5, lac7, lac8 and lac9 are also unable to grow on galactose (Gal-). Since the Gal- and Lac- phenotype co-segregate, they are probably due to a single mutation. Strains bearing mutations in any of the seven complementation groups grow normally on glucose. However, strains bearing mutations in lac3, lac5 and lac6 do not grow on glucose if lactose is also present in the medium. Likewise, strains bearing mutations in lac3 and lac5 do not grow on glucose in the presence of galactose. Complementation groups lac4 and lac5 are loosely linked and map within a cluster of auxotrophic mutations on a chromosome that we designate chromosome 2. The remaining five groups are unlinked. Thus, there is no evidence for clustering of Lac genes into an operon-like regulatory unit.--To further characterize the nature of the Lac- phenotype, the basal and inducible level of beta-galactosidase activity were measured. All mutants had nearly normal basal enzyme levels, except those in lac4, which had barely detectable levels. Inducible enzyme levels varied from barely detectable levels in mutants bearing lac4 mutations up to four-fold inducible levels in strains bearing mutations in other complementation groups. In all cases, however, induction levels were below the 30-fold level obtained in wild-type cells. Three strains bearing lac5 mutations contain increased enzyme activity in the absence of inducer, indicating constitutive synthesis of beta-galactosidase. In summary, these data indicate that several genes are necessary for synthesis of beta-galactosidase activity.

Enzyme Activation↗
PubMed1986

Substrate specificity of endo-beta-galactosidases from Flavobacterium keratolyticus and Escherichia freundii is different from that of Pseudomonas sp.

The substrate specificity of endo-beta-galactosidase of Pseudomonas sp. was found to differ from that of Flavobacterium keratolyticus or Escherichia freundii, based on the following experimental results. The endo-beta-galactosidases from these three bacteria released 6-O-sulfo-GlcNAc beta 1-3Gal as one of the major products from keratan sulfates from different sources. In addition to the sulfated disaccharide, Flavobacterium and Escherichia enzymes produced GlcNAc beta 1-3Gal, which is also an integral repeating unit of keratan sulfate, whereas the Pseudomonas enzyme did not release any non-sulfated disaccharide. Tetrasaccharides were prepared from the teleost skin keratan sulfate by digestion with Pseudomonas enzyme followed by gel filtration on Sephadex G-50 chromatography. A part of the tetrasaccharide fraction was hydrolyzed by Flavobacterium enzyme to produce 6-O-sulfo-GlcNAc beta 1-3Gal and GlcNAc beta 1-3Gal, whereas the fraction was completely resistant to retreatment with the Pseudomonas enzyme. Endo-beta-galactosidases from F. keratolyticus and E. freundii hydrolyzed the internal beta-1,4-galactosyl linkage of various neolacto-type glycosphingolipids to produce glucosylceramides. However, these glycosphingolipids were completely resistant to the Pseudomonas enzyme. These findings clearly show that the sulfation on the N-acetylglucosamine adjacent to galactose in the lactosaminoglycans is essential for expression of the Pseudomonas enzyme, but not for that of the Flavobacterium or Escherichia enzyme.

Chromatography, Thin Layer↗
PubMed1988

Poly-N-acetyllactosamines synthesized by cultured Ehrlich carcinoma cells: application of endo-beta-galactosidase C for analysis of the terminal structure.

Poly-N-acetyllactosamines were prepared from Ehrlich carcinoma cells cultured in the presence of [14C]galactose. Methylation analysis indicated that 31% of the galactose was in the non-reducing end. Of it, 77% was cleaved by alpha-galactosidase, and 56% was released as a disaccharide by endo-beta-galactosidase C. Methylation analysis confirmed that the released disaccharide was mostly Gal alpha 1----3Gal. Therefore, Gal alpha 1----3Gal structure, not Gal alpha 1----3(Gal alpha 1----6)Gal structure, was the major alpha-galactosyl structure in the poly-N-acetyllactosamines synthesized. Furthermore, alpha-galactosidase digestion did not change the content of disubstituted galactosyl residues. Thus, Gal alpha 1----3(Gal alpha 1----6)Gal structure, which was suggested to be the sole non-reducing terminal structure of poly-N-acetyllactosamines of Ehrlich carcinoma cells, was not detected in significant amounts under the present experimental conditions.

Animals↗
PubMed1981

Reversible aggregation and stability of lysosomal acid beta-galactosidase from porcine adrenal cortex.

1. Acid beta-galactosidase [EC 3.2.1.23] of porcine adrenocortical lysosomes, assayed for its activity towards p-nitrophenyl-beta-D-galactopyranoside, showed two activity peaks on gel filtration profile at pH 7.4, one corresponding to a molecular weight of approximately 270,000 (termed form A3) and the other about 65,000 (termed form A1). 2. Another form of acid beta-galactosidase with a molecular weight of about 130,000 (termed form A2) was found when the high speed extract or partially purified form A1 was chromatographed on Sephadex G-150 at pH 4.5. 3. In the presence of 0.1 M NaCl or saturating amounts of substrate at pH 4.5, the high speed extract showed the aggregation of form A2 yielding form A3. Dissociation of form A3 back to form A1 was observed on incubation at 37 degrees C in 0.02 M sodium phosphate buffer, pH 7.4, and that was followed by irreversible enzyme inactivation. 4. Dissociation of form A3 into form A1 and enzyme inactivation in phosphate buffer, pH 7.4, were prevented by addition of 0.1 M NaCl. 5. The interconvertible enzymic forms showed the same pH-activity profiles and Michaelis constants. 6. These results suggest that the lysosomal acid beta-galactosidase in the porcine adrenal cortex exists in vivo as the dimer, and that the dimer may further aggregate into the tetramer.

Adrenal Cortex↗
PubMed1981

Purification and properties of alpha-galactosidase from immature stalks of Saccharum officinarum (sugar cane).

The immature sugar cane stalks studied contained less than 7% sucrose, and showed the activities of enzymes such as invertase, alpha-galactosidase, alpha-mannosidase, beta-N-acetylglucosaminidase, beta-glucosidase, beta-xylosidase, and beta-galactosidase. The alpha-galactosidase was highly purified by ammonium sulfate fractionation, gel filtration on a Sephadex G-100 column, ionexchange chromatography on DEAE-cellulose, and CM-cellulose columns, and heat treatment (60 degrees C, 15 min) in the presence of 0.2 m D-galactose. In polyacrylamide gel electrophoresis, the purified enzyme was homogeneous, having a molecular weight of approximately 46,000. In gelfiltration, it was approximately 47,000. The activity was optimum at pH 4.5 and at 60 degrees C. The purified enzyme hydrolyzed p-nitrophenyl-alpha-D-galactopyranoside (Km, 0.83 mM; Vmax, 25.0 mumol/mg/min), raffinose (Km, 25.9 mM; Vmax, 15.4 mumol/mg/min), and stachyose (Km, 13.0 mM; Vmax 2.7 mumol/mg/min), in addition to melibiose, guar gum, and locust bean gum. The hydrolysis of p-nitrophenyl-alpha-D-galactopyranoside was markedly inhibited by HgCl2, AgNO3, p-chloromercuribenzoate (PCMB), L-ascorbic acid, melibiose, stachyose, and D-galactose. Also the purified enzyme showed a lectin activity with trypsinized erythrocytes.

Chemical Phenomena↗
PubMed1984

Comparison of beta-D-galactosidase from Escherichia coli and horseradish peroxidase as labels of anti-human ferritin Fab' by sandwich enzyme immunoassay technique.

beta-D-Galactosidase from Escherichia coli and horseradish peroxidase were compared as labels of anti-human ferritin Fab' by sandwich enzyme immunoassay technique using fluorogenic substrates for enzyme assay. The anti-human ferritin Fab'-peroxidase conjugates gave lower nonspecific bindings and higher specific bindings than the corresponding Fab'-beta-D-galactosidase conjugates. As a result, the former provided more sensitive dose response curves for human ferritin than the latter. However, the peroxidase conjugates were required in a larger quantity, since peroxidase assay was much less sensitive than beta-D-galactosidase assay.

Antibodies↗
PubMed1986

Acid beta-galactosidase from human fibroblasts. A microscale purification method monitored by a highly sensitive enzyme assay.

A highly sensitive microassay method and a microscale purification system were developed to isolate the residual acid beta-galactosidase in GM1-gangliosidosis fibroblasts. The sensitivity of the microassay system, composed of a 96-well microplate and a microplate fluorometer, was 100-fold higher than that of the conventional system and the response was linear in the pmole range. Acid beta-galactosidase was characterized as a thiol enzyme which was inactivated by a mercuric compound. This enzyme was completely adsorbed on an Hg-agarose column and was easily eluted from the column by 10 mM 2-mercaptoethanol. The microscale purification system using Con A-Sepharose, PAT-Sepharose, and Hg-agarose column chromatography achieved 565- and 7,970-fold purifications of acid beta-galactosidase with an overall yields of 44% and 45% from normal and GM1-gangliosidosis fibroblasts, respectively. The purified enzyme fractions did not contain any other lysosomal enzyme activities except for a small amount of beta-N-acetylhexosaminidase activity.

Chromatography, Agarose↗
PubMed1988

Fitness effects of amino acid replacements in the beta-galactosidase of Escherichia coli.

Two genetic procedures were used to obtain amino acid replacements in the lacZ-encoded beta-galactosidase in Escherichia coli. Amino acid replacements could be obtained without regard to their effects on lactase activity by selecting spontaneous mutations that relieved the strong polarity of six nonsense mutations. When streaked on MacConkey-lactose indicator plates, approximately 75% of these mutants gave strong red lactose-fermenting colonies, and 25% gave white nonfermenting colonies. Mutants from 11 other nonsense codons were isolated directly using MacConkey-lactose indicator plates, on which positive color indication requires only 0.5% of the wildtype lactase activity. Among the total of 17 codons, 25 variant beta-galactosidases were identified using electrophoresis and thermal denaturation studies. The fitness effects of these variant beta-galactosidases were determined using competition experiments conducted with lactose as the sole nutrient limiting the growth rate in chemostat cultures. Three of the replacements were deleterious, one was selectively advantageous, and the selective effects of the remaining 21 were undetectable under conditions in which the smallest detectable selection coefficient was approximately 0.4%/generation.

Alleles↗
PubMed1979

Relationships among raffinose plasmids determined by the immunochemical cross-reaction of their alpha-galactosidases.

Plasmid-encoded alpha-galactosidase served as a marker enzyme for the recognition and comparison of raffinose (Raf) plasmids present in strains of Escherichia coli. Immunochemical relationships were established among Raf plasmids of 39 independent isolates from man and domestic animals (from three continents) by using antiserum against alpha-galactosidase. Immunodiffusion revealed three serological subclasses of alpha-galactosidase, which are correlated with the biological and geographical origin of the host strains. It is concluded that the raf determinants of all Raf plasmids tested have evolved from a common ancestor.

Animals↗
PubMed1979

Heterogeneity and some properties of beta-galactosidase from newborn rat epidermis.

Two forms of beta-galactosidase from newborn rat epidermis could be separated by DEAE-cellulose chromatography. Both enzymes showed similar enzymic properties. They had a pH optimum around 3.5--4.5 and the optimal temperature of these enzymes was approximately 60 degrees C. They were not affected by divalent cations, ethylenediaminetetraacetic acid(EDTA) and 2-mercaptoethanol(2-ME), while rho-chloromercuribenzoic acid (PCMB) was a strong inhibitor for each enzyme. These enzymes showed the same Km value (1.25 x 10(-4) M) towards 4-methylumbelliferyl-beta-D-galactoside. However they had different isoelectric points at pH 6.3 and 9.0, respectively. Six different forms of beta-galactosidase activity were found by using isoelectric focusing. When the crude extract was incubated with neuraminidase before electrofocusing, the acidic forms of the enzyme were largely lost and converted to more basic forms without loss of the total activity. This finding suggests the glycoprotein nature of newborn rat epidermal beta-galactosidase.

Animals↗
PubMed1984

Identification of an essential carboxylate group at the active site of lacZ beta-galactosidase from Escherichia coli.

[3H] Conduritol C cis-epoxide (1,2-anhydro-epi-inositol, I) was synthesized as an active-site-directed inhibitor for lacZ beta-galactosidase from Escherichia coli. A considerable kinetic isotope effect was noted in the reduction by [3H]NaBH4 of the p-benzoquinone-derived precursor for I. Complete loss of beta-galactosidase activity occurred on incorporation of 4 mol I/mol beta-galactosidase tetramer. The inhibitor was very labile in the denatured enzyme at pH greater than 8, implying the formation of an ester bond between I and a carboxylate at the active site. The radioactive material released from the labeled enzyme was identified as allo-inositol. The stereochemistry of the expoxide reaction (trans-diaxial ring opening) is thus the same as for beta-glucosidases with the corresponding epoxides. The binding site for I was identified as Glu-461 by the isolation and partial sequence analysis of a radioactive octapeptide from the cyanogen bromide and pepsin fragments of the labeled enzyme. A failure to determine the N-terminal amino acid of the labeled peptide is ascribed to the great reactivity of the esterified gamma-carboxyl group of its N-terminal Glu-461 which causes rapid cyclisation of this residue to pyroglutamate, even under weakly basic conditions. The participation of the carboxylate of Glu-461 in catalysis is discussed.

Binding Sites↗