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PubMed1995

Hematological reconstitution and gene therapy: retroviral transfer of the bacterial beta-galactosidase activity into human hematopoietic CD34+ cell populations and into T lymphocytes derived from the peripheral blood.

We report the possibility to transfer marker genes coding for beta-galactosidase activity using retroviral vectors into human peripheral blood CD34+ cells, peripheral blood T-lymphocytes and into the growth factor-dependent human hematopoietic cell line TF-1. Using the MFG-nisLacZ and the FLac vector and various packaging cell lines, we demonstrated retroviral transfer and high expression of a bacterial beta-galactosidase activity induced by the nisLacZ gene or the Sh-ble/LacZ gene. Kinetics of expression of the transgenes were analyzed both in primary cells and cell lines. Absence of cytotoxicity related to the expression of the bacterial beta-galactosidase was assessed in both cell types. These results open interesting prospectives for the use of the beta-galactosidase activity to mark and follow the fate of genetically modified cells isolated from patients prior to reimplantation.

Antigens, CD↗
PubMed1995

Identification of the active-site nucleophile in 6-phospho-beta-galactosidase from Staphylococcus aureus by labelling with synthetic inhibitors.

Kinetic parameters for the inactivation of the 6-phospho-beta-galactosidase of Staphylococcus aureus by a series (fluoro, chloro, bromo) of 2,4-dinitrophenyl-2-deoxy-2-halogeno- galactoside-6-phosphates have been determined. These inhibitors function by the formation of a stabilised glycosyl-enzyme intermediate. Inactivation and reactivation studies indicate that the fluoro derivative is formed most rapidly, but is also hydrolysed fastest. The chloro derivative forms the most stable covalent intermediate. HPLC profiles of V8-protease digestion of native and inhibited protein show significant differences, whereas the inhibited 6-phospho-beta-galactosidase and a point mutant of 6-phospho-beta- galactosidase (E375Q) yield the same proteolytic fragments. The suggestion that E375 is derivatised is strengthened by matrix-assisted laser-desorption ionisation mass spectrometry experiments which show that the two peptides, residues 336-375 and 376-383, are not produced, due to the absence of the expected cleavage at residues 375 and 376. The reason for the altered proteolysis pattern of the inhibited protein is blocking of the respective V8 cleavage site due to the chemical reaction of the inhibitor at position 375. Specific modification of the glycosyl bond between the inhibitor and E375 by aminolysis with benzylamine generated a glutamatic-acid-5-benzylamide complex at that position in the peptide. The Edman derivative of the modified E375 appears to be stable and was isolated by Edman degradation of trypsin-digested V8-peptide. It was shown to be identical to an authentic, synthetic sample. From this, it is evident that E375 is the active-site nucleophile of 6-phospho-galactosidase, consistent with previous findings for enzymes in this family.

Amino Acid Sequence↗
PubMed1993

Evaluation of a nude mouse tumor model using beta-galactosidase-expressing melanoma cells.

We developed and evaluated an in vivo athymic nude mouse model for tumor growth, angiogenesis, metastasis, and antineoplastic drug development. Melanoma cell lines expressing beta-galactosidase encoded by the Escherichia coli lac Z gene have been created by infecting an immortal murine melanocyte cell line with a recombinant retrovirus expressing the v-Ha-ras oncogene and lac Z to generate the MRB (melanoma, ras, beta-galactosidase) cell lines. The amelanotic, phorbol ester-independent, transformed melanoma cell lines developed tumors rapidly when injected subcutaneously into nude mice, as well as experimental lung metastases when injected i.v. into the tail vein. beta-galactosidase-expressing subcutaneous tumors and lung metastases stained blue with X-gal. The melanomas produced in nude mice have been characterized by using various histochemical and immunohistochemical staining methods to detect melanoma- and endothelial-cell-specific markers to determine the extent of neovascularization in MRB nude mouse tumors. Optimal staining of endothelial cells involved in tumor angiogenesis was observed by using ADPase activity and antiangiotensin-converting enzyme antibody staining. Attempts at indirect quantification of metastatic tumor cell number within the lung by either beta-galactosidase enzymatic activity or ELISA immunoreactivity were unsuccessful. However, the MRB cell lines should be useful in screening for and studying the mechanisms of action of antineoplastic, antimetastatic, and angiostatic drugs in vivo in athymic nude mice.

Animals↗
PubMed1976

Enzyme-linked immunoassay: conjugation of the Fab' fragment of rabbit IgG with beta-D-galactosidase from E. coli and its use for immunoassay.

1. A method for the conjugation of the Fab' fragment of rabbit IgG with beta-D-galactosidase from Escherichia coli is described. The method consists of two main steps: treatment of the Fab' fragments containing sulfhydryl groups with excess N,N'-o-phenylenedimaleimide, to introduce maleimide residues into the fragments, and then incubation of the dimaleimide-treated Fab' fragments with beta-D-galactosidase, which also contains sulfhydryl groups, to form the rabbit Fab'-beta-D-galactosidase complex. More than 90% of the enzyme used can be converted to the Fab'-enzyme complex, and the complex is readily separated from free Fab' fragments by chromatography on a Sepharose 6B column. 2. The application of the rabbit Fab'-beta-D-galactosidase complex for immunoassay of macromolecular antigens is shown by measuring human IgG by the sandwich method. The rabbit (anti-human IgG) IgG-coupled Sepharose 4B is incubated with human IgG and then with the rabbit (anti-human IgG) Fab'-enzyme complex, and the enzyme activity bound to the Sepharose is measured. In this way it is possible to determine as little as 0.3 fmoles of human IgG.

Animals↗
PubMed1994

Disruption of a recombinant yeast for the release of beta-galactosidase.

A recombinant yeast, Saccharomyces cerevisiae, expressing Escherichia coli beta-galactosidase gene under the control of CYC1 constitutive promoter of the yeast, was disrupted in a continuous flow, high speed, bead mill for the release of intracellular beta-galactosidase (EC 3.2.1.23). Release of the beta-galactosidase activity was characterized with respect to glass bead loading in the grinding chamber (70-85% of chamber volume), diameter of the beads (0.25-0.75 mm), number of passes of the cell slurry through the mill (0-6 passes), flow rate of the slurry (25-250 mL.min-1), cell concentration in the slurry (5-20 gDW.L-1), the agitation rotor speed (1000-4000 rpm) and the pH of the slurry (pH 5-10). The optimal conditions for the release of the enzyme were pH 6.0-9.0, 85% loading of 0.5 mm diameter beads and an agitation speed of 2000 rpm. The enzyme release followed first-order kinetics. For otherwise fixed conditions, the extent of cell disruption increase with increasing bead load, number of passes and agitation rotor speed. Cell concentration did not affect disruption. The release of beta-galactosidase activity declined with increasing flow rate of the cell slurry through the mill, but the disruption rate constant increased with flow rate. Under optimal condition, three passes through the grinding chamber were sufficient to release all of the enzyme. In comparison with disruption in the bead mill, chloroform-sodium dodecyl sulfate induced lysis of cells was ineffective in releasing the enzyme quantitatively.

Biotechnology↗
PubMed1994

Does Beano prevent gas? A double-blind crossover study of oral alpha-galactosidase to treat dietary oligosaccharide intolerance.

BACKGROUND: Beano, an over-the-counter oral solution of alpha-galactosidase, is used to prevent flatus and other gastrointestinal symptoms resulting from a high-fiber diet. The efficacy of this product, however, has not yet been adequately evaluated. METHODS: Nineteen subjects were randomized into two groups and fed test meals of meatless chili. At the first test meal, group 1 received eight drops of alpha-galactosidase solution and group 2 received eight drops of placebo. After the meal, subjects were asked to keep a careful record of gastrointestinal symptoms, including occurrences of intestinal gas passage, for the next 6 hours. One week later, an identical test meal was served to each study subject and the solutions were reversed. Again subjects recorded their symptoms for the next 6 hours. Data were analyzed by means of paired t tests. RESULTS: The number of flatulence events per hour was significantly less in the group treated with alpha-galactosidase than placebo over the 6-hour follow-up period (F = 2.87, P = .016). When the two groups were compared at each follow-up interval, this difference was statistically significant only for the 5th hour after ingesting the test meal (t = 2.19, P = .04). No differences between the two groups were found in the extent of bloating or pain following the meal. CONCLUSIONS: Oral alpha-galactosidase solution is efficacious, at least in some patients, for the prophylaxis of gastrointestinal intolerance of oligosaccharides.

Adult↗
PubMed1994

Hydrolysis of a novel lysosomotropic enzyme substrate for beta-galactosidase within intact cells.

With the goal of improving the detection of lysosomal sphingolipid hydrolases within intact cells, we have recently synthesized a new fluorophor, O-[4-(1-imidazolyl)butyl]-2,3-dicyano-1,4-hydroquinonyl beta-D-galactopyranoside (Im-DCH-beta-Gal). In the present study, we evaluated the interaction of Im-DCH-beta-Gal and its tetraacetate derivative, Im-DCH-beta-Gal(OAc)4, with living human fibroblasts. Im-DCH-beta-Gal was shown to be a specific substrate for human lysosomal beta-galactosidase in cell homogenates. Im-DCH-beta-Gal(OAc)4 was taken up and hydrolyzed by normal fibroblasts under physiological culture conditions. Very little hydrolysis of Im-DCH-beta-Gal(OAc)4 was observed in fibroblasts genetically deficient in lysosomal acid beta-galactosidase or in normal cells pretreated with the lysosomal inhibitors chloroquine and ammonium chloride. Analysis of substrate processing by cells indicated that normal and acid beta-galactosidase-deficient cells showed similar rates of uptake and deacetylation of Im-DCH-beta-Gal(OAc)4, with an 80% decrease in the rate of deglycosylation of substrate by beta-galactosidase-deficient fibroblasts. However, under our conditions, the fluorescent product was not well retained by cells. Our results indicate that this novel class of compounds may be useful in measuring lysosomal enzyme function in intact cells and may have application as a fluorescent marker for genetically altered cells.

Cell Line↗
PubMed1994

Effects of double amino-acid substitution polymorphism in acid beta-galactosidase gene in two inbred strains of mice.

We observed earlier that there are 5 nucleotide polymorphisms in the protein coding sequence of the acid beta-galactosidase gene between the C57BL/6J and DBA/2J strains of mice. Two of them result in amino acid substitutions. Consequences of the difference in the primary amino acid sequence were studied by introducing the two DBA polymorphisms into the C57BL cDNA, individually and in combination, by oligonucleotide-directed mutagenesis and expressing the resultant cDNAs in the COS-1 cell expression system. Introduction of one polymorphism, Asn517-->Asp into the C57BL cDNA, did not alter the acid beta-galactosidase activity in the transfected COS-1 cells, while introduction of Gly539-->Arg completely abolished the catalytic activity. When both polymorphisms were introduced together, as in the DBA mice, however, the acid beta-galactosidase activity was restored to that of the C57BL level. Thus, Asn517-->Asp appears to counteract the activity-abolishing effect of Gly539-->Arg, although it does not by itself raise the catalytic activity. All four types of cDNA generated similarly large amounts of stable mRNA in COS-1 cells. These results do not explain the significantly low acid beta-galactosidase activity in tissues of DBA mice, described earlier and also confirmed in this study.

Animals↗
PubMed1996

Purification and properties of beta-galactosidase from Aspergillus nidulans.

Beta-Galactosidase from mycelial extract of Aspergillus nidulans has been purified by substrate affinity chromatography and used to obtain anti-beta-galactosidase polyclonal antibodies. A. nidulans growing in lactose as carbon source synthesizes one active form of beta-galactosidase which seems to be a multimeric enzyme of 450 kDa composed of monomers with 120 and 97 kDa. Although the enzyme was not released to the culture medium, some enzymatic activity was detected in a cell-wall extract, thus suggesting that it can be an extracellular enzyme. Beta-Galactosidase of A. nidulans is a very unstable enzyme with an optimum pH value of 7.5 and an optimum temperature of 30 degrees C. It was only active against beta-galactoside substrates like lactose and p-nitrophenyl-beta-D-galactoside (PNPG).

Aspergillus nidulans↗
PubMedDate not supplied

[The cloning and expression of the beta-galactosidase gene of Candida pseudotropicalis yeasts in Escherichia coli cells].

The gene encoding the beta-galactosidase of the yeast Candida pseudotropicalis was cloned on YEp13 shuttle vector as the XhoI-fragment of chromosomal DNA of about 9.5 kb. SalGI-fragment of 7.5 kb with the beta-galactosidase gene was subcloned from the pG2 hybrid plasmid obtained into the pBR322 plasmid and then shortened to give 5.2 kb via deletion on XhoI site. This plasmid constructed was designated pBG2-1. In DNA/DNA hybridization studies the appearance of one XhoI-fragment and seven EcoRI-fragments in DNA C. pseudotropicalis hybridized to the DNA cloned fragment and the absence of hybridization to DNA Escherichia coli were shown. The beta-galactosidase activity of cells of transformants of E. coli was lower than the activity of prototrophic strain. The beta-galactosidase biosynthesis in transformants was insignificantly induced by lactose and IPTG and was not repressed by glucose.

Candida↗
PubMed1997

[Cloning and expression of the gene for thermostable beta-galactosidase from Thermoanaerobacter ethanolicus in Escherichia coli: purification and properties of the product].

An anaerobic thermophilic bacterium Thermoanaerobacter ethanolicus 39E (Clostridium thermohydrosulfuricum 39E) gene library was constructed in E. coli. Recombinant plasmid (pUT50) containing the thermostable beta-galactosidase was isolated by direct selection of clones for enzyme activity using 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal) and mapping procedures were carried out. The beta-galactosidase was purified from cell extracts of E. coli. Physicochemical characteristics of the recombinant beta-galactosidase were determined. The enzyme has two optimum pH values: 5.3 and 6.0, the temperature optimum is 75-80 degrees C. The molecular weight of beta-galactosidase was determined by PAG electrophoresis: about 83 kDa.

Bacteria, Anaerobic↗
PubMed1964

BETA-GALACTOSIDASE: INACTIVATION OF ITS MESSENGER RNA BY ULTRAVIOLET IRRADIATION.

A brief exposure of Escherichia coli cells to an indutcer for beta- galactosidase results in the production of a messenger RNA which subsequently expresses itself as beta-galactosidase. Ultraviolet irradiation of cells after exposure to the inducer results in a decrease in the amount of beta-galactosidase formed. The messenger RNA formed during the brief exposure to inducer is evidently inactivated by the ultraviolet radiation. The decay of messenger RNA activity in irradiated cells has the same kinetics as that observed in unirradiated cells.

Chloramphenicol↗
PubMed1965

PUROMYCIN: EFFECT ON MESSENGER RNA SYNTHESIS AND BETA-GALACTOSIDASE FORMATION IN ESCHERICHIA COLI 15T.

Incubation of Escherichia coli 15T(-) in the presence of puromycin inhibits the inducible formation of beta-galactosidase to a greater extent than it inhibits protein synthesis. In E. coli 33.00 beta-galactosidase formation is also more sensitive to the presence of puromycin than is protein synthesis. In the presence of glycerol (but not in its absence) puromycin prevents the production of messenger RNA for beta-galactosidase, presumably as a result of catabolite repression.

Escherichia coli↗
PubMed1965

BETA-GALACTOSIDASE OF STREPTOCOCCUS LACTIS.

Citti, J. E. (Oregon State University, Corvallis), W. E. Sandine, and P. R. Elliker. beta-Galactosidase of Streptococcus lactis. J. Bacteriol. 89:937-942. 1965.-Synthesis of beta-galactosidase by several strains of Streptococcus lactis was induced by lactose. The rate of hydrolysis of o-nitrophenyl-beta-d-galactopyranoside was used to measure enzyme activity. The enzyme of all but one strain was unstable when whole cells were sonic-treated or treated with toluene; the enzyme of one strain of S. lactis was stable to these treatments, which resulted in at least a fivefold increase in activity over that found in whole cells. The optimal assay conditions for toluene-treated cells of this strain involved incubation at 37 C in pH 7.0 sodium phosphate buffer. Lactose was the most effective inducer of enzyme synthesis. Methyl-beta-d-thiogalactopyranoside, isopropyl-beta-d-thiogalactopyranoside, and galactose were also inducers of the enzyme, but were not as effective as lactose. Melibiose, maltose, and calcium lactobionate were poor inducers of enzyme synthesis. Exogenously supplied glucose repressed enzyme synthesis. The means of control of induced beta-galactosidase synthesis in S. lactis was similar to that in Escherichia coli.

Disaccharides↗
PubMed1982

Human beta-galactosidase and alpha-neuraminidase deficient mucolipidosis: genetic complementation analysis of the neuraminidase deficiency.

Human beta-galactosidase and alpha-neuraminidase deficient mucolipidosis [ML(gal-neur-)] is an inherited lysosomal enzymopathy which recently was designated as a sialidosis. We analyzed the neuraminidase deficiency of this disorder with genetic complementation analyses using a heterokaryon enrichment procedure. The genetic defects of two apparent variants of this disorder complemented the defects of the neuraminidase deficiency diseases, sialidosis I and mucolipidosis I, resulting in the restoration of neuraminidase activity in heterokaryons. The neuraminidase deficiency, therefore, may not be the primary defect in ML(gal-neur-) and is not an appropriate test for determining carrier status. The clinical and biochemical characteristics of this disorder suggest that a post-translational or processing event for these enzymes may be defective. The defect, however, is different from I-cell disease and pseudo-Hurler polydystrophy, two disorders of post-translational lysosomal enzyme biosynthesis, since complementation studies demonstrated recovery of intracellular beta-galactosidase and alpha-neuraminidase levels in heterokaryons. The lack of human beta-galactosidase expression in man-mouse somatic cell hybrids formed from fibroblasts of the infantile onset type disorder suggests that the defect is not corrected by the mouse genome. The ML(gal-neur-) disorder therefore appears to be a distinct subtype of the inherited neuraminidase deficiencies in which the defect mat occur in a post-translational or regulatory step which coordinately affects the expression of lysosomal beta-galactosidase and alpha-neuraminidase.

Animals↗
PubMed1995

Arylsulfatase A and beta-galactosidase activities in leukocytes and lymphocytes from normal and psychiatric subjects. Effects of blood-processing delay and interleukin-2 stimulation.

Arylsulfatase A (ASA) and cerebroside-beta-galactosidase activities in leukocytes serve as a diagnostic tool for determining the presence of metachromatic leukodystrophy and globoid cell leukodystrophy, respectively. It has not been demonstrated whether a delay in blood processing and the presence of mixed cell types in different proportions in leukocytes affect the activities of the two enzymes in these cells. We have in the present study determined the specific activity in leukocytes and lymphocytes (T-cells) prepared from blood samples processed immediately after, 4, and 24 h after collection. In order to determine whether the enzyme activities in lymphocytes reflect expression of genetic trait, and not environmental or "state" influence, the activities of the two enzymes in interleukin 2-stimulated T-cells and resting T-cells were compared. A delay of up to 24 h in blood processing did not significantly change the specific activities of the two enzymes in both leukocytes and lymphocytes. The specific activity of ASA and beta-galactosidase in lymphocytes was 1.4-1.8 times that in leukocytes. The activities of the two enzymes in interleukin 2-stimulated T-cells did not differ from those in resting T-cells. These results indicate that blood-processing delay had no significant effects on ASA and beta-galactosidase activity. The data further indicate that the ASA and beta-galactosidase activity in interleukin 2-stimulated T-cells was not significantly different from resting lymphocytes from either normal or psychiatric subjects exposed to various medications. The activity levels in lymphocytes from psychiatric subjects thus reflect expression of genetic trait, rather than environmental or state influence.

Adult↗
PubMed1978

A benign deficiency of typeB beta-galactosidase in human liver.

The type A or 'acid' and type B or 'neutral' beta-galactosidase activities have been measured in post-mortem liver samples from individuals dying of non-genetic diseases and patients dying of ganglioside storage disease other than GM1 gangliosidosis. The type A activities fell within the established normal range in all samples. The type B activities showed a biomodal distribution suggesting the occurrence of two distinct populations of human individuals. The greater proportion had activities within the range 11.67 pkat/mg of protein (+/- 3.33, S.D.), while others had lower activities in the range 0.48 pkat/mg of protein (+/- 0.38, S.D.). No clinical symptoms were associated with the much lower type B beta-galactosidase activities and it appears that this beta-galactosidase deficiency could be found in the original tissues. Methods of screening for type B beta-galactosidase deficiency are described and the significance of this enzyme deficiency is discussed.

Chromatography, DEAE-Cellulose↗
PubMed1982

Km defect in neuraminidase of dysmorphic type sialidosis with and without beta-galactosidase deficiency.

Kinetic studies of 4-methylumbelliferyl neuraminidase activity were carried out in cultured skin fibroblasts from patients with various disorders of neuraminidase deficiency. Cell extracts from two patients with dysmorphic type sialidosis of infantile onset, with isolated deficiency of neuraminidase activity, and three patients with dysmorphic type sialidosis of juvenile onset, with combined deficiency of neuraminidase and beta-galactosidase activities, demonstrated 7-12 times higher apparent Km values than those of normal controls (1.0-1.5 mmol/l as compared with 0.12-0.15 mmol/l). The apparent Ki values for N-acetylneuraminic acid and colominic acid were also increased in the dysmorphic type (7-15 and 7-11 times the normal values, respectively). In contrast, in the normomorphic type, normal apparent Km and Ki values were found for 4-methylumbelliferyl neuraminidase activity in fibroblasts from one patient with isolated neuraminidase deficiency and two patients with combined deficiency of neuraminidase and beta-galactosidase. The altered kinetics in the dysmorphic cases indicates a primary defect in neuraminidase with a secondary deficiency of beta-galactosidase in patients with combined deficiency. It is not clear if the primary defect in the normomorphic cases involves a defect in neuraminidase other than a Km defect or if neuraminidase or both neuraminidase and beta-galactosidase deficiencies are secondary to another defect as yet undetermined.

Cell Line↗