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Results for “GALACTOSIDASE”

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PubMed1995

Kinetics of expression of inducible beta-galactosidase in murine fibroblasts: high initial rate compared to steady-state expression.

We have constructed a murine fibroblast cell line in which synthesis of beta-galactosidase can be induced by incubation with isopropyl-beta-D-thiogalactopyranoside (IPTG). This was obtained by transfection by both a plasmid expressing lacI and a second plasmid expressing lacZ from a modified simian virus 40 (SV40) promoter containing a lac operator. We have measured the induction kinetics as well as the basal and induced differential rate of synthesis of beta-galactosidase. The steady-state rate of synthesis is tenfold higher in the presence than in the absence of inducer; we calculate an average of 1200 lacZ polypeptides are synthesized per minute per cell in the induced cultures. However, immediately after induction, the rate of accumulation of beta-galactosidase is up to 50-fold higher than the basal level. Based on our measurements of stability of beta-galactosidase, we suggest that induction may result in a subsequent down-modulation of the transcriptional activity from the induced gene. We hypothesize this inhibition may result from structural changes in DNA components, such as nucleosomes.

Animals↗

PubMed1998

Folding and association of beta-Galactosidase.

beta-D-Galactosidase from Escherichia coli is one of the largest tetrameric enzymes known at present. Although its physiological importance, the regulation of its synthesis, its enzymatic properties and its structure are well established, little is known about the stability and the folding pathway of this enzyme. Here we show that the overall folding mechanism of chemically denatured beta-galactosidase consists of three stages: (i) formation of elements of secondary structure; (ii) collapse to subdomains and structured monomers; (iii) association to the native quaternary structure via dimeric intermediates. The first rate-limiting step is the association of structured monomers to form dimers in a bi-molecular reaction, with a rate constant of 4.3x10(3) M-1 s-1 at 20 degreesC. The second rate-limiting uni-molecular folding step leads to dimers which are competent for further association, with a rate constant of 0.5x10(-3) s-1 at 20 degreesC. Tetramers form from these dimers in a fast reaction. By determining a similar mechanism for alpha-complementation of beta-galactosidase fragments it could be confirmed that beta-galactosidase follows a consecutive bi-uni-molecular mechanism of folding and association.

Dimerization↗

PubMed2002

Adenovirus-transduced lung as a portal for delivering alpha-galactosidase A into systemic circulation for Fabry disease.

Gene therapy efforts have focused primarily on the use of either the liver or skeletal muscle as depot organs for the production of a variety of therapeutic proteins that act systemically. Here we examined the lung to determine whether it could function as yet another portal for the secretion of proteins into the circulation. Fabry disease is caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A, resulting in the abnormal deposition of the glycosphingolipid globotriaosylceramide (GL-3) in vascular lysosomes. Pulmonary instillation of a recombinant adenoviral vector (Ad2/CMVHI-alpha(gal)) encoding human alpha-galactosidase A into Fabry mice resulted in high-level transduction and expression of the enzyme in the lung. Importantly, enzymatic activity was also detected in the plasma, liver, spleen, heart, and kidneys of the Fabry mice. The detection of enzymatic activity outside of the lung, along with the finding that viral DNA was limited to the lung, indicates that the enzyme crossed the air/blood barrier, entered the systemic circulation, and was internalized by the distal visceral organs. The levels of alpha-galactosidase A attained in these tissues were sufficient to reduce GL-3 to basal levels in the lung, liver, and spleen and to approximately 50% of untreated levels in the heart. Together, these results suggest that the lung may be a viable alternate depot organ for the production and systemic secretion of alpha-galactosidase A for Fabry disease.

Adenoviridae↗

PubMed1995

beta-Galactosidase transgene expression in transplanted rabbit retinal pigment epithelial cells in vivo.

BACKGROUND: Intraocular transplantation of genetically modified cells that release a particular substance could have a major impact on the treatment of various ocular diseases. We studied the expression of the reporter gene beta-galactosidase (lacZ) in transplanted retinal pigment epithelial (RPE) cells in vivo. METHODS: RPE cells from pigmented rabbits were transduced with the beta-galactosidase gene in a retroviral vector. Cells were then assayed for gene expression and transplanted subretinally into the eyes of New Zealand White rabbits. RPE cells that were transduced with a similar vector without the beta-galactosidase gene were used as controls. Rabbits were killed on days 1, 7, and 21 and the eyes processed for transmission electron microscopy RESULTS: Neomycin-resistant rabbit RPE cells that showed beta-galactosidase activity were generated within 2-5 weeks. After transplantation, viable RPE cells that expressed the transgene and that phagocytosed rod outer segments were observed on days 1, 7, and 21 CONCLUSIONS: The results show that generation of genetically modified RPE cells is feasible and that the transplanted cells remain viable and continue to express the transgene in the subretinal space of the host animal for at least 21 days. Transplantation of such genetically modified RPE cells could provide a new tool for studying retinal diseases and, potentially, for correcting metabolic abnormalities in retinal degenerations and dystrophies.

3T3 Cells↗

PubMed1992

Proteolytic response to the expression of an abnormal beta-galactosidase in Escherichia coli.

Because induction of proteolytic activity and stress-response proteins can significantly affect expression levels in recombinant Escherichia coli, the influence of low-level expression of a mutant beta-galactosidase was investigated. A single copy of the well-characterized CSH11 mutant of the lacZ gene was integrated into the chromosome. Induction of expression of the mutant beta-galactosidase caused a measurable increase in ATP-dependent intracellular proteolytic activity but resulted in no significant change in ATP-independent proteolytic activity. Growth at temperatures above 40 degrees C resulted in a significant decrease in the level of ATP-independent proteolytic activity compared to growth at 37 degrees C, and the ATP-dependent activity increased 2.5-fold from 30 to 42 degrees C. Synthesis of stress-response proteins was evident in two-dimensional gel electrophoresis analysis of proteins in the strain expressing the abnormal beta-galactosidase at 37 degrees C, but no such response was evident when mutant beta-galactosidase expression was induced at 30 degrees C. In separate experiments, stress proteins were overexpressed by inducing expression of the htpR gene on a plasmid. Resulting increases in stress-protein levels correlated with an increase in ATP-dependent proteolytic activity with no significant change in the intracellular ATP-independent proteolytic activity. These data suggest that even very low levels of abnormal protein can substantially influence protease levels and stress response in E. coli. These responses were reduced by induction at lower temperatures.

Adenosine Triphosphate↗

PubMed1994

Improved in situ beta-galactosidase staining for histological analysis of transgenic mice.

The present study describes a novel method for the histochemical demonstration of beta-galactosidase activity on tissue sections. We have replaced 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) with 5-bromo-indolyl-beta-o-galactopyranoside (Bluo-Gal) as a chromogenic substrate for the bacterial beta-galactosidase (lacZ). After beta-galactosidic cleavage, Bluo-Gal precipitates in form of fine birefringent crystals, whereas X-gal gives rise to an amorphous precipitate. Upon microscopic examination under polarized light, the crystals emit a strong signal consisting of yellow reflected light. This property of Bluo-Gal results in greatly enhanced sensitivity of the staining method for beta-galactosidase and allows for optimal morphological resolution. To exemplify the applications of this technique, the expression is demonstrated in transgenic mice of beta-galactosidase driven by a fragment of the human tissue-type plasminogen activator promoter.

Animals↗

PubMed1976

Generalized gangliosidosis: acid beta-galactosidase deficiency with early onset, rapid mental deterioration and minimal bone dysplasia.

This report concerns a 3-month-old girl with rapidly progressive psychomotor retardation, hepatomegaly, vacuolated lymphocytes, minimal bone dysplasia and normal excretion of acid mucopolysaccharides. A deficiency of acid beta-galactosidase was demonstrated in isolated leucocytes and in a liver biopsy. The diagnosis of generalized gangliosidosis due to deficiency of beta-galactosidase was also based on the absence of the enzyme activity from cultured fibroblasts. The diagnosis was confirmed on autopsy at 16 months by typical histology, electron microscopy and biochemistry of the organs. beta-galactosidase deficiency has been demonstrated in various clinical conditions ranging from generalized gangliosidosis with severe mental retardation to clinical pictures resembling Morquio's disease and normal intelligence. The heterogeneity of the clinical manifestations in beta-galactosidase deficiency could be explained by different residual activities of a structurally mutated enzyme towards its various substrates.

Bone Diseases, Developmental↗

PubMed1996

Location of the beta-galactosidase of the yeast Kluyveromyces marxianus var. marxianus ATCC 10022.

During the growth of Kluyveromyces marxianus var. marxianus ATCC 10022 on lactose, peaks of glucose, but not beta-galactosidase activity, were detected in culture medium. Harvested and washed whole cells produced glucose and galactose from lactose, or ortho-nitro-phenol from the chromogenic substrate ortho-nitro-phenyl-beta-D-galactopyranoside (ONPG), indicating that beta-galactosidase is physically associated with cells. ONPG hydrolysis by whole cells presented a monophasic kinetics (Km 36.6 mM) in lactose exponential growth phase cells, but a biphasic kinetics (Km 0.2 and 36.6 mM) in stationary growth phase cells. Permeabilization with digitonin or disruption of cells from both growth phases led to monosite ONPG hydrolysis (Km 2.2 to 2.5 mM), indicating that beta-galactosidase is not located in the periplasm. In addition, the energy inhibitors fluoride or arsenate, as well as the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) prevented ONPG hydrolysis by whole cells. These findings indicate that energy coupled transmembrane transport is the rate-limiting step for intracellular ONPG cleavage. The taxonomic and physiologic implications of the exclusive intracellular location of beta-galactosidase of K. marxianus var. marxianus ATCC 10022 are discussed.

Arsenates↗

PubMed1976

Effects of the Bgs locus on mouse beta-galactosidase.

The Bgs locus determines tissue levels of beta-galactosidase in the mouse, so that enzyme levels are twice as high in mice carrying the Bgsh allele as in mice carrying the Bgsd allele (Felton et al., 1974). By immunotitration with antiserum to purified beta-galactosidase, we have found that the Bgs locus influences the amount of enzyme protein present in the tissues. We have utilized recombinant inbred lines derived from a cross between C57BL/6J and DBA/2J mice to confirm the location of the Bgs locus on chromosome 9. The inhibition of mouse beta-galactosidase by the active-site-directed reagent N-bromoacetyl-beta-D-galactosylamine has been investigated. beta-Galactosidase from the high and low Bgs strains has identical affinity for this inhibitor.

Acetylgalactosamine↗

PubMed1997

Beta-galactosidase as a marker of HSP70 promoter induction in Dunning R3327 prostate carcinoma cells.

Hyperthermia is known to improve the response of tumors to radiation or chemotherapeutic treatment when combined in multimodal strategies. The cellular response to hyperthermia is associated with the synthesis of heat shock proteins (HSP). To study the stress response in prostate cancer we have developed a clone of Dunning R3327 rat prostate carcinoma cells stably transfected with a gene construct containing the E. coli beta-galactosidase gene driven by the Drosophila HSP70 promoter. The measurement of beta-galactosidase serves as a rapid and semiquantitative assay of HSP70 gene activation. The Dunning cell clone showed evidence of incorporation of the HSP70/beta-galactosidase construct within the genomic DNA by Southern blot analysis. When compared to mock-transfected control cells, the clone showed minimal baseline beta-galactosidase activity, which significantly increased following a hyperthermic stress. The time course of beta-galactosidase elevation following heat stress paralleled the time course of cellular HSP70 elevation by Western blot analysis. These stably transfected Dunning R3327 cells may provide a useful tool to study the effects of hyperthermia, radiation, and chemotherapeutic agents on the cellular stress response and in the establishment of HSP70 as a marker of cellular resistance in the multimodal treatment of prostate cancer.

Adenocarcinoma↗

PubMed1994

Expression of beta-galactosidase and luciferase in insect cell lines infected with a recombinant AcMNPV.

An AcMNPV recombinant (Ac-gal-luc) carrying the beta-galactosidase and luciferase genes under the control of the p10 and polyhedrin promoters, respectively, was used to study expression in nine insect cell lines. All AcMNPV-permissive cell lines expressed both reporter genes with the coleopteran cell line, Anthonomus grandis (AGE), producing the highest concentrations of beta-galactosidase (5.0 x 10(6) pg/ml) and luciferase (2.67 x 10(3) pg/ml). Both enzymes were detected as early as 12 h postinoculation in lysate samples of the AGE cell line. Helicoverpa armigera (HA), a nonpermissive cell line, expressed beta-galactosidase at 72 h postinoculation at a concentration of 3.5 x 10(3) pg/ml. However, expression of luciferase was not detected. Expression of luciferase and beta-galactosidase was also not detected in the nonpermissive Helicoverpa zea (HZ) cell line.

Animals↗

PubMed1999

Characterization and some reaction-engineering aspects of thermostable extracellular beta-galactosidase from a new Bacillus species.

A new strain of Bacillus sp. was isolated from a hot water spring in India. This strain generated a high activity of extracellular beta-galactosidase at 37 degrees C in shake flasks. The beta-galactosidase activity was found to increase continuously but the production rate was slower than with some other organisms reported in the literature. There were noteworthy differences in the time-domain profiles of bacterial concentration and beta-galactosidase activity when the starting concentration of substrate (glucose) was tripled from 10 g/L. These differences may be explained in terms of the relative rates of enzyme synthesis and its diffusion across the cell wall. The enzyme produced by this organism is more stable than other beta-galactosidases; its half-life is 408 h at 50 degrees C and 94 h at 55 degrees C, while the reported enzymes showed perceptible loss of activity within 2 h.

Bacillus↗

PubMedDate not supplied

Treatment of lactose intolerance with exogenous beta-D-galactosidase in pellet form.

The effectiveness of a new beta-D-galactosidase pellet formulation in the treatment of lactose intolerance was studied. The encapsuled beta-D-galactosidase (lactase) pellets were first tested in vitro for their enzymatic activity within an environment simulating gastric conditions and subsequently within an environment simulating duodenal conditions. Effectiveness was measured by the % of glucose formed by hydrolysis of lactose. The pellets were found to retain their enzymatic activity in gastric pH conditions (mean 69 +/- 1 mg/dl glucose) and were found to hydrolyse lactose in human duodenal fluid (106.35 +/- 1 mg/dl). Finally the effectiveness of the new lactase formulation on glucose absorption was studied in 8 lactose intolerant subjects in a randomized, double blind, crossover trial. After fasting, the subjects were given one capsule containing 100 u/ml beta-galactosidase (i.e. 10 pellets of 10 u/ml each) or one capsule containing placebo pellets, followed by 100 g lactose dissolved in water. The washout period between lactose challenges was one week. Plasma glucose concentrations were measured before and at intervals after the challenges and the subjects completed symptom questionnaires every eight hours for 24 hours. Results showed a statistically significant increase in plasma glucose levels 30, 60, 90 and 120 min after lactose ingestion (repeated measures analysis of variance, p<0.01). Subjective ratings of the severity of abdominal cramping, belching, flatulence, vomiting and diarrhoea were significantly decreased following ingestion of the lactase pellets and lactose (no incidence of diarrhoea) compared with after ingestion of placebo and lactose. The results of the study were considered to be very promising as the beta-D-galactosidase formulation (which was produced at very low cost and with great ease) resisted inactivation in the stomach, effectively transformed lactose to glucose in vivo and reduced symptoms of lactose intolerance.

Adult↗

PubMed2005

Secreted beta-galactosidase from a Flavobacterium sp. isolated from a low-temperature environment.

The bacterial strain Flavobacterium sp. 4214 isolated from Greenland was found to express beta-galactosidase (EC 3.2.1.23) at temperatures below 25 degrees C. A chromosomal library of Flavobacterium sp. 4214 was constructed in Escherichia coli, and the gene gal4214-1 encoding a beta-galactosidase of 1,046 amino acids (114.3 kDa) belonging to glycosyl hydrolase family 2 was isolated. This was the only gene encoding beta-galactosidase activity that was identified in the chromosomal library. Expression levels in both Flavobacterium sp. 4214 and in initial recombinant E. coli strains were insufficient for biochemical characterization. However, a combination of T7 promoter expression and introduction of an E. coli host that complemented rare transfer RNA genes yielded 15 mg of beta-galactosidase per liter of culture. Gal4214-1-His protein was found to be active in monomeric conformation. The protein was secreted from the cytoplasm, probably through an N-terminal signaling sequence. The Gal4214-1-His protein was found to have optimum activity at a temperature of 42 degrees C, but with short-term stability at temperatures above 25 degrees C.

Amino Acid Sequence↗

PubMed1996

The effect of cations on the hydrolysis of lactose and the transferase reactions catalysed by beta-galactosidase from six strains of lactic acid bacteria.

beta-Galactosidases from Lactobacillus delbruekii subsp. bulgaricus 20056, Lb. casei 20094, Lactococcus lactis subsp. lactis 7962, Streptococcus thermophilus TS2, Pediococcus pentosaceus PE39 and Bifidobacterium bifidum 1901 were partially purified. The rate of hydrolysis of lactose given by the predominant beta-galactosidase activity from each of the bacteria studied was in all cases enhanced by Mg2+, while the effect of K+ and Na+ differed from strain to strain. The beta-galactosidases from all strains also catalysed trans-galactosylation reactions. The types of oligosaccharides produced appeared to be very similar in each case, but the rates of their production differed. All the beta-galactosidases were also capable of hydrolysing galactosyl-lactose although, unlike the other bacteria studied, Lb. delbruekii subsp. bulgaricus 20056 and Lc. lactis subsp. lactis 7962 were unable to utilise galactosyl-lactose as a carbon source for growth.

Cations↗

PubMed2002

The beta-galactosidase activity in Kluyveromyces marxianus CBS6556 decreases by high concentrations of galactose.

In this paper we report on the effect of different concentrations of lactose and galactose in the production of beta-galactosidase by Kluyveromyces marxianus CBS6556. The results clearly demonstrate a decrease in enzyme specific activity during cultivation at high concentrations of L-lactose or D-galactose, despite the fact that these carbohydrates are normally used for induction of the beta-galactosidase activity. Therefore, maximum induction of beta-galactosidase in K. marxianus batch cultures was obtained at low concentrations of the inducer carbohydrates, in the range between 0.5 to 15 mM. Those informations can help to design low cost medium with higher beta-galactosidase productivity by K. marxianus cells.

Culture Media↗

PubMed2006

Effects of carbon source and Vitreoscilla hemoglobin (VHb) on the production of beta-galactosidase in Enterobacter aerogenes.

At fixed concentration (0.5%), lactose and galactose acted as inducers while glucose and other tested carbon sugars showed repression effects on beta-galactosidase production in Enterobacter aerogenes strain. The expression of Vitreoscilla hemoglobin gene (vgb) in this bacterial strain managed to overcome the repression effects as well as improving the induction of beta-galactosidase formation by carbon sources. In parallel, the bacterial O(2) consumption was increased correspondingly to the vgb induction of beta-galactosidase synthesis. When Enterobacter aerogenes strains were grown at the incubation temperature 42 degrees C, about 5-fold higher enzyme productivity was obtained than with a similar incubation at 37 degrees C. The bacterial growth expressed as biomass yield had a different optimum temperature and was not influenced to the same extent by variations in the carbon sources. These data are discussed in terms of proposed enhancement in beta-galactosidase productivity by vgb expression as well as its significance to improve the technology of whey processing.

Bacterial Proteins↗

PubMed1999

Alpha-galactosidase of Bifidobacterium adolescentis DSM 20083.

Bifidobacterium adolescentis was grown anaerobically in medium enriched with alpha-D-galactosides. alpha-Galactosidase (EC 3.2.1. 22) was released from the cells by ultrasonic treatment and purified 36-fold by ultrafiltration, ammonium-sulphate precipitation, anion-exchange chromatography, and size-exclusion chromatography. Two protein bands were consistantly observed after sodium-dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoretically homogeneous alpha-galactosidase was only obtained by electroelution. The enzyme had an apparent molecular mass of 344 kDa and 79 kDa as judged by size-exclusion chromatography and SDS-PAGE, respectively. Activity-staining after nondenaturing SDS-PAGE indicated an apparent molecular mass of 145 kDa. Thus, a tetrameric structure of the protein is suggested. The alpha-galactosidase showed optimal activity at pH 5.5 and 55 degrees C. Lower pH values and higher temperatures rapidly inactivated alpha-galactosidase. The enzyme hydrolyzed specifically alpha-galactosidic linkages, and alpha-(1-3)-linkages were hydrolyzed at a higher rate compared to alpha-(1-6)-linkages. Hydrolysis of galactosides followed normal saturation kinetics; KM-values for p-nitrophenyl-alpha-galactopyranoside (p-NPG) and raffinose were calculated with 0.957 mM and 4.12 mM, respectively.

Bifidobacterium↗