Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The fruit extracts of ripening cv. Harumanis mango contained a number of glycosidases and glycanases. Among the glycosidases, beta-D-galactosidase (EC 3.2.1.23) appeared to be the most significant. The enzyme activity increased in parallel with increase in tissue softness during ripening. Mango beta-galactosidase was fractionated into three isoforms, viz. beta-galactosidase I, II and III by a combination of chromatographic procedures on DEAE-Sepharose CL-6B, CM-Sepharose and Sephacryl S-200 columns. Apparent Km values for the respective beta-galactosidase isoforms for p-nitrophenyl beta-D-galactoside were 3.7, 3.3 and 2.7 mM, and their Vmax values were 209, 1024 and 62 nkat mg-1 protein. Optimum activity occurred at ca pH 3.2 for beta-galactosidase I and II, and pH 3.6 for beta-galactosidase III. Mango beta-galactosidase and its isoforms have galactanase activity, and the activity of the latter in the crude extracts generally increased during ripening. The close correlation between changes in beta-galactosidase activity, tissue softness, and increased pectin solubility and degradation suggests that beta-galactosidase might play an important role in cell wall pectin modification and softening of mango fruit during ripening.
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PMID:beta-Galactosidase and its significance in ripening mango fruit. 776 93

The dismal results of conventional therapy for primary malignant brain tumors has justified exploring gene therapy approaches for this disease. Transduction of animal brain tumor models in vivo has been reported previously with retroviruses and herpes viruses. Because adenoviruses have the advantage of transducing quiescent and actively dividing tumor cells, they may prove to be more effective in such therapy. We used a replication-deficient recombinant adenovirus bearing the Escherichia coli beta-galactosidase gene in a rat C6 glioma tumor model. Transduced cells were detected by X-5-bromo-4-chloro-3-indolyl beta-D-galactoside staining to reveal beta-galactosidase activity. Initial experiments in vitro showed 50% and 90% transduction at vector titers of approximately 10(7) and 10(8) plaque-forming units/ml, respectively. Although no cytopathic effects were seen at 10(7) plaque-forming units/ml, more than 50% reduction in tumor cell growth was noted at 10(8) plaque-forming units/ml both in vitro and in vivo. Stereotactic delivery of the recombinant adenovirus into the frontal lobe of normal rat brains resulted in intense staining of all cell types, that is, neurons, astrocytes, and ependymal cells. Stereotactic injection into C6 glioma brain tumors in rats stained 25 to 30% of the tumor cells. We conclude that adenovirus vectors can be used to transfer genes to central nervous system tumors in vivo. Using stereotactic delivery, adenovirus vectors can transfer genes into the central nervous system intended for tumor therapy.
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PMID:Stereotactic delivery of a recombinant adenovirus into a C6 glioma cell line in a rat brain tumor model. 783 41

We describe here a simple and efficient transfection method for transient expression of cloned genes in cell lines and primary cultured cells. The method involves the use of DEAE-dextran to target DNA to the cellular endocytotic pathway and the use of a human adenovirus to ensure efficient lysis of endosomal vesicles. The procedure allows effective delivery of DNA into the cytoplasm and, therefore, results in a higher fraction of cells expressing exogenous proteins. Using this method, we routinely obtain 60%-90% of COS cells or Chinese hamster ovary cells expressing beta-galactosidase, as determined by in situ staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). We have also obtained much improved levels of expression in cells that are difficult or impossible to use in transient expression assays, such as rat-1 fibroblasts or primary osteoblast cultures. We successfully used the method to express heteromeric proteins that require subunit assembly for proper function. The method also proved effective to express functions in which the exogenous protein needs to couple to the endogenous cellular machinery. Thus, this transient transfection method should prove valuable for many functional studies in a broad variety of cell lines and primary cultures.
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PMID:Adenovirus-mediated transfection of cultured cells. 798 Sep 40

A kinetic assay for the expression of beta-galactosidase in cells transfected with the LacZ gene was developed using a 96-well-plate format. The assay involves solubilization of the cells followed by measuring hydrolytic rates of o-nitrophenyl beta-D-galactoside on a standard 96-well-plate reader without other manipulations. The protocol requires only that reagent be added sequentially to the wells at ambient temperatures, thus permitting a semiautomated or fully automated determination of reporter expression. The rates of chromophore development were found to be linear over a 6-log enzyme concentration range, from 0.001 to 100 mU. Additionally, the use of kinetic data avoids the complications of non-enzymatic, background optical density.
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PMID:Kinetic determination of cellular LacZ expression. 806 Jun 77

For the hydrolysis of the two glycosidic bonds of fluorescein di-beta-D-galactoside (FDG) by beta-galactosidase from Escherichia coli, small [Hofmann, J. & Sernetz, M. (1983) Anal. Biochem. 131, 180-186] to dramatic [Huang, Z. (1991) Biochemistry 30, 8535-8540] deviations from simple stepwise substrate-intermediate-product kinetics have been reported. Intermediate channelling, a preferred hydrolysis of the intermediate fluorescein mono-beta-D-galactoside (FMG) formed from FDG at the active site and thus in a favourable position for further reaction, has been postulated. As there were reasons to doubt the previous findings and conclusions, the hydrolysis experiments have been repeated at initial FDG concentrations of 7-200 microM, following the concentrations of FDG, FMG and fluorescein with a reliable method, quantitative HPLC, to completion of the reaction. The transient appearance of substantial amounts of the intermediate FMG also in experiments with 200 microM FDG already rules out the existence of the most efficient intermediate channelling deduced by Huang (1991) from measurements of the initially developing fluorescence, incorrectly ascribed to fluorescein. Redetermination of the Michaelis constants for FDG and FMG led to much higher values than those reported previously. Fitting the progress curves by means of nonlinear regression combined with numerical integration of the rate equations resulted in good fits of the normal stepwise substrate-intermediate-product mechanism, without any necessity of assuming a more complex course of the reaction. So one of the rare examples of the hydrolysis of two bonds at a single enzyme-substrate encounter has been invalidated.
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PMID:No intermediate channelling in stepwise hydrolysis of fluorescein di-beta-D-galactoside by beta-galactosidase. 820 Mar 55

The absorption maximum of p-nitrophenol upon mixing with molar equivalents of alpha-cyclodextrin (alpha-CD) or hydroxypropyl-beta-cyclodextrin (HPB) showed nearly 5 nm shift to the longer wavelength region, indicative of complex formation, while beta-cyclodextrin (beta-CD), gamma-cyclodextrin (gamma-CD) and hydroxyethyl-beta-cyclodextrin (HEB) produced only a marginal shift of about 1-2 nm, suggestive of a weaker interaction. It has been shown by circular dichroism spectral studies that the aglycon part of p-nitrophenyl-beta-D-glycoside (PNPG) is also encapsulated by alpha-cyclodextrin. The encapsulated form of PNPG could be hydrolyzed by beta-galactosidase, the temperature and pH-optima for hydrolysis of anchored substrates being essentially identical to that of free substrate. However, small but consistent increase in Km values were obtained for alpha-cyclodextrin-, HEB- and HPB-anchored substrates. The kcat values also registered an increase for the HEB- and HPB-anchored substrates. However, there was no increase in kinetic efficiency (kcat/Km) of enzyme. The inhibition noted at higher concentrations of HEB- and gamma-cyclodextrin-anchored PNPG but not with o-nitrophenyl-beta-D-galactoside (ONPG)-cyclodextrin mixture suggests that PNPG-cyclodextrin complexes were responsible for the inhibition. Taken together, these results suggest that the enzyme catalyses the hydrolysis of anchored substrates.
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PMID:Evidence for beta-galactosidase catalyzed hydrolysis of paranitrophenyl-beta-D-galactopyranoside anchored in cyclodextrins. 827 24

We have investigated whether three-dimensional cultivation of cells to multicell spheroids influences the expression of a transfected gene. Ltk- cells (mouse fibroblasts, thymidine kinase negative) have been transfected with a bacterial lacZ gene which was coupled to a beta-actin promoter. The transfected cells synthesize beta-galactosidase, a cytoplasmic enzyme which can easily be stained for histology with 5-bromo-4-chloro-3-indoxyl beta-D-galactoside and for cytometry with fluorescein di-(beta-D-galactopyranoside). As we have shown with monolayer cells, beta-galactosidase is produced independently of cell density, medium condition, and cell cycle. In multicell spheroids, however, the portion of producing cells was reduced from approximately 98% to approximately 2% within a week. This reduction is also independent of cell density, medium condition, and cell cycle. Nonproducing multicell spheroid cells, however, regained their ability to synthesize beta-galactosidase within a few days when the cells were recultivated as monolayers. Since the lacZ gene was not lost, its expression might have been regulated by its beta-actin promoter. We, therefore, investigated whether the endogenous synthesis of beta-actin was similarly regulated. A correlation between the distinct reduction in beta-galactosidase-producing cells and filamentous or total actin concentration was not unequivocally observed.
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PMID:Beta-galactosidase activity in transfected Ltk- cells is differentially regulated in monolayer and in spheroid cultures. 831 68

In the present report, we established a K562 cell line useful for an enzyme release assay of human natural killer (NK) activity. Human myelogenous leukemia cell line, K562, was transfected with a plasmid carrying Escherichia coli beta-galactosidase (beta-gal) gene. A colony that permanently expresses the enzyme activity was isolated, and designated K562/Zneo. Incubation of K562/Zneo cells (1 x 10(4)) with nonadherent human peripheral blood lymphocytes (PBL) resulted in the release of beta-gal activity depending on the incubation time and the number of effector cells. Released beta-gal activity was assayed sensitively by using 4-methylumbelliferyl-beta-D-galactoside, a fluorescent substrate. The cytolytic activity of PBL was augmented significantly when the cells were preincubated with interleukin-2 for 20 h. This enzyme release assay showed a comparable sensitivity to that of 51Cr release assay. Thus, K562/Zneo cell line is thought to be useful for the nonradioactive assay of human NK and lymphokine-activated killer activities.
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PMID:Enzyme release assay of human NK cell activity using beta-galactosidase-expressing K562 target cell line. 836 May 3

We have combined a receptor-mediated DNA delivery system with the endosomal lysis ability of adenovirus and shown that DNA can be delivered into primary hepatocytes, resulting in a high level of gene expression. When asialoorosomucoid conjugated with poly(L-lysine) was used to deliver the Escherichia coli beta-galactosidase gene into primary hepatocytes through binding with the hepatic asialoglycoprotein receptor, only a low level of beta-galactosidase was detectable, with less than 0.1% of the hepatocytes being transfected. This level of activity can be greatly enhanced by the cointernalization of the DNA.protein complex with a replication-defective adenovirus, resulting in 100% of the hepatocytes staining blue with 5-bromo-4-chloro-3-indolyl beta-D-galactoside. Quantitative analysis of beta-galactosidase expression also showed a 1000-fold enhancement of activity. To test the applicability of this DNA delivery system for the correction of phenylketonuria, a metabolic disorder that causes severe mental retardation in children, we have delivered the human phenylalanine hydroxylase (PAH) gene to hepatocytes derived from a PAH-deficient mouse strain and demonstrated complete reconstitution of enzymatic activity. This method shows great promise for efficient gene delivery to the liver for correction of hepatic disorders.
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PMID:Hepatic gene therapy: adenovirus enhancement of receptor-mediated gene delivery and expression in primary hepatocytes. 838 12

The cysteine-rich trefoil motif of rat intestinal trefoil factor (rITF) was cloned and expressed in Escherichia coli. A 270-bp cDNA fragment including the signal sequence and the trefoil motif was cloned into the expression vector pAX5+ to direct the expression of a beta-galactosidase collagen-hinged fusion protein in E. coli. Cultures harbouring the recombinant plasmid produced a soluble novel protein with a molecular mass of 134.5 kDa, as predicted for the trefoil-motif-containing fusion protein. Purification of the rITF moiety was achieved by p-aminophenyl-thio-beta-D-galactoside(APTG)-affinity chromatography, collagenase digestion of the hybrid molecule, and removal of the beta-galactosidase-hinge molecule by a further APTG-affinity step. It was demonstrated that intrachain disulphide-bond formation in rITF occurred during the procedure, so no refolding steps were required. Analysis by immunoblotting revealed that the fusion protein and the cleaved trefoil-motif-containing protein were recognised by an antibody raised against the chemically synthesised peptide. The trefoil motif present in the fusion protein was used to localise putative trefoil-binding sites in sections of frozen rat tissue. Binding was demonstrated using the beta-galactosidase portion of the fusion protein as a reporter moiety, either directly with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside, or indirectly using a monoclonal antibody to beta-galactosidase and indirect immunohistochemistry. Binding sites were localised to the foveolar and surface epithelium of rat stomach, the collecting ducts of the kidney and within colonic crypts. The presence of a trefoil motif was necessary for binding. The use of beta-galactosidase fusion proteins for histochemical localisation of peptide-binding sites should prove more generally useful.
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PMID:Expression and purification of a trefoil peptide motif in a beta-galactosidase fusion protein and its use to search for trefoil-binding sites. 844 92


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