Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

A 3.3-kilobase DNA complementary to human microtubule-associated protein 2 (MAP2) was sequenced by the dideoxy method. The 3' end terminates at an internal EcoRI site before the polyA tail. Due to the arrangement of the cDNA insert in the lambda gt11 vector, the MAP2 fragment is not fused to beta-galactosidase when expressed. The Chou Fasman algorithm for the initial 58 amino acids from the first in-frame methionine predicts an alpha helix. Beyond this point, a series of turns is predicted until amino acid 160. The frequent presence of basic residues in proximity to serines or threonines is consistent with multiple phosphorylation sites. The minimum specificity determinant for Ca2+/calmodulin-dependent kinase is repeated 13 times. The sequence of a region containing a MAP2 epitope that is shared with the Alzheimer neurofibrillary tangle was determined by DNase treatment of the cDNA and antibody selecting the small resultant clones in a lambda gt11 sublibrary. Likewise, a MAP2 epitope that is not shared with the neurofibrillary tangle also has been located. Both epitopes are in the projection portion of the molecule. A bovine MAP2 cyanogen bromide fragment, which contains the epitope shared with the neurofibrillary tangle, is partially insoluble under aqueous conditions, probably due to the aggregation of oppositely charged residues. Thus, rapid cleavage of MAP2 to small peptides is probably necessary in vivo to prevent the aggregation of larger cleavage fragments.
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PMID:Partial sequence of MAP2 in the region of a shared epitope with Alzheimer neurofibrillary tangles. 245 76

A method was developed for determining the specific activity of bacterial beta-galactosidase (EC 3.2.1.23) during growth of Streptococcus thermophilus and Lactobacillus bulgaricus in skim milk. Individual and mixed strain cultures of S. thermophilus (St 3642, St14485) and L. bulgaricus (Lb11842, Lb880) were examined for growth (OD at 600 nm and viable cell counts), acid production, and beta-galactosidase activity (expressed as a function of recoverable TCA-precipitable cellular protein). Cultures were inoculated into 10% skim milk (2% inoculum) and incubated at 40 degrees C for 12 h. Aliquots were removed at 2-h intervals and diluted with ice cold EDTA, pH 12. The EDTA chelates calcium and solubilizes milk protein, allowing separation of the bacteria by centrifugation. Cells were then washed twice with 20 mM phosphate buffer and disrupted by sonication. Cell debris and intact cells were removed by centrifugation and the cell-free extract evaluated for beta-galactosidase activity using o-nitrophenyl-beta-D-galactopyranoside as substrate. Specific activities ranged from 0 to 6 units/mg protein. This simple and reproducible method is applicable for enzyme assays and measurement of cellular components where contamination by milk proteins is a potential problem.
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PMID:A method for determining beta-galactosidase activity of yogurt cultures in skim milk. 249 15

An efficient, simple, and reproducible DNA-mediated gene transfer procedure has been developed for primary cultures of adult rat hepatocytes. Calcium phosphate-DNA precipitate is formed in complete culture medium during 5 hr incubation with cells. Unabsorbed precipitate is then washed out, and 40 hr later gene expression is measured. Under optimal conditions, up to 20-25% of cells in cultures transfected with the beta-galactosidase (lacZ) gene stain positively for this activity, and cells transfected with the chloramphenicol acetyl transferase (CAT) gene, fused to a strong promoter, express CAT activities of 10-14 nmoles/min per mg protein. Five conditions were optimized based on transfection efficiency, CAT expression, and cell viability. (i) Medium composition: the presence of protein, such as fetal bovine serum or bovine serum albumin, in the medium was essential. (ii) Cell substratum: tissue culture plastic was superior to calf skin collagen and Matrigel. (iii) Cell density: 0.5-1.0 X 10(6) cells/60-mm dish were superior to higher densities. (iv) Duration of exposure to calcium phosphate-DNA: 5-8 hr was better than shorter or longer times. (v) Length of time hepatocytes were maintained in culture before initiating transfection: 2-3 days was superior to earlier times. This procedure was successful with reporter genes linked to three different eukaryotic promoters. These included a chimeric promoter containing the polycyclic aromatic hydrocarbon-responsive enhancer of the cytochrome P450c gene (CYP1A1), which was shown to confer upon the CAT gene responsiveness to polycyclic aromatic hydrocarbons comparable to that of the native P450c gene. This transfection procedure should be of considerable use for the study of liver-specific gene expression in primary hepatocyte cultures.
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PMID:Efficient DNA-mediated gene transfer into primary cultures of adult rat hepatocytes. 250 73

Endothelial injury has been proposed as a feature of a wide variety of vascular diseases, and release of endothelial lysosomal hydrolases could contribute to the pathological changes seen. We have determined the relative activities of 14 glycosidases, two esterases and four peptide hydrolases in human umbilical vein endothelial cells and investigated whether known agonists of endothelial function, or materials known to modulate hydrolase secretion in other phagocytic cells, influenced the activity or secretion of these enzymes by human umbilical vein endothelial cells. Hexosaminidase, beta-galactosidase, beta-glucuronidase and alpha-iduronidase accounted for most of the measured glycosidase activity. Acid phosphatase activity greatly exceeded arylsulphatase activity, and most of the measured peptidase activity was due to acid peptidases. Optimum pH and apparent Km values were determined for the most abundant hydrolases. Exposure of human umbilical vein endothelial cells to bradykinin, thrombin or interleukin-1 resulted in negligible release of either hexosaminidase or lactate dehydrogenase (LDH), in contrast to phorbol myristate acetate, which caused a parallel, dose-dependent release of both enzymes. Treatment of these cells with calcium ionophore A23187, trypsin or platelet-activating factor, caused less than 10% release of either hexosaminidase or LDH. Agents known to modulate lysosomal enzyme secretion by other phagocytic cells failed to induce selective secretion of lysosomal enzymes by human umbilical vein endothelial cells.
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PMID:Lysosomal hydrolases of human vascular cells: response to agonists of endothelial function. 264 39

Efficient transfection of Spodoptera frugiperda cells with Autographa californica nuclear polyhedrosis virus (AcNPV) DNA has been carried out using the technique of electroporation. The efficiency of transfection was monitored by assaying the extracellular virus in cell culture supernatants 3 days post-electroporation. Maximum titres of 2 x 10(9) p.f.u./ml AcNPV were obtained when using a pulse length of 7.7 ms and a field strength of 500 V/cm. This compared with a titre of 2 x 10(6) p.f.u./ml AcNPV using the standard calcium phosphate transfection method. Cotransfections of wild-type AcNPV DNA and the transfer plasmid pAcRP23-lacZ were also performed using electroporation and gave beta-galactosidase recombinant virus titres of 5 x 10(4) p.f.u./ml; this compared with 5 x 10(2) p.f.u./ml using the calcium phosphate method. The maximum proportion of recombinant virus, 2.9%, was obtained by harvesting the transfection medium after 2 days, and using a pulse length of 2.8 ms and a field strength of 750 V/cm. We therefore conclude that electroporation provides a very efficient method for the transfection of insect cells with baculovirus DNA.
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PMID:Efficient transfection of insect cells with baculovirus DNA using electroporation. 269 34

In this new colorimetric assay for Factor XIII in plasma, 5-(biotinamido)pentylamine is used as the amine substrate. Factor XIII, a zymogen, is transformed by thrombin and Ca2+ to active Factor XIIIa, and the incorporation of 5-(biotinamido)pentylamine into N,N-dimethylcasein is used to measure catalytically active Factor XIIIa. The biotinylated enzymatic product is immobilized onto 96-well microtiter plates, complexed with streptavidin-beta-galactosidase, and the absorbance at 405 nm is monitored for production of p-nitrophenol from p-nitrophenyl-beta-D-galactopyranoside. Concentrations of N,N-dimethylcasein, 5-(biotinamido)pentylamine, Ca2+, and thrombin were chosen to allow near-maximum velocity of amine incorporation. A linear relationship was obtained between assay product and plasma volume, from 0.5 to 50 microL of plasma. Results correlated well (r greater than 0.924) with those from the most frequently utilized radiometric filter-paper assay for Factor XIII. The method appears to be ideal for routine diagnostic estimation of Factor XIII in plasma because of its simplicity, its lack of use of radioisotopes, and its potential for assay of large numbers of samples by use of microtiter plates and automated plate readers.
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PMID:Colorimetric assay of blood coagulation factor XIII in plasma. 289 56

The binding sites in fibrinogen for Factor XIII were localized using an immunoblotting technique. Platelet Factor XIII bound to fibrinogen and to plasmin degradation products of fibrin(ogen) including Fragments: X, D1-D3, and D-dimer, but did not bind to Fragment E. Binding of Platelet Factor XIII was independent of calcium ions but could be inhibited by the presence of 0.5 M NaCl. Binding could also be inhibited by preincubating Factor XIII with a 100-fold molar excess of fibrinogen but not by 100-fold molar excess of Fragment E. Binding of Factor XIII to fibrinogen was specific, since several other proteins tested (ovalbumin, bovine serum albumin, alpha 2-macroglobulin, beta-galactosidase, fructose kinase, lactic dehydrogenase, triose phosphate isomerase, fumarase and pyruvate kinase) did not bind Factor XIII. Furthermore, binding was not observed either when Factor XIII was left out or when antiFactor XIII antiserum was substituted with nonimmune serum. When fibrinogen was reduced prior to electrophoresis, Factor XIII bound to the A alpha and B beta chains of fibrinogen and des A,B fibrinogen, the B beta-chain of Fragment X, but not the gamma-chains. Localization of the Factor XIII binding sites to the carboxy terminal segments of the A alpha and B beta chains in the Fragment D-domain of fibrinogen could have important physiological consequences.
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PMID:Factor XIII binds to the A alpha- and B beta- chains in the D-domain of fibrinogen: an immunoblotting study. 295 7

Depletion of macrophages from human peripheral blood mononuclear cells (PBMC) caused a marked decrease in galactose oxidase and sodium periodate, but not a calcium ionophore, stimulated Interferon-gamma (IFN-gamma) production. Reconstitution of such depleted cultures with galactose oxidase treated macrophages, but not lymphocytes, restored IFN-gamma levels to those of control nonfractionated PBMC. Thus, galactose oxidase seemed to act on macrophages which in turn stimulated lymphocyte production of IFN-gamma. Unlike human cells which have terminal galactose residues on glycoproteins, murine cell glycoproteins terminate their oligosaccharide component in the order N-acetyl-neuraminic acid followed by D-galactose, N-acetyl-glucosamine, and glycoprotein. Galactose oxidase or sodium periodate only activated murine macrophages to stimulate lymphocyte IFN-gamma production after exposing D-galactose residues by the removal of the terminal N-acetyl-neuraminic acid residues with neuraminidase. Removal of such exposed terminal galactose residues with beta-galactosidase inhibited the effect of galactose oxidase on murine macrophages. Taken together, these results strongly suggest that oxidation of terminal galactose residues on macrophages is the initial site of action of galactose oxidase and sodium periodate. Studies with Boyden chambers have shown that galactose oxidase-treated macrophages released a soluble factor which stimulates lymphocyte production of IFN-gamma. Based on these findings, it appears that the oxidation of terminal galactose residues on the surface of macrophages leads to the induction and transmission of a soluble signal for lymphocyte production of IFN-gamma.
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PMID:Generation of a soluble IFN-gamma inducer by oxidation of galactose residues on macrophages. 299 10

A cDNA portion coding for one of the repetitive regions of pig heart calpastatin (107 kDa) was subcloned into E. coli plasmid pUC119 to express the portion of the proteinase inhibitor gene in bacteria. The expressed protein was a chimaeric protein whose calpastatin segment (130 amino acid residues) was fused with an amino-terminus portion (7 amino acid residues) of beta-galactosidase. The chimaeric protein could inhibit proteolytic activity of calpain (Ca2+-dependent cysteine proteinase), and maintained properties of the authentic calpastatin concerning inhibition specificity and heat stability. These findings led us to conclude that the repetitive region is a functional unit of the proteinase inhibitor.
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PMID:Repetitive region of calpastatin is a functional unit of the proteinase inhibitor. 303 Mar 19

In order to know if the beta-galactosidase of the rat epididymal fluid, as other secreted acid hydrolases, carries a marker in its molecule, we studied the binding of this enzyme to cellular membranes of the epididymal tissue. The binding, like that mediated by the phosphomannosyl receptor, was saturable, did not require calcium, had a Kd in the nM range and was inhibited by phosphatase or metaperiodate treatment of the enzyme. However fructose 6-phosphate derivates were more effective competitive inhibitors than mannose 6-phosphate. The binding capacity of the membranes were extractable with Triton X-100 and incorporable into liposomes. Trypsin inhibited the binding capacity of Triton extracts but it did not affect the affinity of intact cellular membranes for beta-galactosidase. The results suggest that a phosphorylated carbohydrate of the enzyme is bound by a recognizing site of the cellular membranes different from the phosphomannosyl receptor.
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PMID:beta-Galactosidase from rat epididymal fluid is bound by a recognition site attached to membranes of the epididymis different from the phosphomannosyl receptor. 303 84


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