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)

Cell-enzyme-linked immunosorbent assay (cell-ELISA) is a technique for the rapid, convenient, and quantitative detection of molecules expressed on the cell surface. Here we present an evaluation of beta-galactosidase as an antibody-tag for cell-ELISA. In contrast to substrates for horseradish peroxidase (HRP) and alkaline phosphatase, murine splenocytes do not hydrolyze the beta-galactosidase substrate chlorophenolred-beta-D-galactopyranoside (CPRG). beta-Galactosidase-antibody conjugates show much lower background binding to murine T cells than conjugates with HRP or alkaline phosphatase. We describe step-by-step procedures for direct and indirect beta-galactosidase based cell-ELISA to quantitate the expression of molecules on the surface of unfixed, live cells. Variations of the basic protocol are suitable for adherent and non-adherent cells, large scale screening for expression of cell surface molecules, and the screening of hybridomas for production of antibodies to cell surface epitopes. Since relatively few beta-galactosidase conjugated antibodies are commercially available, we describe an efficient method to couple beta-galactosidase to antibodies using a novel water soluble heterobifunctional crosslinker, sulfosuccinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (sulfo-SMCC). We demonstrate the utility of this method by conjugating F(ab')(2) fragments of an anti-B7-2 antibody, and using this conjugate to assay B7-2 on Fc-receptor bearing cells.
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PMID:Cell-ELISA using beta-galactosidase conjugated antibodies. 1066 80

Glycosyl-hydrolytic enzymes from suspension-cultured carrot (Daucus carota L. cv. Kintoki) cells grown in calcium (Ca2+)-deficient and normal liquid media were studied after extraction successively by K-phosphate (pH 7.0) and Na-acetate (pH 5.2) containing 3 M LiCl. The same activities were detected in two protein fractions from control and Ca2+-deprived cells. The specific activities of alpha-galactosidase and polygalacturonase decreased under Ca2+ deprivation, but beta-galactosidase activity in the buffer-soluble protein from Ca2+-deprived cells increased 1.7-fold compared to control cells. Upon ion exchange and size-exclusion chromatography the fraction (Ca-Ia-I) in the buffer-soluble protein from Ca2+-deprived cells represented beta-galactosidase activity associated with a galacturonic acid-rich polysaccharide peak, whereas the corresponding fraction could hardly be detected in the buffer-soluble protein from control cells. Several of the same glycosidase activities were detected in the extract solubilized with cyclohexane-trans-1,2-diaminetetra-acetate (CDTA) from active cell walls of Ca2+-deprived cells as in the extract of control cells, but the beta-galactosidase activity was considerably reduced under Ca2+ deprivation. Following the same chromatography the fraction (CDTA-Ca-1) of beta-galactosidase activity in the extract solubilized with CDTA from active cell walls of Ca2+-deprived cells was also completely overlapping with the peak of galacturonic acid-rich polysaccharide. The molecular mass of fractions Ca-Ia-I and CDTA-Ca-1 was 300 kDa, and the polysaccharides in these two fractions were composed of approximately equal amounts of rhamnosyl and galacturonosyl residues. These results suggest that the increase of beta-galactosidase in the buffer-soluble protein fraction from Ca2+-deprived cells is the result of solubilization of a part of the acidic pectic polymer-bound beta-galactosidase due to the structural changes in the cell walls that occur during Ca2+ deprivation.
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PMID:Pectin-bound beta-galactosidase present in cell walls of carrot cells under the different calcium status. 1190 68

Pectic polysaccharides solubilized in vivo during ripening, were isolated using phenol, acetic acid, and water (PAW) from the outer pericarp of kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang and A.R. Ferguson var deliciosa ;Hayward') before and after postharvest ethylene treatment. Insoluble polysaccharides of the cell wall materials (CWMs) were solubilized in vitro by chemical extraction with 0.05 molar cyclohexane-trans-1,2-diamine tetraacetate (CDTA), 0.05 molar Na(2)CO(3), 6 molar guanidinium thiocyanate, and 4 molar KOH. The Na(2)CO(3)-soluble fraction decreased by 26%, and the CDTA-soluble fraction increased by 54% 1 day after ethylene treatment. Concomitantly, an increase in the pectic polymer content of the PAW-soluble fraction occurred without loss of galactose from the cell wall. The molecular weight of the PAW-soluble pectic fraction 1 day after ethylene treatment was similar to that of the Na(2)CO(3)-soluble fraction before ethylene treatment. Four days after ethylene treatment, 60% of cell wall polyuronide was solubilized, and 50% of the galactose was lost from the CWM, but the degree of galactosylation and molecular weight of pectic polymers remaining in the CWMs did not decrease. The exception was the CDTA-soluble fraction which showed an apparent decrease in molecular weight during ripening. Concurrently, the PAW-soluble pectic fraction showed a 20-fold reduction in molecular weight. The results suggest that considerable solubilization of the pectic polymers occurred during ripening without changes to their primary structure or degree of polymerization. Following solubilization, the polymers then became susceptible to depolymerization and degalactosidation. Pectolytic enzymes such as endopolygalacturonase and beta-galactosidase were therefore implicated in the degradation of solubilized cell wall pectic polymers but not the initial solubilization of the bulk of the pectic polymers in vivo.
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PMID:Cell Wall Dissolution in Ripening Kiwifruit (Actinidia deliciosa) : Solubilization of the Pectic Polymers. 1666 51