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)

Comparing the marker enzymes horseradish peroxidase (HRP), alkaline phosphatase (AP) and beta-galactosidase (beta Gal) in IgG-coupled form with respect to their temperature-dependent kinetics over a period of 22 h the temperature of 37 degrees C warrants highest substrate turnover for all enzymes at all reaction times using fluorogens. Also applying chromogens the optimum temperature for beta Gal is 37 degrees C and depends for HRP and AP on the reaction time. The substrate turnover of HRP using ABTS as chromogen is much higher compared to the other enzymes--both related to mol enzyme (molar activity) and to gram enzyme (specific activity). The turnover decreases for all enzymes in different degrees after coupling to IgG. The turnover of fluorogenic substrates is lower for all enzymes than the turnover of chromogenic substrates but due to the more sensitive detection of fluorogenic products the detection limits for all conjugates were lowered too--especially for beta Gal-IgG by a factor of 333 compared to the colorimetric procedure. In a 2-site binding enzyme immunoassay for alpha-1-fetoprotein (AFP) the detection limit for AFP was reduced by a factor of 2 only by the fluorimetry compared to the colorimetry with all 3 marker enzymes. The HRP-IgG conjugates warranted lowest detection limits for AFP (0.5-1 microgram/1), highest analytical sensitivity (slope of standard curves) at shortest periods of substrate reaction compared to the other enzymes.
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PMID:Which of the commonly used marker enzymes gives the best results in colorimetric and fluorimetric enzyme immunoassays: horseradish peroxidase, alkaline phosphatase or beta-galactosidase? 392 20

Cathepsin D was visualized in free pulmonary alveolar macrophages (AM), in oil-induced peritoneal macrophages (MN) and in rabbit pulmonary and dermal BCG lesions with unlabeled antibodies and the peroxidase-antiperoxidase (PAP) complex. Large amounts of cathepsin D were present in AM and lower amounts in MN. In the lung this enzyme was richest in the alveolar macrophages that accumulated around the BCG lesions. In the dermal lesions, cathepsin D was in highest concentration in macrophages at the border of the necrotic (liquefying) centers. It was also found in high concentration in keratinizing cells of the dermal epithelium and hair follicles. It did not, however, increase appreciably in many of the activated macrophages that stained intensely for the lysosomal enzyme beta-galactosidase. In fact, many epithelioid cells with high beta-galactosidase activity contained no visible cathepsin D. This proteinase does not, therefore, seem to be primarily involved in the lymphocyte-mediated macrophage activation associated with acquired cellular resistance to tubercle bacilli. It is probably more involved with cell autolysis, with the digestion of ingested necrotic debris and, in all likelihood, with the process of liquefaction, the most adverse event in the pathogenesis of tuberculosis in man.
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PMID:The role of cathepsin D in the pathogenesis of tuberculosis. A histochemical study employing unlabeled antibodies and the peroxidase-antiperoxidase complex. 480 13

Histochemical procedures for PMN granule enzymes were carried out on smears prepared from normal rabbit bone marrow, and the smears were examined by light microscopy. For each of the enzymes tested, azo dye and heavy metal techniques were utilized when possible. The distribution and intensity of each reaction were compared to the distribution of azurophil and specific granules in developing PMN. The distribution of peroxidase and six lysosomal enzymes (acid phosphatase, arylsulfatase, beta-galactosidase, beta-glucuronidase, esterase, and 5'-nucleotidase) corresponded to that of azurophil granules. Progranulocytes contained numerous reactive granules, and later stages contained only a few. The distribution of one enzyme, alkaline phosphatase, corresponded to that of specific granules. Reaction product first appeared in myelocytes, and later stages contained numerous reactive granules. The results of tests for lipase and thiolacetic acid esterase were negative at all developmental stages. Both types of granules stained for basic protein and arginine. It is concluded that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appears to be restricted to one of the granules. The findings further indicate that azurophil granules are primary lysosomes, since they contain numerous lysosomal, hydrolytic enzymes, but the nature of specific granules is uncertain since, except for alkaline phosphatase, their contents remain unknown.
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PMID:Differences in enzyme content of azurophil and specific granules of polymorphonuclear leukocytes. I. Histochemical staining of bone marrow smears. 487 49

Four proteins, which have been designated A, B, C and D, have been purified from human parotid saliva. These proteins are the major constituents of parotid saliva which migrate rapidly to the anode in polyacrylamide electrophoresis at pH9.5. Gel filtration and polyacrylamide electrophoresis were employed in the purification procedures. After purification all four preparations were tested for homogeneity by electrophoresis at pH2.8 and 9.5, by isoelectric focusing in the pH range 3-10, by immunodiffusion, and by sedimentation in the analytical ultracentrifuge. None of the proteins showed significant activity in assays for amylase, acid and alkaline phosphatase, protease, lysozyme, ribonuclease, peroxidase, beta-glucuronidase, beta-galactosidase, iron-binding activity and esterase. No cross-reactions were detected with antisera specific for lactoferrin and 15 serum proteins. All four proteins were rich in glutamic acid, proline and glycine and were lacking completely the sulphur-containing amino acids. Proteins A and C contained no threonine or tyrosine. Carbohydrate could be demonstrated only in protein A at a concentration of 4% of the total protein.
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PMID:Purification and partial characterization of four proteins from human parotid saliva. 500 93

A mutant strain of Escherichia coli (E. coli ML-35) was used to follow the kinetics of phagocytosis, perforation of the bacterial cell envelope, and inactivation of bacterial proteins by human neutrophils. This particular E. coli mutant strain has no lactose permease, but constitutively forms the cytoplasmic enzyme beta-galactosidase. This implies that the artificial substrate ortho-nitrophenyl-beta-D-galactopyranoside cannot reach the beta-galactosidase unless the bacterial cell envelope has been perforated. Thus, the integrity of the E. coli envelope can be measured simply by the activity of beta-galactosidase with this substrate. Indeed, ingestion of E. coli ML-35 by human neutrophils was followed by perforation of the bacteria (increase in beta-galactosidase activity). Subsequently, the beta-galactosidase activity decreased due to inactivation of the enzyme. With a simple mathematical model and a curve-fitting computer program, we have determined the first-order rate constants for phagocytosis, perforation, and beta-galactosidase inactivation. With 32 normal donors, we found an interdonor variation in these rate constants of 20% to 30% (SD) and an assay variance of 5%. The perforation process closely correlated with the loss of colony-forming capacity of the bacteria. This new assay measures phagocytosis and killing in a fast, simple, and accurate way; it is not hindered by extracellular bacteria. Moreover, this method also measures the postkilling event of inactivation of a bacterial protein, which permits a better detection of neutrophils deficient in this function. The assay can also be used for screening neutrophil functions without the use of a computer program. A simple calculation suffices to detect neutrophil abnormalities. Neutrophils from patients with chronic granulomatous disease (CGD) showed an impaired rate of perforation and thus also of inactivation. Neutrophils from myeloperoxidase-deficient patients or from a patient with the Chediak-Higashi syndrome only showed a retarded inactivation of beta-galactosidase, but normal ingestion and perforation. The role of myeloperoxidase in the killing process is discussed. Although myeloperoxidase does not seem to be a prerequisite for perforation, it probably plays a role in bacterial destruction by normal cells, because the inactivation of bacterial proteins seems strictly myeloperoxidase dependent.
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PMID:Kinetics and mechanism of the bactericidal action of human neutrophils against Escherichia coli. 608 60

BCG lesions were produced in the skin of rabbits, and biopsies were performed at 7, 21, and 42 days, when they were developing, maximal in size, and almost healed, respectively. Tissue sections were prepared and stained histochemically for several enzymes. The percentage of cells stained for a given enzyme and the distribution of such cells within lesions of various ages were determined. Seven-day BCG lesions contained few esterase- and beta-galactosidase-positive macrophages, but 21-day lesions contained many, especially in the viable and nonviable tuberculous granulation tissue at the edge of the now prominent caseous necrotic center. Both 7-day and 21-day lesions contained many acid phosphatase- and cathepsin-D-positive macrophages, which were numerous in the more peripheral parts of the lesion, where little or no necrosis was present. Enzyme patterns in 42-day lesions resembled those in 21-day lesions. The role of each of these enzymes in the development and regression of the BCG lesion is unknown. Nonetheless, these studies clearly demonstrate that this macrophage population is heterogeneous and that macrophages carry out different functions in different parts of the lesion at different times. Histochemical techniques were developed to stain two enzymes in the same tissue section. The first stain usually contained a naphthol substrate and produced a red color; the second stain contained an indoxyl substrate and produced a blue color. A cell staining with both was colored purple. The peroxidase-antiperoxidase immunocytochemical technique for cathepsin D (producing a red color) was also employed. 1) Red esterase (hydrolyzing naphthol AS-D acetate) and beta-galactosidase, and 2) red esterase and blue esterase (hydrolyzing 5-bromo-4-chloro-indoxyl acetate), probably the same enzyme, were usually present in the same macrophage. In contrast, each of the following enzyme pairs was usually present in a different macrophage: 3) cathepsin D and beta-galactosidase, 4) cathepsin D and blue esterase, 5) acid phosphatase and beta-galactosidase, and 6) acid phosphatase and blue esterase. Roughly 10% of the macrophages stained for one enzyme existed side by side with macrophages stained for a different enzyme. These results suggest that local macrophage activation is under two levels of control. The first, macrolocal control, would determine the overall enzyme distribution in the lesion; whereas the second, microlocal control, would determine enzyme distribution on a cell-by-cell basis, ie, how two neighboring macrophages can each be rich in a different enzyme.
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PMID:Macrophage functional heterogeneity in vivo. Macrolocal and microlocal macrophage activation, identified by double-staining tissue sections of BCG granulomas for pairs of enzymes. 615 72

The binding and internalization of a model lysosomal enzyme, beta-galactosidase, was visualized by use of rabbit anti-beta-galactosidase and goat anti-rabbit IgG; the second antibody was labeled with rhodamine or fluorescein (for detection by fluorescence) or with horseradish peroxidase (for electron microscopy). Chinese hamster ovary cells were incubated with beta-galactosidase at 4 degrees C, and then were washed and sequentially incubated in the cold with the two antibodies. The beta-galactosidase was found primarily in coated pits. The binding of the enzyme was completely inhibited by 5 mM mannose 6-phosphate. After the reaction with enzyme and antibodies, the cells were warmed to 37 degrees C; within 1 minute, the beta-galactosidase--antibody complex had begun to move to uncoated vesicles (receptosomes). After 8 min, the beta-galactosidase--antibody complex was seen in receptosomes near tubular elements in the Golgi/GERL area, within such tubular elements and at times, in vesicular elements that may correspond to coated structures of the GERL system. After 15 min, the enzyme--antibody complex was found in lysosomes near the Golgi/GERL are and a half-hour later it was in lysosomes distributed throughout the cytoplasm. Double-label experiments using beta-galactosidase and gold/alpha 2-macroglobulin showed the presence of the two ligands in the same coated pits and receptosomes. Thus, the pathway for internalization of beta-galactosidase via the mannose 6-phosphate receptor is similar to the pathway established for other ligands such as low density lipoprotein and alpha 2-macroglobulin.
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PMID:Morphologic study of the internalization of a lysosomal enzyme by the mannose 6-phosphate receptor in cultured Chinese hamster ovary cells. 627 98

Enzyme/anti-enzyme antibody soluble immune complexes were prepared with monoclonal mouse antibodies (MA) which were directed against peroxidase (PO) and beta-galactosidase (GAL). These enzyme monoclonal antibody complexes (EMAC) functioned as markers to quantify mouse antibodies using an enzyme immunoassay which incorporated an anti-mouse Ig as the bridge between the EMAC and the specific antibody bound to an antigen immobilized on a polystyrene plate. EMAC prepared with PO (PO-MAC) or with GAL (GAL-MAC) were both effective in quantifying polyclonal as well as monoclonal mouse antibodies, and gave sensitivity equal or superior to that obtained with covalent enzyme/anti-mouse Ig conjugates. The smaller amount of antibodies detected with EMAC depended on the affinity of both the antibody tested and the monoclonal antibody used to prepare EMAC. This method is an improvement on the 'antibody bridge' method, because EMAC can be prepared easily by simply mixing the enzyme and the MA at the appropriate amounts 2 h prior to use. In addition, EMAC can be prepared using crude preparations of enzyme and unpurified ascitic fluids containing the MA, thus decreasing the cost of the test considerably.
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PMID:Enzyme/anti-enzyme monoclonal antibody soluble immune complexes (EMAC): their use in quantitative immunoenzymatic assays. 640 22

Antibodies conjugated to enzymes such as horseradish peroxidase or alkaline phosphatase are widely used to detect antibody binding to individual cells or tissue sections through the deposit of insoluble colored reaction products. However, the presence of endogenous enzyme activity, especially in lymphomyeloid cell populations, necessitates the use of inhibitors which can be shown to decrease the sensitivity of the assay, a particular problem when monoclonal antibodies are used. As no endogenous beta-galactosidase activity can be demonstrated in lymphomyeloid cells, a cytoimmunoenzyme assay based on this enzyme should provide a preferable system for cytological investigations of antigens in these cells. Such as assay was developed using azo coupling of 2 alternative substrates with different diazonium salts to produce rapid and specific precipitation of reaction products of different colors. The sensitivity of the beta-galactosidase cytoimmunoenzyme assay was shown to be comparable with that of assays using FITC or peroxidase coupled antibodies.
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PMID:A beta-galactosidase linked immunoassay for the analysis of antigens on individual cells. 640 7

beta-galactosidase is a ubiquitous lysosomal hydrolase that specifically cleaves terminal beta-galactosyl residues from glycoproteins, glycosaminoglycans, oligosaccharides, and glycolipids. To study the intracellular distribution of this enzyme, we prepared a specific polyclonal antibody to lysosomal beta-galactosidase by immunizing rabbits with a highly purified preparation of beta-galactosidase from rat liver. Using this antibody we employed an immunocytochemical technique (protein A coupled to horseradish peroxidase and diaminobenzidine cytochemistry) and showed that beta-galactosidase is present in all hepatocytes of the rat liver. All types of lysosomes, the rough endoplasmic reticulum, and the specialized region of smooth endoplasmic reticulum known as GERL showed immunoreactivity. This in situ distribution suggests that these organelles are involved in the biosynthesis and intracellular sorting of this lysosomal enzyme.
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PMID:Immunocytochemical localization of lysosomal beta-galactosidase in rat liver. 641 69


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