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

An enzyme-linked immunosorbent assay (ELISA) for insulin was developed. Anti-insulin antibody was bound to the bottom of 96-well microtiter plates. Insulin conjugated to beta-galactosidase was used as a label and methyl umbilliferyl beta-D galactoside was used as an enzyme substrate. To estimate insulin, relative fluorescence was measured with a fluorescent microtiter plate reader. Unknowns from an insulin release experiment yielded results comparable to those obtained with our enzyme-immunoassay (EIA) and a conventional radioimmunoassay (RIA). The insulin ELISA is suitable for research purposes in which samples contain solutions of physiological salts and albumin, but not for samples containing serum. The insulin ELISA is as sensitive as the insulin RIA and has several advantages over the standard insulin RIA. These include (1) avoidance of hazards and inconvenience of handling radioactivity, (2) not requiring a separate test tube for each sample, (3) stability of the enzyme-labeled insulin (greater than 18 months), (4) short time period required for the assay (less than 6 hours), and (5) the possibility of long-term storage (at least 3 months) of antibody-coated microtiter plates.
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PMID:A rapid ELISA for measuring insulin in a large number of research samples. 265 25

The hybridoma, 62H3, which secretes a monoclonal IgG2b with anti-HLA-DR specificity, was expanded in pristane-primed BALB/c mice and the antibody was isolated from the ascitic fluid by affinity chromatography on Protein A-Sepharose. The purified IgG2b antibody was tested by an enzyme immunoassay for antibody activity against a panel of 40 self and non-self antigens. It was found to react strongly with beta-galactosidase, actin, glutamate dehydrogenase, rabbit and human IgG and di- and trinitrophenyl groups; and moderately with tubulin, insulin and phosphorylcholine; but it did not react with various other self and non-self antigens, such as DNA, albumin, keyhole limpet hemocyanin, hen lysozyme and horseradish peroxidase. Fab and Fc fragments were prepared from this IgG2b by papain proteolysis. The Fab fragment possessed the same spectrum of polyreactivities as the native IgG2b, whereas no activity was detected with the Fc fraction. In order to investigate the properties of the antigen binding site, the actin, TNP and rabbit IgG antibody activities were studied in more detail by enzyme immunoassay, Western blot and immunocytochemistry. The monomolecular nature of this multireactivity was confirmed by immunoabsorption analysis. Furthermore, 62H3 monoclonality was also verified by comparative isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with other monospecific antibodies. The dissociation constants (Kd) of antigen-antibody equilibria in solution were measured. The Kd for actin was 1.11 +/- 0.24 x 10(-5) M and the Kd for TNP-BSA was 8.7 +/- 0.51 x 10(-7) M. No interaction with rabbit IgG could be detected in solution. These findings raise the question of the possible implication in autoimmune pathology or in normal physiology of IgG class polyspecific antibodies with solid-phase restricted cross-reactive rheumatoid factor activity.
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PMID:Immunochemical studies of a murine polyreactive IgG2b autoantibody with rheumatoid factor activity. 277 Jul 48

The cold agglutinin isolated from the albumin gland of the snail Achatina fulica was modified with various chemical reagents in order to detect the amino acids and/or carbohydrate residues present in its carbohydrate-binding sites. Treatment with reagents considered specific for modification of lysine, arginine and tryptophan residues of the cold agglutinin did not affect the carbohydrate-binding activity of the agglutinin. Modification of tyrosine residues showed some change. However, modification with carbodiimide followed by alpha-aminobutyric acid methyl ester causes almost complete loss of its binding activity, indicating the involvement of aspartic acid and glutamic acid in its carbohydrate-binding activity. The carbohydrate residues of the cold agglutinin were removed by beta-elimination reaction, indicating that the sugars are O-glycosidically linked to protein part of the molecule. Removal of galactose residues from the cold agglutinin by the action of beta-galactosidase indicated that the galactose molecules are beta-linked. These carbohydrate-modified glycoproteins showed a marked change in agglutination property, i.e. they agglutinated rabbit erythrocytes at both 10 degrees C and 25 degrees C, indicating that the galactose residues of the glycoprotein play an important role in the cold-agglutination property of the glycoprotein. The c.d. data showed the presence of an almost identical type of random-coil conformation in the native cold agglutinin at 10 degrees C and in the carbohydrate-modified glycoprotein at 10 degrees C and 25 degrees C. This particular random-coil conformation is essential for carbohydrate-binding property of the agglutinin.
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PMID:Studies on chemical modification of cold agglutinin from the snail Achatina fulica. 311 67

The expression vector lambda gt11Amp3 has been used to construct a cDNA library from rat liver polyadenylated RNA. Clones expressing antigenic determinants for rat albumin, transferrin, transthyretin, apolipoprotein E and apolipoprotein AII have been identified. Albumin clones containing cDNA inserts ranging from 0.9 kb to 1.9 kb were further identified by restriction mapping and nucleic acid sequencing. The largest insert contained the entire coding sequence for albumin. Characterization of the expressed proteins by acrylamide gel electrophoresis followed by immunological detection indicated that the proteins were produced as hybrids linked to the bacterial beta-galactosidase. A cDNA library for human liver polyadenylated RNA has also been constructed. Clones expressing antigenic determinants for human serum albumin, transferrin and apolipoproteins AI, AII, AIV and E have been isolated and their identity established by nucleotide sequencing and restriction mapping. Both rat and human serum protein cDNA clones are currently being used to study the tissue specific expression of serum proteins and for the isolation and characterization of the corresponding genes.
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PMID:Isolation and characterization of genes for blood proteins. 330 67

Conditions for recovery of small amounts of proteins (1-50 micrograms) from disulfide crosslinked polyacrylamide gels have been examined. Procedures were developed for solubilization and precipitation of Coomassie blue-stained protein bands excised from gels after electrophoretic separations. The precipitated protein was then resolubilized for use in peptide mapping, amino acid analyses, or microsequencing. The amino acid compositions of standard proteins (bovine albumin, ovalbumin, phosphorylase b, and beta-galactosidase) isolated by this method were in good agreement with the values for the corresponding conventionally purified proteins. Sequencing was done with high repetitive yield on samples of 100 pmol or below. The method has been successfully applied to several proteins and protein fragments.
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PMID:Isolation of proteins for peptide mapping, amino acid analyses, and sequencing using disulfide crosslinked polyacrylamide gels. 340 32

Two antibodies were prepared for use in a sandwich enzyme immunoassay of human IgG. Completely purified guinea pig anti-human IgG was labelled with beta-D-galactosidase (EC 3.2.1.23), using a heterobifunctional cross-linker named GMBS. Partially purified anti-human IgG was immobilized on a new solid support: Amino-Dylark balls. Optimal conditions for immobilizing the antibody, using glutaraldehyde as the coupling reagent, were studied in detail. With the enzyme-labelled antibody and the solid-phase anti-human IgG, a sandwich enzyme immunoassay of human IgG with a lower limit of detection at 10.5 pM (0.3 ng/tube) was developed. A comparative study of the EIA method and a laser nephelometric method showed a good correlation. The specificity of the assay was excellent: all 4 types of IgG tested showed the maximum 0.0001%; human IgA, IgM and albumin possessed the maximum 0.54% in their cross-reactivity values with human IgG.
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PMID:A new solid support for sandwich enzyme immunoassays of human immunoglobulin G. 393 19

1. alpha-Mannosidase from jack-bean meal was purified 150-fold. beta-N-Acetyl-glucosaminidase and beta-galactosidase were removed from the preparation by treatment with pyridine. Zn(2+) was added during the purification to stabilize the alpha-mannosidase. 2. At pH values below neutrality, alpha-mannosidase undergoes reversible spontaneous inactivation at a rate dependent on the temperature, the degree of dilution and the extent of purification. The enzyme is also subject to irreversible inactivation, which is prevented by the addition of albumin. 3. Reversible inactivation of alpha-mannosidase is accelerated by EDTA and reversed or prevented by Zn(2+). Other cations, such as Co(2+), Cd(2+) and Cu(2+), accelerate inactivation; an excess of Zn(2+) again exerts a protective action, and so does EDTA in suitable concentration. 4. Neither Zn(2+) nor EDTA has any marked effect in the assay of untreated enzyme. In an EDTA-treated preparation, however, Zn(2+) reactivates the enzyme during assay. 5. It is postulated that alpha-mannosidase is a dissociable Zn(2+)-protein complex in which Zn(2+) is essential for enzyme activity.
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PMID:Purification and properties of alpha-D-mannosidase from jack-bean meal. 497 51

1. alpha-d-Mannosidase from rat epididymis was purified 300-fold. beta-N-Acetyl-glucosaminidase and beta-galactosidase were removed from the preparation by treatment with pyridine. Zn(2+) was added during the purification to stabilize the alpha-mannosidase. 2. Mammalian alpha-mannosidase is most stable at pH6. At lower pH values it undergoes reversible spontaneous inactivation. The enzyme is also subject to irreversible inactivation, which is delayed by the addition of albumin. 3. Reversible inactivation of alpha-mannosidase is accelerated by EDTA and reversed or prevented by Zn(2+). Other cations, such as Co(2+), Cd(2+) and Cu(2+), accelerate inactivation and the action of a toxic cation can be prevented by Zn(2+) or by EDTA in suitable concentration. 4. The enzyme is stabilized by substrate and neither Zn(2+), EDTA nor a toxic cation has more than a small effect in the assay of an untreated preparation. The addition of Zn(2+) is necessary, however, for a constant rate of hydrolysis during prolonged incubation of the enzyme with substrate. In an EDTA-treated preparation, Zn(2+) reactivates the enzyme during the assay. 5. Evidence is presented that alpha-mannosidase is a dissociable Zn(2+)-protein complex, in which Zn(2+) is essential for enzyme activity.
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PMID:Purification and properties of alpha-D-mannosidase from rat epididymis. 498 Mar 9

The effect of various macromolecules on the activity of several hydrolases was studied. Dilution of partially purified acid beta-galactosidase from ileal mucosa of suckling rats resulted in a decrease of specific activity. The relationship between specific activity and dilution of the enzyme suggests a dissociation of the enzyme. This could be prevented by addition of several proteins tested. However, addition of DNA to the assay mixture for acid beta-galactosidase caused an inhibition. This inhibition could be prevented by addition of proteins. Other polynucleotides and tRNA also exert an inhibitory effect that is prevented by albumin, but nucleotides have no effect. This inhibition occurs maximally at a low pH (3.0-4.0); no inhibition is observed at pH5.5. A similar pH-dependent inhibition by DNA was also found with various other acid hydrolases.
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PMID:Effects of proteins and polynucleotides on the activity of various hydrolases. 507 27

The chromogenic substrate o-nitrophenyl-beta-d-galactopyranoside (ONPG) was hydrolyzed by lactose-positive Neisseria. Eight strains of pharyngeal origin were examined. In culture reactions, seven strains resembled Neisseria meningitidis with the exception that they produced acid from 1% (w/v) lactose. An eighth strain (V8) differed in that it did not form acid from maltose or from 1% lactose. However, acid formation was observed in 10% lactose cultures of strain V8, suggesting that entry of lactose occurred by passive diffusion, rather than as a result of permease activity. The enzymes which hydrolyzed ONPG were produced constitutively by the cells of all eight strains. Thus, specific activity in these strains was not increased by prior exposure to lactose, or to two other possible inducers, isopropyl-beta-d-thiogalactoside or methyl-beta-d-thiogalactoside. Study of cell-free extracts of one strain showed that the enzyme was heat-labile, having a half-life of 10 min at 45 C. The enzyme was unstable at low protein concentrations, but it was protected completely or partially when albumin or manganous ions were added. The enzyme appeared to be a typical beta-galactosidase: alpha-galactosides (melibiose and p-nitrophenyl-alpha-d-galactopyranoside) were not hydrolyzed, activity against ONPG was not dependent upon inorganic phosphate, and galactose was released by cleavage of ONPG. ONPG hydrolysis provided a simple and rapid method for detecting lactose-positive Neisseria.
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PMID:Galactosidase activity of lactose-positive Neisseria. 563 31


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