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PubMed1983

Improved purification of beta-galactosidase from rabbit brain: two separable fractions share kinetic and structural properties.

beta-Galactosidase has been purified from rabbit brain by a procedure which gives substantially greater purification and yield than the previously reported method. The enzyme was solubilized in 4% aqueous butanol and purified by a procedure which included affinity chromatography on columns of concanavalin A - Sepharose (ConA-Sepharose) and aminophenylthiogalactoside Sepharose (APT galactoside-Sepharose). The activity was resolved by DEAE-Sepharose chromatography into two fractions; the first did not bind to the column and was eluted in the unbound fraction, while the second bound to the column and could be eluted with a salt gradient. The unbound and bound fractions were purified 7600-fold and 4900-fold, respectively, by the newly developed procedure. Both gave two closely migrating protein bands on polyacrylamide gel electrophoresis and the major contaminating protein was beta-hexosaminidase in each case. The enzyme in the unbound fraction had an isoelectric point (pI) of 6.7 and an apparent molecular weight by gel filtration of 114 000 +/- 10 000. The enzyme that bound to DEAE-Sepharose at pH 8.0 and comprised about 60% of the total acid beta-galactosidase activity of rabbit brain eluted from Sephacryl S-200 as a single peak with apparent molecular weight 140 000 +/- 10 000. The two fractions displayed similar relative specific activities towards the synthetic substrates and ganglioside GM1 and lactosyl ceramide. Neither enzyme hydrolyzed galactosyl ceramide. We have immunological evidence which suggests that the two fractions of beta-galactosidase are also structurally related.

Acid Phosphatase↗
PubMed1979

Development of jejunoileal differences of activity of lactase, sucrase and acid beta-galactosidase in isografts of fetal rat intestine.

Jejunal and ileal segments from preterm rat fetuses were implanted under the kidney capsula of adult rats. Sucrase, lactase and acid beta-galactosidase activities were determined in the isografts at different times after implantation, and in corresponding segments developing in situ. Whereas fetal intestine contains considerable activity of acid beta-galactosidase and lactase, no sucrase activity is detectable. Similarly -- as in situ -- 4 weeks after the implantation the jejunal segment exhibited higher activity of sucrase and lactase than the ileal segment. Acid beta-galactosidase was more active in ileal than in jejunal segments -- both growing in situ as well as isografts. Experiments have thus demonstrated that the expression of the jejunoileal gradient of activity of the 3 enzymes studied does not depend on direct contact with food or gastric, pancreatic and biliary juices. This gives validity to the suggestion that the gradient may already be programmed in fetal intestinal tissue, but other factors active in situ might be responsible for its magnitude.

Animals↗
PubMed1980

Selective noncompetitive assimilation of bovine testicular beta-galactosidase and bovine liver beta-glucuronidase by generalized gangliosidosis fibroblasts.

Bovine liver beta-glucuronidase and testicular beta-galactosidase were assimilated by generalized gangliosidosis fibroblasts at respectively rates of 90 and 464 times the rate of assimilation of horseradish peroxidase. Assimilation of either of the two enzymes by the fibroblasts was saturable, suggesting the participation of receptor-mediated adsorptive endocytosis for internalization. The rate of assimilation of either enzyme was not affected by high levels of the other enzyme, suggesting that distinct receptors for each enzyme occur on the fibroblasts' cell surface. Furthermore, although assimilation of beta-galactosidase was inhibited by mannose, methyl mannosides, mannosyl alpha 1 leads to 2 mannose, and mannose-6-phosphate, these compounds did not detectably inhibit the assimilation of beta-glucuronidase. These results suggest that testicular beta-galactosidase was assimilated by the well-established phosphomannosyl recognition system. However, liver beta-glucuronidase was assimilated by a distinct, noncompeting, and as yet undefined, recognition system.

Animals↗
PubMed1990

Combined beta-galactosidase and immunogold/silver staining for immunohistochemistry and DNA in situ hybridization.

A combination of beta-galactosidase enzyme and the immunogold/silver staining method was studied for evaluation of double-staining experiments. Applications are shown for immunohistochemical double staining using two monoclonal antibodies and for combined immunohistochemistry and DNA in situ hybridization in one tissue section. The following advantages for the present double-staining method were evaluated: superior sensitivity of the immunogold/silver staining method for at least one epitope, which also allows detection of biotinylated DNA probes. The structure of the indolyl precipitate after revelation of beta-galactosidase activity did not show a concealing effect during a sequential double-staining method, as compared with the visualization of peroxidase with diaminobenzidine. These factors, and the sharply contrasting colored reaction products of beta-galactosidase (blue-green) and the immunogold/silver staining method including silver enhancement (brown-black), allow clear distinction of mixed-stained cell constituents.

Antibodies, Monoclonal↗
PubMed1980

Practical enzyme immunoassay for plasma cortisol using beta-galactosidase as enzyme label.

Enzyme immunoassay for cortisol was developed using beta-galactosidase as an enzyme label and m-maleimidobenzoyl derivatives of cortisol, i.e. cortisol-21-m-maleimidobenzoate (CT-MB) and cortisol-21-hemisuccinate conjugated with m-maleimidobenzoic acid through p-phenylenediamine linkage (CHS-MB), as the haptens coupled to sulfhydryl groups of the enzyme. This enzyme-coupling procedure was highly efficient; over 90% of the enzyme was labeled while full enzyme activity was retained. CHS-MB-beta-galactosidase conjugate showed high immunoreactivity to antibody produced against cortisol-21-hemisuccinate-bovine serum albumin but showed poor displacement with the added cortisol. CT-MB-beta-galactosidase conjugate, however, showed not only a high immunoreactivity to the antibody but also displaced well with cortisol, showing maximum sensitivity of 1 microgram/dl with a 20-microliter sample size. Modification around the linkage of cortisol derivative resulted in high sensitivity to cortisol. Cross-reactions to cortisol-21-acetate, cortisone, and corticosterone were 150%, 10%, and 7%, respectively. The accuracy, precision and correlation with RIA of this method were satisfactory for clinical application.

Galactosidases↗
PubMed1981

Tissue distribution of unentrapped or liposome-entrapped 131I-labeled beta-galactosidase injected into rats.

Either unentrapped (free) or liposome-entrapped 131I-labeled beta-galactosidase was injected into rats from tail veins. Tissue distribution and intracellular localization of the radioactivity of both sources were compared. The half-life of liposome-entrapped enzyme in the circulation was much longer than that of the free enzyme. The radioactivity removed from the circulation was recovered primarily in the liver, and to a lesser extent in almost all tissues studied. A small but significant uptake of liposome-entrapped enzyme by the brain was also observed. Uptake of liposome-entrapped enzyme was greater thant that of free enzyme in the spleen, heart, lungs and brain, excepting the liver and kidneys. Subcellular fractionation showed distribution of the radioactivity of liposome-entrapped enzyme favoring the mitochondrial-lysosomal fraction from these tissues except the heart. There was a difference in the pattern of intracellular distribution of the radioactivity in the brain of rats between the administration of free enzyme and that of liposome-entrapped enzyme. These findings suggest that when liposomes containing beta-galactosidase were injected into rats from the tail veins, they would penetrate the blood-brain barrier and would reach the lysosomes in the central nervous system tissue more effectively than the free enzyme itself. Beta-galactosidase; liposome; enzyme replacement therapy; rat tissues, 131I.

Animals↗
PubMed1985

Complementation analysis of beta-galactosidase deficiency by means of histochemical method.

Cultured skin fibroblasts from 4 beta-galactosidase-deficient patients with different clinical features were hybridized and beta-galactosidase activities were measured using 4-methylumbelliferyl (4MU)-derivate and by means of indigogenic method. Compared with the assays of beta-galactosidase activity using 4MU-derivate, more clear-cut evidence was obtained in the indigogenic method for a judgement of complementation.

Cells, Cultured↗
PubMed1989

A method for determining beta-galactosidase activity of yogurt cultures in skim milk.

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.

Animals↗
PubMed1979

Improved procedure for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide.

The procedures for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide were improved in several respects as compared with the previous methods (Eur. J. Biochem. 62, 285--292, 1976; J. Immunol. 116, 1554--1560, 1976). Maleimide residues were efficiently introduced into antibodies under an atmosphere of nitrogen; the average number of maleimide residues introduced into IgG and Fab' antibodies were 0.78 (0.65--0.86) and 0.86 (0.80--0.95) per molecule, respectively. The conjugation with the enzyme was performed at 4 degrees C at pH 6.5 for 15 or more hours. The conjugates were almost completely separated from unreacted IgG and Fab' by gel filtration. When the recoveries of IgG, Fab', and beta-D-galactosidase in the conjugates were 23-29, 35-44, and 99%, respectively, the average numbers of IgG and Fab' molecules conjugated with the enzyme were 1.5-1.7 and 2.1-2.8 per molecule, respectively. There was no significant impairment of beta-D-galactosidase activity or the activity of anti-human IgG antibody to bind to human IgG upon conjugation. However, the conjugate preparation was heterogeneous, and one-third of each preparation consisted of aggregated conjugates less useful in sandwich enzymoimmunoassay than the remaining material. The conjugate with Fab' antibody gave lower control values in sandwich enzymoimmunoassay with silicone rubber as a solid phase than that with IgG antibody.

Animals↗
PubMed1979

DNA-directed in vitro synthesis of beta-galactosidase. Purification and characterization of stimulatory factors in an ascites extract.

Previous studies have described a partially defined system for the DNA-directed in vitro synthesis of beta-galactosidase (Kung, H.F., Redfield, B., Treadwell, B.V., Eskin, B., Spears, C., and Weissbach, H. (1977) J. Biol. Chem. 252, 6889-6894). An Ehrlich ascites extract was shown in these in vitro studies to acylate Escherichia coli tRNA with 13 amino acids, and the ascites extract was used in place of the corresponding 13 E. coli aminoacyl-tRNA synthetases. The present studies indicate that the ascites extract is supplying an additional protein factor, besides the aminoacyl-tRNA synthetases, that stimulates the DNA-directed synthesis of beta-galactosidase. The protein factor has been highly purified and may be functioning by protecting mRNA against degradation. In addition, NAD or T4 DNA ligase stimulates the synthesis of beta-galactosidase in the partially defined system.

Amino Acyl-tRNA Synthetases↗
PubMed1990

A case of GM1-gangliosidosis type I: glycosphingolipid profiles of urine and transformed lymphocytes and beta-D-galactosidase activities in peripheral lymphocytes, cultured skin fibroblasts and transformed lymphocytes.

A female infant with early-onset GM1-gangliosidosis type I was investigated. The lymphocytes, transformed lymphocytes and cultured skin fibroblasts of the patient were demonstrated to have severe beta-D-galactosidase deficiency. The beta-D-galactosidase activities of these cells from the patient's father and mother were at the lower limit of the normal range. The oligosaccharide accumulation in urine of the patient showed the typical type I GM1-gangliosidosis pattern, but no GM1 ganglioside was detected in the patient's urine or transformed lymphocytes. The clinical features were compatible with infantile GM1-gangliosidosis. The mixture of homogenates from the cultured fibroblasts or transformed lymphocytes of the patient and controls showed no complementation of beta-D-galactosidase activity against the controls.

Cells, Cultured↗
PubMed1979

[Molecular forms of human leukocytic alpha-galactosidase and N-acetyl-alpha-galactosaminidase].

1. Normal human leukocytes present three molecular forms of alpha-galactosidase (EC 3.2.1.22) separated using electrofocusing: a new major form IV (pI 4.0) characteristic of leukocytes and two forms that exist in other tissues, form I (pI 4.5) and form II (pI 4.0). 2. Normal human leukocytes present only one molecular form (pI 4.5) of N-acetyl-alpha-galactosaminidase (EC 3.2.1.49) corresponding to alpha-galactosidase form II. 3. In leukocytes from patients with Fabry disease, the electrofocusing shows that the alpha-galactosidase lacking corresponds to forms I and IV, while the residual activity corresponds to form II (or N-acetyl-alpha-galactosaminidase).

Acetylgalactosamine↗
PubMedDate not supplied

[Beta-galactosidase from E. coli as a marker in immunoenzyme analysis].

In the present review the authors analyze factors influencing sensitivity of the enzyme immunoassay (EIA). beta-Galactosidase from E. coli was chosen as a marker enzyme. Physico-chemical properties of the enzyme, detection methods and various ways of obtaining chemical conjugates with antigens and antibodies are discussed. Some examples of using beta-galactosidase as a label in homogeneous and heterogeneous EIA are given which enables different compounds to be analyzed with a high sensitivity. New approaches employing gene engineering for constructing "fusions" between beta-galactosidase and antigens (alternative to chemical conjugates) are discussed that can be used in the near future.

Escherichia coli↗
PubMed1989

[Molecular characteristics of beta-galactosidase secreted by Penicillium canescens].

Extracellular beta-galactosidase from P. canescens culture medium was purified by ion-exchange chromatography on DEAE and CM-Sepharose CL-6B and gel filtration. The enzyme active form was shown to be a monomer with a molecular weight of about 120 kDa; the isoelectric point is 6.7 and the sedimentation coefficient is 6.5. In terms of physico-chemical and catalytic properties, the purified enzyme is similar to beta-galactosidases of other fungi of genus Penicillium. The amino acid composition and the NH2-terminal sequence of 24 residues non-homologous to the corresponding sequences of bacterial and yeast beta-galactosidases were determined.

Amino Acid Sequence↗
PubMed1989

Influence of detergents and pH on the isolation, purification and molecular properties of alpha-galactosidase-C2 from germinating guar (Cyamopsis tetragonolobus).

alpha-Galactosidase was isolated from germinating guar. The extract also contained small amounts of alpha-mannosidase and beta-mannosidase activities. The fractionation of the enzyme extract with ammonium sulphate (75% saturation) resulted in the appearance of all the three enzymes in a floating lipid complex. The inclusion of detergents such as Triton X-100 and sodium deoxycholate in the extraction medium failed to prevent the appearance of these enzymes in the floating lipid complex. However, by using acetone powder of the seedlings, alpha-galactosidase could be sedimented with ammonium sulphate. The presence of detergents in the extraction medium affected the molecular properties of the enzyme. Using a set of carefully selected conditions alpha-galactosidase was purified to apparent homogeneity. Analytical ultracentrifugation and gel filtration studies of the purified enzyme showed association-dissociation phenomenon as a function of pH and temperature. The effect of pH on the association-dissociation indicates the predominance of electrostatic interactions in the association of subunits.

Chemical Phenomena↗
PubMed1987

In vitro generated human monoclonal trinitrophenyl-specific B cell lines. Evidence that human and murine anti-trinitrophenyl monoclonal antibodies cross-react with Escherichia coli beta-galactosidase.

Stable human antigen-specific monoclonal B cell lines were established without prior in vivo immunization. This was accomplished by expanding the anti-trinitrophenyl (TNP) B cells in vitro with the antigen TNP-Brucella abortus and then immortalizing them with Epstein-Barr virus. Five anti-TNP clones were selected by sequential limiting dilution. All five anti-TNP clones secreted IgM kappa antibodies. When tested against a panel of self and environmental antigens, all five anti-TNP clones exhibited cross-reactivity with an Escherichia coli-derived beta-galactosidase. To determine whether this was a more general phenomenon, a panel of murine monoclonals were tested and found to bind to beta-galactosidase. It is therefore possible that human and murine anti-TNP beta cell responses reflect reactivity against an environmental antigen, namely an epitope present on E. coli-derived beta-galactosidase. This approach of expanding human antigen-specific B cells by antigen stimulation in vitro, with a T-independent hapten-carrier conjugate before Epstein-Barr virus transformation, may prove useful in the development of human monoclonals for therapeutic purposes.

Animals↗
PubMed1988

The membrane domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase confers endoplasmic reticulum localization and sterol-regulated degradation onto beta-galactosidase.

A hybrid gene has been constructed consisting of coding sequence for the membrane domain of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase linked to the coding sequence for the soluble enzyme Escherichia coli beta-galactosidase. Expression of the hybrid gene in transfected Chinese hamster ovary cells results in the production of a fusion protein (HMGal) which is localized in the endoplasmic reticulum. The fusion protein contains the high-mannose oligosaccharides characteristic of HMG-CoA reductase. Importantly the beta-galactosidase activity of HMGal decreases when low density lipoprotein is added to the culture media. Therefore, the membrane domain of HMG-CoA reductase is sufficient to determine both correct intracellular localization and sterol-regulation of degradation. Mutant fusion proteins which lack 64, 85, or 98 amino acid residues from within the membrane domain of HMG-CoA reductase are found to be localized in the endoplasmic reticulum and to retain beta-galactosidase activity. However, sterol-regulation of degradation is abolished.

Animals↗
PubMed1986

Enhanced thermal stability of lysosomal beta-D-galactosidase in parenchymal cells of tumour bearing mice.

The thermal stability of the enzyme beta-D-galactosidase varies among different organs in normal C57Bl/6 mice, and increases in the same organs in mice with Lewis Lung carcinoma. Thermal stability of this enzyme is also increased by treatment of the mice with cell-free extracts of tumour cells or with inflammatory compounds such as carrageenan or orosomucoid. After desialylation, orosomucoid more effectively increases the heat stability of the enzyme. By contrast talc, which has no galactosyl groups, is without effect on the stability of the enzyme in vivo. Macrophages of tumour bearing mice release into the culture medium a more heat resistant enzyme than macrophages from control mice. In both cases the heat resistance of the secreted enzyme is higher when fetal calf serum is present in the culture medium. Bovine serum does not modify the thermal stability of beta-D-galactosidase in this system. Incubation of lysosomal fractions of various organs with the synthetic beta-D-galactosidase substrate, p-nitrophenyl-galactopyranoside, also strongly increases the heat resistance of the enzyme. The results suggest that one factor influencing the heat resistance of this enzyme may be complex formation between the enzyme and its substrates, an example of substrate protection of the enzyme. This may not be the only factor involved in enzyme stabilization in vivo.

Animals↗