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PubMed1985

Purification of hybrid beta-galactosidase proteins encoded by phi X174 E phi lacZ and Escherichia coli prlA phi lacZ: a general method for the isolation of lacZ fusion polypeptides produced in low amounts.

A facile immunoaffinity chromatography method is described for the purification of lacZ fusion gene products. The method is general for any molecule antigenically related to beta-galactosidase and involves only a single step. We report its use to purify the products of lacZ fusions with a bacteriophage gene, phi X174E, and an Escherichia coli chromosomal gene, prlA. The hybrid protein products of both of these genes are membrane bound and present in very low molar amounts with respect to total cellular protein. Evidence is presented that substrate-affinity chromatography is not applicable to the isolation of low-level fusion proteins such as these.

Bacteriophage phi X 174↗
PubMed1985

An assessment of acid-base balance and activity of lysosomal enzymes: mannosidase, b-galactosidase and b-glucosidase in CSF and arterial blood in early and later phases of the clinical course of cerebral contusion.

Results of the examination of the acid-base balance and the concentration of enzymes - mannosidase, b-galactosidase and b-glucosidase in 61 patients with cerebral contusion are presented. Examination was carried out on an average 11, 15 and 39 days after injury in groups of patients characterised by various clinical states. Examination showed that, in contradistinction to the acid-base balance, enzymal changes in CSF are less evident than in blood. On the base of the results presented it would appear that the level of b-glucosidase in CSF changes in accordance with the level in the blood whereas the other enzymes show a certain autonomy of the CSF in this field.

Acid-Base Equilibrium↗
PubMed1985

Development of enzyme immunoassay for chromomycin A3 and olivomycin using beta-D-galactosidase as a label.

An antibody specific for chromomycin A3 (CHM; byname, toyomycin) was produced in sufficiently high titer in rabbits by immunization with a CHM-bovine serum albumin conjugate, prepared using diazotized p-aminobenzoic acid as a coupling agent. CHM was also coupled with beta-D-galactosidase (EC 3.2.1.23) using diazotized m-aminobenzoic acid and was used as a tracer. With these reagents, a double-antibody enzyme immunoassay for CHM and for the CHM homologue olivomycin was developed which was highly sensitive and accurate enough to measure as little as 10 and 50 pg of each drug per assay tube, respectively. The enzyme immunoassay did not cross-react with mithramycin and drugs commonly used with CHM in combination chemotherapy for cancer treatment. Using this assay, drug levels were easily determined in blood and urine of rats following administration of CHM in a single dose of 2.0 mg/kg i.v. The sensitivity and specificity of the enzyme immunoassay for CHM and olivomycin should provide a valuable new tool for use in pharmacokinetic and toxicity studies of these drugs.

Animals↗
PubMed1982

Repertoires of T cells directed against a large protein antigen, beta-galactosidase. I. Helper cells have a more restricted specificity repertoire than proliferative cells.

The proliferative and helper T cell repertoires were compared in the CBA/J mouse for the response to the large protein antigen, tetrameric beta-galactosidase (GZ = 1021 a.a/monomer). The systems assessed the ability of cyanogen bromide (CB) peptides of GZ to: 1) prime for a T cell proliferative response to GZ; or 2) generate T cell help, measured by the production of anti-FITC PFC in the in vitro response to GZ-FITC. Priming for in vitro proliferation was attempted with 11 CB peptides comprising 70% of the GZ molecule. Strong priming was found with five peptides and intermediate priming was found with four other peptides; two peptides were without effect (CB-20 = a.a 767-862, and CB-4 = a.a. 188-202). Despite this indication of generally dispersed recognition of GZ epitopes, only two CB peptides, CB-2 (a.a. 3-92) and CB-10 (a.a. 378-418) were able to induce a T helper cell response. The surprising dearth of helper T cell-inducing epitopes may be peculiar to the limited fluorescein (FITC) substitution on GZ-FITC (17-25 FITC residues per tetramer) or it may reflect the constraints involved in T cell recognition required for T-B collaboration. Also considered was the possibility that the helper T cell repertoire might be distinct from the proliferative repertoire, the latter reflecting DNA synthesis and recruitment by other functional T cell subpopulations.

Animals↗
PubMed1983

Sensitive enzyme immunoassay for the quantification of bleomycin using beta-D-galactosidase as a label.

A sensitive and specific enzyme immunoassay (EIA) for an anticancer drug, bleomycin (BLM), has been developed, which allows measurement of as little as 25 pg of the antibiotic/tube. An antibody to BLM was obtained by immunizing rabbits with an antigen prepared by conjugating BLM with mercaptosuccinylated bovine serum albumin via N-(gamma-maleimidobutyryloxy)succinimide as a coupling agent. Enzyme labeling of BLM was performed using beta-D-galactosidase (beta-Gal; EC 3.2.1.23) via N-(m-maleimidobenzoyloxy)succinimide. Free and antibody-bound BLM-beta-Gal conjugates were separated by precipitation of the antibody-bound fraction with a second antibody (anti-rabbit IgG). Displacement of BLM-beta-Gal by unlabeled BLM when plotted as a logit-log function was linear over a concentration range of 10 pg-1 ng. The antibody distinguished alterations in the terminal structure of BLM, showing 277% cross-reaction with BLM B2, 4.6% with A2, 0.42% with A2'-b, 0.13% with A5, and 0.07% with the second-generation analog peplomycin. The EIA is free from interference by other anticancer drugs. Using this assay, drug levels were easily determined in tissues of rats following sc administration at a dose of 500 micrograms/kg. The sensitivity and specificity of the EIA should provide a useful tool for developing pharmacokinetic and toxicity studies of BLM.

Animals↗
PubMed1981

The specificity of induction of alpha-galactosidase from Saccharomyces carlsbergensis.

A number of sugars and derivatives have been tested for their ability to induce the synthesis of alpha-galactosidase from Saccharomyces carlsbergensis. Besides galactose and the substrates of the enzyme melibiose, raffinose and stachyose, D-galacturonic acid, L-arabinose, D-tagatose, methyl-alpha-D-galactoside, lactose and isopropyl-beta-D-thiogalactoside were able to act as inducers. Of these, methyl-alpha-D-galactoside, lactose, isopropyl-beta-D-thiogalactoside and L-arabinose have been shown to be gratuitous inducers with which kinetic studies of induction have been carried out. Lactose was the most efficient inducer, giving a maximal differential rate of synthesis of the enzyme of 110 mU/10(7) cells at a concentration of 180 mM, followed by L-arabinose (60 mU/10(7) cells at 40 mM), isopropyl-beta-D-thiogalactoside (43 mU/10(7) cells at 60 mM) and methyl-alpha-D-galactoside (25 mU/10(7) cells at 150 mM). The concentration of inducer required to obtain half-maximal induction was similar for lactose, L-arabinose and isopropyl-beta-D-thiogalactoside and about 5-fold higher for methyl-alpha-D-galactoside. The property of the compounds to act as inducers was compared to their ability to interact with the enzyme and the results discussed in terms of the molecular structures which are recognized by the enzyme and by the induction machinery.

Carbohydrates↗
PubMed1983

Activator protein for the degradation of globotriaosylceramide by human alpha-galactosidase.

An activator protein which stimulates the degradation of globotriaosylceramide by human hepatic alpha-galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was isolated from human liver and purified some 1300-fold. The purified activator was heat stable up to 95 degrees C, its molecular weight was estimated at 20,000 by gel filtration. Ampholyte displacement chromatography resolved the activating factor into two fractions with isoelectric points at pH 4.6 and pH 4.8, respectively, with otherwise identical properties. The protein did not stimulate the hydrolysis of the water-soluble 4-methylumbelliferyl-alpha-galactoside.

Chymotrypsin↗
PubMed1983

A novel proteolytic activity apparently initiating degradation of beta-galactosidase nonsense fragments in in vitro extracts of Escherichia coli.

Our previous in vivo studies demonstrated that large premature fragments of beta-galactosidase are degraded in Escherichia coli by a common pathway, and the initial event appears to be a site-specific cleavage (McKnight, J. L., and Fried, V. A. (1981) J. Biol. Chem. 256, 9652-9661). We now have developed a cell-free system that retains the specificity of this early cleavage event. Immunochemical techniques were used to isolate and quantitate the polypeptide substrate and products in pulse-chase experiments. The in vitro system has an activity that quantitatively converts the prematurely terminated A polypeptide of the lacZ non-sense mutant CSH-10 to the 90-kilodalton common B polypeptide intermediate observed in vivo. The activity is localized in the cytoplasm since the cleavage reaction is not affected by osmotic shock of whole cells or removal of the membrane fraction from cell-free extracts. The lon mutation capR9, which blocks this degradation pathway in vivo, does not affect the initial cleavage event in cell-free extracts of CSH-10 carrying this mutation. The in vitro cleavage event in extracts of lon+ CSH-10 or the isogenic lon- mutant is not stimulated by addition of ATP, not inhibited by depletion of ATP pools by hexokinase-2-deoxyglucose treatment, and not inhibited by EDTA or phenylmethylsulfonyl fluoride. These results suggest that the ATP-dependent proteolytic activity of the lon gene product does not directly catalyze this primary cleavage event.

Escherichia coli↗
PubMedDate not supplied

Isolation and properties of beta-galactosidase of a strain of Lactobacillus helveticus isolated from natural whey starter.

beta-Galactosidase has been isolated from Lactobacillus helveticus of a strain isolated from natural starters for the manufacture of Argentine hard cheeses and its properties have been studied. The enzyme was purified 14-fold (by chromatography on DEAE-cellulose and Sepharose 6B-DEAE-cellulose columns and by affinity chromatography in agarose-p-aminophenyl-beta-D-thiogalactoside). The purified extract exhibited a single band following polyacrylamide gel electrophoresis. Maximum enzymatic activity was observed at 42 degrees C and pH 6.5 in 50 mM phosphate buffer. At pH values substantially different from the optimum, a positive cooperativity between substrate molecules was observed. The Km's for o-nitrophenylgalactoside (ONPG) and ONPG + 10 mM of lactose were 4.46 X 10(-5) and 8.9 X 10(-5) M, respectively. Glucose, galactose, galactose 6-phosphate, and lactate acted as noncompetitive inhibitors; MgCl2 protected the enzyme from thermal denaturation. The activation energy of enzymatic hydrolysis of ONPG was 11,400 cal/mol. The Mr was estimated to be 250,000. It is an oligomeric enzyme made of 4 subunits of Mr 65,000.

Animals↗
PubMed1984

Effect of glycolipids contained in liposomes on the incorporation of beta-galactosidase into specific organs.

A series of glycolipids were examined to find a system capable of targeting liposomes into specific organs of rats. Sulfatide was found to be the best among the components of liposomes examined for delivering the entrapped enzyme, beta-galactosidase from Aspergillus oryzae, into the brain and liver; gangliosides, for the spleen; and sphingomyelin, for the lung. To introduce the enzyme into the liver, galactocerebroside was far better than glucocerebroside. These data suggest that the sugar residues of glycolipids function to target the liposomes into specific organs.

Animals↗
PubMedDate not supplied

Alteration of the substrate specificity of Aspergillus oryzae beta-galactosidase by modification with polyethylene glycol.

beta-Galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) purified from Aspergillus oryzae was modified with 2,4,6-trichloro-s-triazine derivatives of polyethylene glycol (activated BPEG) having molecular weights of 600, 1500, 2000, and 4000. Polyethylene glycol derivatives were attached to 6 of the 12 amino groups exposed on the surface of the enzyme. Upon modification, the enzymatic activity for a water-soluble substrate, o-nitrophenyl beta-D-galactopyranoside, was reduced with increasing molecular weight of the activated BPEG. On the contrary, the enzymatic activity for another substrate, 4-methylumbelliferyl beta-D-galactopyranoside, was increased upon modification. The Michaelis constants of native and modified enzymes for these two substrates were virtually the same. The effect of the modification was more marked in the enzymatic hydrolysis of the beta-galactosidic bond of amphipathic substrates. A fluorescent analog of naturally occurring galactocerebroside, 1-O-galactosyl-2-N-(1-dimethylaminonaphthalene-5-sulfonyl)-sphingosine, was hydrolyzed more rapidly by the modified enzyme than by the native one. The enzyme modified with activated BPEG of 1500 Da had the highest activity for this substrate. The beta-galactosidic bond of the terminal galactose of GM1-ganglioside (II3NeuAcGgOse4Cer, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminosyl)-galactosyl -glucosylceramide) was cleaved by the modified but not by the native enzyme.

Aspergillus↗
PubMed1984

The distributive pattern of beta-galactosidase in the forebrain and mid-brain of the mouse.

The present study demonstrates the distribution pattern of beta-galactosidase (BGL) in the forebrain and mid-brain of the mouse. Mild enzyme activity was observed in the neurons of the forebrain and mid-brain. Interestingly, strong enzyme activity in some nuclei was due to the presence of myelinated fibers. Most of the fiber tracts and commissures showed strong enzyme activity. The involvement of BGL in myelin metabolism is discussed.

Animals↗
PubMed1983

Kinetic properties of beta-galactosidase in Morquio B disease.

Some kinetic Properties of beta-galactosidase in crude extracts of cultured fibroblasts obtained from patients with Morquio B disease, GM1-gangliosidosis and from the controls were compared. The main defect in Morquio B seems to be the lowering of enzyme affinity to the substrates, while in the case of GM1-gangliosidosis of infantile type 1 a dramatic decrease of Vmax value concomitant with normal KM value was observed. Based on these observations possible interpretations of the clinical picture are discussed.

Fibroblasts↗
PubMed1983

[Obtaining beta-galactosidase using whey].

The search of a microorganism with the ability to produce the enzyme beta-galactosidase was undertaken according with the requirements of the market, in economical, technological and sanitary terms. The process consisted of recovery and use of the effluent from milk and cheese used to feed pigs, producing at the same time different types of contamination; once investigated and adjusted the technological variables to produce the enzyme, and selected the most convenient microorganism for such purpose the acting upon the extraction, conservation and purification of the product were adjusted. Comparative results of conversion were obtained with different test, at laboratory scale and industrial plants, in similar conditions to those obtained with importation products.

Animals↗
PubMed1978

Feline GM1 gangliosidosis: characterization of the residual liver acid beta-galactosidase.

The residual liver acid beta-galactosidase (beta-gal) activity from a case of feline GM1 gangliosidosis was partially purified and characterized with respect to its pH optimum, kinetic properties, thermostability, isoelectric point, molecular weight, and antigenicity. In comparison to the normal enzyme, the mutant enzyme had the same pH optima for the three substrates tested, a reduced Km for 4-methylumbelliferyl-beta-gal, elevated Km's for GM1 and asialofetuin (ASF), and increased thermolability. In addition, the mutant beta-gal had a higher isoelectric point, a reduced molecular weight, and appeared to be antigenically different from normal. The results suggest that the mutation in the Birmingham GM1 cat is structural and that the residual enzyme activity is a structurally altered acid beta-gal. The apparent lack of antigenic identity between the mutant and normal enzymes, in contrast to the situation in many human GM1 patients, is most unusual.

Animals↗
PubMed1979

Aflatoxin B1 effect on beta-D-galactosidase in some starter cultures.

Six starter cultures were examined for their ability to induce beta-D-Galactosidase activity. The maximum (713 x 10(-4) units/ml) and minimum (140 x 10(-4) units/ml) enzyme activity was shown by Lactobacillus bulgaricus and Streptomyces thermophilus, respectively. The maximum inhibition by aflatoxin B1 of induction and activity of enzyme in vitro was observed at 60 micrograms/ml in all cultures. However, low levels of the toxin stimulated induction of the enzyme in Streptococcus lactis and S. thermophilus.

Aflatoxins↗
PubMed1978

Methionine 500, the site of covalent attachment of an active site-directed reagent of beta-galactosidase.

The site of attachment to beta-galactosidase of the active site-directed inhibitor, beta-D-galactopyranosylmethyl p-nitrophenyl triazene, was determined. When the enzyme is completely inactivated, 1 mol of the galactopyranosylmethyl group is bound per mol of monomer with retention of the tetrameric structure. After reaction with the [14C]methyl reagent, labeled peptides were isolated and analyzed. The radioactive label was found to be covalently bound to methionine residue 500.

Amino Acid Sequence↗
PubMed1978

Amino acid sequence of beta-galactosidase. V. Isolation and sequences of chymotryptic peptides.

The amino acid sequence of 72 chymotryptic peptides isolated from 14C-, 3H-labeled carboxymethyl-beta-galactosidase has been determined. A variety of techniques were used in the isolation procedures including separation by solubility, size, and ion exchange and paper chromatography. These peptides contain approximatley 500 amino acids, range in size from 2 to 26 residues, and give overlaps with tryptic peptides of 16 to 55 residues. Peptides from this digest and those reported earlier from tryptic digests account together for the sequence of about 600 of the 1021 residues in the subunit.

Amino Acid Sequence↗