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PubMed1990

Partial deletion of human alpha-galactosidase A gene in Fabry disease: direct repeat sequences as a possible cause of slipped mispairing.

A partial deletion involving exon 3 associated with a single base change (A to C) was found in the alpha-galactosidase A gene of a hemizygous male Fabry patient and his mother, a heterozygous proband. This 402-bp deletion was flanked by 6-bp direct repeat sequences, and the intervening portion was found to have unique complementary sequences. These specific structures may have promoted "slipped mispairing" in this family.

Base Sequence↗
PubMed1990

Presence of an endo-beta-galactosidase degrading the linkage region between the chondroitin sulfate chain and core peptide of proteoglycan.

Pyridylamino chondroitin sulfate, of which the reducing terminal xylose was coupled with a fluorescent 2-aminopyridine, was incubated at pH 4.0 with an extract from the mid-gut gland of Patnopecten. The high- and low-molecular-weight products were separated by ethanol precipitation, and identified by high-performance liquid chromatography analysis. The enzyme was found to expose a galactose residue at the reducing terminus of chondroitin sulfate, and also released the pyridylamino disaccharide, galactosylxylose, from the reducing terminal site of pyridylamino chondroitin sulfate. These results suggest that endo-beta-galactosidase activity, which hydrolyzes the galactosylgalactose linkage of peptidochondroitin sulfate, is present in the mid-gut gland of Patnopecten.

Aminopyridines↗
PubMed1983

Selective depolymerisation of keratan sulfate: production of radiolabelled substrates for 6-O-sulfogalactose sulfatase and beta-D-galactosidase.

Keratan sulfate (KS) was N-deacetylated with anhydrous hydrazine and then degraded with nitrous acid, and the products were reduced with NaBT4. Radiolabelled disaccharides constituted at least 76% of the total oligosaccharide fraction. Three major disaccharides were isolated and identified. Of the total disaccharide isolated from bovine intervertebral-disc and human costal-cartilage, 91 and 79%, respectively, was identified as a disulfated disaccharide, O-(beta-D-galactopyranosyl 6-sulfate)-(1 leads to 4)-2,5-anhydro-D-[1-3H]mannitol 6-sulfate (Gal6S-anM6S). The disaccharide fraction isolated from bovine-cornea KS contained only 14% of Gal6S-anM6S. The yield of monosulfated disaccharide, identified as O-beta-D-galactopyranosyl-(1 leads to 4)-2,5-anhydro-D-[1-3H]mannitol 6-sulfate, was 9, 17, and 84% of the total KS-disaccharide fraction isolated from intervertebral disc, costal cartilage, and cornea, respectively. For each of the KS type studied, the yield of unsulfated disaccharide was less than 4% of the total disaccharide-fraction. The tetrasaccharides were fractionated, on the basis of their sulfate content, into at least four species by paper electrophoresis, and some tentative structures are proposed. Disaccharide and tetrasaccharide species were evaluated as substrates for beta-D-galactosidase and 6-O-sulfogalactose sulfatase.

Animals↗
PubMed1986

Synthesis of 2,6-anhydro-S-[ethylmercury(II)]-1-thio-D-glycero-L-manno-heptitol+ ++ and bis(2,6-anhydro-1-thio-D-glycero-L-manno-heptitol)mercury(II), and the study of their interaction with beta-D-galactosidase from E. coli.

Two competitive inhibitors of beta-D-galactosidase activity, namely, 2,6-anhydro-S-[ethylmercury(II)]-1-thio-D-glycero-L-manno-heptitol (4) and bis(2,6-anhydro-1-thio-D-glycero-L-manno-heptitol)mercury(II) (6), with inhibition constants of 8.0 X 10(-4) M and 1.9 X 10(-4) M, were synthesized. Compound 6 was incorporated into the crystalline enzyme by cocrystallization. The stoichiometry of the enzyme-inhibitor complex was 1:4, corresponding to one molecule of inhibitor per active site of the enzyme. Compound 4 was found to be unstable against X-ray irradiation, whereas compound 6 was submitted to X-rays for several days without any radiation damage.

Alkylmercury Compounds↗
PubMed1982

A sandwich enzyme immunoassay of human growth hormone in serum using anti-human growth hormone IgG-beta-D-galactosidase conjugate.

A sandwich enzyme immunoassay for the measurement of human growth hormone in serum was developed using antibody IgG-beta-D-galactosidase conjugate and antibody IgG-coated silicone rubber rods. The sensitivity of the assay was 4 pg/tube, which corresponded to 0.4 ng/ml, when 10 microliters of serum was used. The assay system showed little cross-reaction to other human pituitary hormones including prolactin, thyroid-stimulating, follicle-stimulating and luteinizing hormones and human chorionic gonadotropin. The coefficients of variation for different levels of serum hGH ranged from 5.8-9.3% and 6.7-14.5%, for within and between run, respectively. The regression equation and coefficient for correlation to radioimmunoassay were gamma (EIA) = 1.06 kappa (RIA) -0.22 and 0.97 (n = 46), respectively.

Animals↗
PubMed1982

Development of a highly sensitive sandwich enzyme immunoassay for human ferritin using affinity-purified anti-ferritin labelled with beta-D-galactosidase from Escherichia coli.

A highly sensitive sandwich enzyme immunoassay of human ferritin was developed. Polystyrene balls were coated with rabbit anti-human ferritin IgG by physical adsorption, and rabbit anti-human ferritin Fab' was purified by affinity chromatography and labelled with beta-D-galactosidase from Escherichia coli. Using the anti-ferritin-coated polystyrene balls and labelled anti-ferritin, the sensitivity obtained was 23 fg (0.05 amol) of ferritin per tube. The range of serum ferritin levels that could be determined using 0.1 microliter of serum was 0.23-4500 ng/ml, and even 2.3 pg/ml was measurable by using 10 microliter of serum. The coefficients of within-assay (n = 25) and between-assay (n = 10) variations were 5.9-8.8%. The regression equation and coefficient of correlation to a radioimmunoassay were Y(RIA) = 0.92 X(EIA) + 3.0 and 0.99 (n = 78), respectively. The corresponding sandwich radioimmunoassay was less sensitive, partly because the specific radioactivity of 125I-labelled anti-ferritin IgG used was not sufficiently high.

Antibody Specificity↗
PubMed1982

Colorimetric assays for N-acetyl-beta-D-glucosaminidase and beta-D-galactosidase in human urine using newly-developed omega-nitrostyryl substrates.

(1) The synthesis of 2-methoxy-4-(2'-nitrovinyl)-phenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (MNP-GlcNAc) and 2-methoxy-4-(2'nitrovinyl)-phenyl beta-D-galactopyranoside (MNP-Gal) as substrates for the assay of NAG and beta-d-galactosidase are described. (2) beta-Glycosidase activities were determined in random urine samples from normal males and females aged between 12 and 87 years and patients with renal disease. (3) Both the MNP N-acetylglucosaminide and MNP galactoside were stable indefinitely, if stored in the solid state at 4 degree C in the dark. (4) The effect of urinary inhibitors was minimized by diluting the urine in the assay procedure. A simple assay procedure has been developed using MNP substrates. A good correlation was found with established assays using 4-methylumbelliferyl and p-nitrophenyl glycosides. (5) The assay was readily automated and a good correlation was found between the automated and manual methods. (6) The assay of urinary glycosidase activity with MNP substrates is simple to perform and has been used successfully in the clinical chemistry laboratory.

Acetylglucosaminidase↗
PubMed1986

The N-terminal 21 amino acids of a 70 kDa protein of the yeast mitochondrial outer membrane direct E. coli beta-galactosidase into the mitochondrial matrix space in yeast cells.

The intracellular location of fusion proteins was investigated in yeast cells. They consisted of the N-terminal 21, 61 or 292 amino acids of the 70 kDa protein of the yeast mitochondrial outer membrane and an enzymatically active E. coli beta-galactosidase. The hybrids containing 61 or 292 residues of the 70 kDa protein, as well as the original 70 kDa protein, were localized on the outer membrane in a tightly membrane-bound form. In contrast, the other hybrid was exclusively localized in the mitochondrial matrix space as a soluble protein.

Cytosol↗
PubMed1990

A case of Fabry's disease in a patient with no alpha-galactosidase A activity caused by a single amino acid substitution of Pro-40 by Ser.

We analyzed a male patient with Fabry's disease who had no activity of the lysosomal hydrolase alpha-galactosidase A (alpha-GalA) and female members of his family. We cloned a cDNA that encoded the mutant alpha-GalA, determined its nucleotide sequence, and found two nucleotide differences between the mutant and the wild-type cDNAs. Although one difference was silent, the other difference, a C-to-T transition at nucleotide number 118, resulted in an amino acid substitution of Pro-40 by Ser. A transient expression assay demonstrated that this missense mutation was the cause of the deficiency of alpha-GalA activity in the patient. In vitro mutagenesis experiments demonstrated that Pro-40 is critical for the appearance of alpha-GalA activity.

Base Sequence↗
PubMed1982

beta-Fucosidase, beta-glucosidase and beta-galactosidase activities associated in bovine liver.

1. beta-D-Fucosidase, beta-D-glucosidase and beta-D-galactosidase activities from bovine liver are associated in a single peak in isoelectric focusing. The isoelectric point is 4.35 for all these activities, suggesting that they are catalyzed by the same enzyme. 2. This enzyme shows the optimal pH in the range 4.5-6.5 for all the above mentioned activities. 3. The Km and Vmax are 0.26 mM and 31 mU mg-1, 0.10 mM and 24 mU mg-1, and 0.30 mM and 20 mM mg-1 for the p-nitrophenyl-fucoside, -glucoside and -galactoside, respectively. The glucoside derivative is the best substrate, with a Vmax/Km value of 0.24 ml . mg-1 . min-1. 4. The Lineweaver-Burk profiles are convex upward in most cases, suggesting a substrate-activation model, and the presence of more than one binding site in the enzyme. 5. The Ki for all the activities were determined with D-fucose, glucose and galactose as inhibitors. D-Fucose is the strongest inhibitor. The inhibition is competitive in all cases.

Animals↗
PubMed1986

Aggregation-dissociation and stability of acid beta-galactosidase purified from porcine spleen.

Sucrose gradient centrifugation of the monomeric form (A1) of porcine spleen beta-galactosidase showed a pH-dependent equilibrium between monomer at neutral pH (pH 7.0) and dimer at acidic pH (pH 5.4-3.0), independent of ionic strength. While the oligomeric form (Ao), which was hardly dissociated under physiological conditions, was dissociated only with some protein denaturing agents into similar catalytic subunit to the A1. Both the A1 and Ao were equally active and stable at acidic pH, in the physiological condition inside lysosome (around pH 4.6).

Animals↗
PubMed1987

The inactivation of beta-galactosidase (E. coli) by the carbodiimide reaction.

When beta-galactosidase reacted with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC), activity was lost. The inhibitor, isopropyl-beta-D-galactopyranoside (IPTG), decreased inactivation. Of 3 nucleophiles tested, incorporation was only decreased in the protected (IPTG added) enzyme when sulfanilic acid was the nucleophile but HPLC profiles of tryptic peptides were identical in protected and unprotected enzyme (except for magnitude). There were also no differences (except for magnitude) of HPLC profiles after 10 and 90 min of reaction and between active (soluble) and inactive (precipitated) enzyme. The data indicate that inactivation is not caused by reaction with a specific active site group. Inactivation probably occurs when a combination of groups are reacted.

Binding Sites↗
PubMed1981

An immunoenzymatic assay for the detection and quantitation of platelet antibodies: the platelet beta-galactosidase test (PGT).

An immunoenzyme assay for detection of platelet antibodies and suitable for routine use is described. Purified rabbit IgG anti-human IgG antibodies are conjugated to beta-galactosidase with meta-maleimidobenzoyl-hydroxysuccinimide ester as a bifunctional reagent and o-nitrophenyl-beta-galactopyranoside as a substrate to evaluate the enzymatic activity of the labeled antiglobulin. The sera of 26 patients suffering from various diseases (acute leukemia, aplastic anemia and systemic lupus erythematosus) and 40 control subjects were assayed with the enzyme-labeled reagent and, for comparison, with an indirect immunofluorescence technique. Half of these patients had never been transfused. Platelet antibodies were detected by both assays in all the transfused patients except one, and in 3 out of 13 non-transfused patients. The sera of all the control subjects were negative. Quantitation of platelet antibodies was obtained by a sensitive antiglobulin absorption technique. A method for standardization of the reagents allowing comparison of results obtained in the same patient at different times and suitable for long-term follow-up studies is also described.

Absorption↗
PubMed1985

Which of the commonly used marker enzymes gives the best results in colorimetric and fluorimetric enzyme immunoassays: horseradish peroxidase, alkaline phosphatase or beta-galactosidase?

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.

Alkaline Phosphatase↗
PubMed1983

Calcium-independent adhesion of extra-embryonic endoderm cells from the early chick blastoderm is inhibited by the blastoderm beta-D-galactoside-binding lectin and by beta-galactosidase.

Extra-embryonic endoderm cells from gastrulating chick embryos possess Ca2+-dependent and Ca2+-independent adhesive mechanisms. These cells also contain an endogenous beta-D-galactoside-binding lectin and cell surface receptors bearing galactose groups. The endogenous lectin inhibits cellular adhesion. To test whether the adhesive interactions involving lectin and galactose molecules are part of the Ca2+-independent or Ca2+-dependent adhesive mechanism, dissociated cells which were preincubated in beta-galactosidase were allowed to aggregate in the presence and absence of Ca2+ ions. Significant decreases in adhesion were observed in both cases. Cells were also allowed to aggregate in the presence and absence of Ca2+ ions when blastoderm lectin was present in the medium. Adhesion was decreased in both cases. The results suggest that cell surface galactose groups and the beta-D-galactoside-binding lectin are involved in Ca2+-independent adhesion.

Animals↗
PubMed1981

Enzyme immunoassay of cyclic adenosine 5', 5' - monophosphate (AMP) using beta-D-galactosidase as label.

Succinyl cyclic adenosine monophosphate (AMP) was synthesized and coupled to beta-D-galactosidase using 1-ethyl-3-(3-dimethylaminopropyl)carbodimide. An antiserum to cyclic AMP was raised in rabbits by immunization with a succinyl cyclic AMP-human serum albumin conjugate. An enzyme immunoassay of cyclic AMP was successfully performed via the competitive binding procedure using the succinyl cyclic AMP-enzyme conjugate and the antiserum to cyclic AMP. The sensitivity of this assay has been increased several hundredfold by prior 2'-O-succinylation of cyclic AMP as already described by Cailla et al. in the radioimmunoassay of cyclic nucleotides. The assay system makes it possible to ascertain values as low as 6 fmol of cyclic AMP/tube. Human plasma cyclic AMP could be accurately determined by this method without requiring a deproteinizing reagent as the first step of assay. The concentration of TCA-extracted cyclic AMP from various tissues of mice was determined by both enzyme immunoassay and radioimmunoassay. There was a good correlation between the values for cyclic AMP determined by the two methods (Y = 1.01X - 0.15, r = 0.996, n = 39). Succinylated nucleotides and nucleosides such as cyclic GMP, ATP, ADP, and AMP, adenosine, adenine, GTP, GDP, GMP, guanosine, and guanine had no effect on the immunoassay of cyclic AMP.

Animals↗
PubMed1985

Action of beta-galactosidase on novel synthetic macromolecular substrates. A processive enzymic reaction controlled by coulombic interactions.

Macromolecular beta-galactosidase substrates were prepared by attaching o-nitrophenyl-beta-galactoside to carboxymethyldextran with positively charged linking groups. Almost all of the substituents were susceptible to enzymic hydrolysis by two distinct pathways. Under some conditions, there was random reaction to give a soluble product. In other conditions, in the initial stages of the reaction, most of the substituents of some, but not all, of the substrate polymers were hydrolyzed to give a product which precipitated as a second aqueous phase. Kinetics of hydrolysis were studied with respect to charge and molecular weight of both the enzyme and substrate. Factors that caused a decrease in Km favored formation of the second phase product. The reaction has similarities to the processive catalytic reactions found in naturally occurring enzyme systems with polymeric charged substrates.

Dextrans↗
PubMed1988

Cloning and expression of the phospho-beta-galactosidase genes on the lactose plasmid and the chromosome of Lactobacillus casei C257 in Escherichia coli.

A 4.7 kb BglII fragment of lactose plasmid pLY101 of Lactobacillus casei C257, which hybridized weakly to a fragment including a streptococcal phospho-beta-galactosidase (P-beta-gal) gene, was cloned into plasmid vectors of Escherichia coli. Transformant cells expressed P-beta-gal activity, confirming the presence of a P-beta-gal gene on pLY101. Deletion mapping showed that the structural gene of P-beta-gal was located on the 2.4 kb PvuI-EcoRV fragment. The direction of transcription was determined using an expression vector. L. casei MSK248 is a non-lactose-utilizing, pLY101-cured derivative of C257 and no P-beta-gal activity was detected. From MSK248, spontaneous lactose-utilizing revertants having P-beta-gal activity were isolated. Using the pLY101-derived P-beta-gal gene as a probe, a homologous sequence was detected in the chromosomal DNAs of MSK248 and MSK256, and cloned into plasmid vectors of E. coli. Both transformants carrying the homologous sequence from MSK248 and MSK256 expressed P-beta-gal activity. Restriction maps of the chromosomal P-beta-gal genes from MSK248 and MSK256 were identical with that derived from pLY101.

Chromosome Mapping↗