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PubMed1978

Amino acid sequence of beta-galactosidase. VII. Isolation of the 24 cyanogen bromide peptides.

All of the 24 cyanogen bromide peptides of beta-galactosidase have been isolated in pure form. Of these 8 ranged in size from 2 to 5 residues and were purified by paper electrophoresis. The 16 large peptides, from 23 to 119 residues, were chromatographed at pH 5.0 on a carboxymethyl-cellulose column in 0.02 M ammonium acetate buffer containing 8 M urea. A number of peptides were obtained in pure from following Sephadex G-50 or G-75 gel filtration. Others were separated on sulfopropyl-Sephadex or diethyl-(2-hydroxylpropylaminoethyl)-Sephadex. There large peptides were obtained in over 50% yield and several others were obtained in more than 25% yield.

Amino Acid Sequence↗
PubMed1978

Amino acid sequence of beta-galactosidase. X. Sequence of the COOH-terminal segment, CNBr peptides 18 to 24, residues 654 to 1021.

The sequence of the COOH-terminal third (omega) of beta-galactosidase is presented. The size of the 7 cyanogen bromide peptides of this segment is larger on the average, about 52 amino acid residues as compared to an average size of 42 for cyanogen bromide peptides in the whole molecule. Tyrosine, phenylalanine, and valine are low in this segment whereas alanine and lysine are high. This region has a slight excess of basic groups.

Amino Acid Sequence↗
PubMed1978

Amino acid sequence of beta-galactosidase. XI. Peptide ordering procedures and the complete sequence.

The amino acid sequence of beta-galactosidase has been determined. The monomer contains 1,021 amino acid residues in a single polypeptide chain and has a molecular weight of 116,349. All 80 tryptic peptides as well as all 24 CNBr peptides have been isolated in pure form. Evidence is presented for the ordering of the CNBr peptides. The sequence determination was aided by analysis of cyanogen bromide peptides obtained from a polypeptide fragment produced by a lacZ termination mutant strain.

Amino Acid Sequence↗
PubMed1977

Demonstration of "hetero-beta-galactosidase" "in situ".

A method for the histochemical demonstration of "hetero-beta-galactosidase" was elaborated. The enzyme is demonstrated in cryostat sections by the semipermeable membrane technique. Pairs of membranes--one pre-washed in saline--are used. The most sensitive method is post-coupling demonstration with 6-Br-2-naphthyl-beta-D-glucoside. The incubation time must be short, to avoid diffusion. The method allows cellular localization. The method with alpha-naphthyl-beta-D-glucoside and hexazonium-p-rosaniline is less sensitive, but localization is better. Indigogenic methods are the least sensitive. The enzyme is localized in the supranuclear zone of differentiated enterocytes of the human, monkey and rabbit small intestine, with maximum activity in the jejunum. The activity of the enzyme is low in patients with coeliac sprue, in the active phase of the disease. In isolated lactase deficiency it is normal. In the kidney, the enzyme is localized chiefly in the cytoplasm of the proximal tubule cells.

Animals↗
PubMed1977

Macular cherry-red spots and beta-galactosidase deficiency in an adult. An autopsy case with progressive cerebellar ataxia, myoclonus, thrombocytopathy, and accumulation of polysaccharide in liver.

An adult patient with macular cherry-red spots, a gargoyle-like physical appearance, cerebellar ataxia, myoclonus, convulsive seizures, and pyramidal tract signs showed a profound deficiency of beta-galactosidase in liver and brain. Thrombocytopathy of undetermined etiology was evident since childhood, and the patient died of intracranial bleeding at age 22. Cerebral ganglioside pattern was normal. Hepatic mucopolysaccharides were not increased. GM1-gangliosidosis and mucopolysaccharidosis were ruled out by those analytical data. However, a large amount of amylopectin-like polysaccharide was found to be accumulated in liver. Hepatocyte contained numerous inclusion bodies with granulofibrillary structure similar to Lafora bodies, corpora amylacea, and inclusion bodies in glycogenosis type IV. This case seems to represent a new inborn metabolic disease closely related to GM1-gangliosidosis and mucopolysaccharidosis. The primary metabolic defect is not known at present.

Adult↗
PubMed2001

High beta-galactosidase and ganglioside GM1 levels in the human parotid gland.

BACKGROUND: Ganglioside G(M1) is a membrane glycolipid typical of nerve cell membranes, where it partakes in neurotransmitter release and is catabolized by the lysosomal beta-galactosidase (GM1ase) (EC 3.2.1.23). After demonstrating a novel degenerative disease of the parotid gland in mice deficient in GM1ase, mimicking the human storage disease GM(1) gangliosidosis, we studied GM1ase and ganglioside G(M1) content in the human parotid glands. STUDY DESIGN: Levels of GM1ase and ganglioside G(M1) were determined in samples of parotid tissues and neighboring muscle (as a negative control) for 3 subjects. Tissues were also processed for histochemical demonstration of GM1ase. RESULTS: The mean specific activity of GM1ase was more than 6-fold higher in the healthy human parotid tissues (1.4 +/- 0.5 nmol of 4-methylumbelliferone per minute per milligram of protein) relative to the neighboring muscle tissue (0.23 +/- 0.07 nmol of 4-methylumbelliferone per minute per milligram of protein). Activity of GM1ase was histochemically localized mainly to striated duct and acinar cells of the parotid gland. Ganglioside G(M1) content in the parotid gland was on average 30-fold higher relative to muscle. CONCLUSIONS: Our results are consistent with previous findings reported in the mouse and the rabbit, and probably reflect a general property of the mammalian parotid glands. The novel mechanism we previously proposed for the mouse parotid saliva secretion, mimicking neurotransmitter release in ganglioside G(M1)-containing nerve cells, is probably applicable also to the human parotid gland. Similarly, the human parotid gland is probably also severely affected in GM(1) gangliosidosis.

Adult↗
PubMed1997

Limitations of nls beta-galactosidase as a marker for studying myogenic lineage or the efficacy of myoblast transfer.

BACKGROUND: Nuclear localizing beta-galactosidase (nls beta-gal) is used as a marker for studying myoblast cell lineage and for evaluating myoblast survival after myoblast transfer, a procedure with potential use for gene complementation for muscular dystrophy. Usefulness of this construct depends on the establishment of the extent to which nls beta-gal or its mRNA may be translocated from the nucleus that encodes it to other non-coding myonuclei in hybrid myofibers and the ease with which the encoding and non-coding myonuclei can be distinguished. Previous in vitro studies (Ralston and Hall 1989. Science, 244:1066-1068) have suggested limited translocation of the fusion protein. We re-examined the extent to which nls beta-gal is translocated in hybrid myofibers, both in vitro and in vivo, and evaluated the extent to which one can rely on histochemistry to distinguish encoding from non-coding nuclei in these myofibers. METHODS: Myotubes formed in co-cultures of a myoblast line (MM14 cells), stably transfected with a construct consisting of a nls beta-gal under the control of the myosin light chain 3F promoter and 3' enhancer (3FlacZ10 cells), and [3H]-thymidine-labeled parental MM14 cells (plated at ratios of 1:6 or 1:20, respectively) were reacted with X-gal. After autoradiography, the distance over which nls beta-gal was translocated in hybrid myotubes was determined. In vivo translocation of nls beta-gal was evaluated by injecting [3H]-thymidine-labeled 3FlacZ10 myoblasts into the regenerating extensor digitorum longus muscle of immunosuppressed normal and mdx (dystrophin deficient) mice. Sections stained with X-gal and subjected to autoradiography permitted determination of the extent of nls beta-gal translocation in hybrid myofibers. RESULTS: In vitro: All nuclei in > 92% of hybrid myotubes showed evidence of nls beta-gal after exposure to X-gal, suggesting extensive translocation. Within hybrid myotubes, MM14-derived myonuclei approximately 350 microns from a 3FlacZ10-derived myonucleus showed evidence of nls beta-gal. In vivo: Similar translocation of nls beta-gal was observed in vivo. One week after myoblast transfer, donor-derived myonuclei were distinguishable from host-derived myonuclei containing nls beta-gal by the greater accumulation of reaction product in donor myonuclei after X-gal staining. However, 2 weeks after injection, host myonuclei often contained a significant amount of nls beta-gal, and accumulation of reaction product could not be used as the criterion for identification of donor myonuclei. CONCLUSIONS: Translocation of nls beta-gal (or its mRNA) is significantly greater than previously reported (Ralston and Hall 1989), resulting in large numbers of nls beta-gal positive non-coding myonuclei in hybrid myofibers. One week after myoblast transfer, distinguishing between nls beta-gal encoding and non-coding myonuclei in hybrid myofibers after X-gal staining of sectioned muscle is feasible; however, by 2 weeks, nls beta-gal increases in host myonuclei, making identification of donor-derived myonuclei problematic. Translocation of nls beta-gal to non-coding myonuclei in hybrid myofibers must be considered when nls beta-gal is used for studies of myogenic lineage or the efficacy of myoblast transfer therapy, particularly if long-term survival of hybrid myotubes is required.

Animals↗
PubMed1999

Tolerance of Escherichia coli beta-galactosidase C-terminus to different-sized fusions.

The tolerance of the beta-galactosidase C-terminus to foreign protein fusions has been explored by using different-sized derivatives of the chimeric protein LACVP1. While the molecular mass of the partner domain shows a minor influence on protein toxicity for the producing E. coli cells, it dramatically affects the proteolytic susceptibility of the whole fusion. Surprisingly, the observed structural modulation of proteolysis is not an all-or-nothing process, but it exhibits a continuous effect concomitantly with the length of the fusion. The conformational effects caused by increasingly sized partners seem to progressively expose cryptic protease target sites, initiating a proteolytic cascade that dramatically reduces the yield of the recombinant protein.

Amino Acid Sequence↗
PubMed2000

Enhanced growth of Sf-9 cells to a maximum density of 5.2 x 10(7) cells per mL and production of beta-galactosidase at high cell density by fed batch culture.

Significant improvement in cell growth and protein production has been achieved in Sf-9 insect cell cultures using pulse additions of multicomponent nutrient feed concentrates (Bédard et al., 1994; Chan et al., 1998). The present work focuses on investigating an alternative feeding strategy wherein the nutrients are fed in a semi continuous manner. Fed batch culture experiments were carried out to compare the two different feeding strategies, pulse and semi continuous and a process developed to achieve a cell density of 5.2 x 10(7) cells/mL of Sf-9 cells in a 3.5 L bioreactor. Production of recombinant protein beta-galactosidase was carried out by infecting the cells with baculovirus at a MOI of 10 at cell densities of 17 x 10(6)cells/mL. Specific productivity could be maintained at cell densities as high as 14 x 10(6) cells/mL. The results presented indicate that the feeding method can provide significant improvements in the performance with a reduction in the amount of total nutrients added. On-line monitoring of the culture using the capacitance probe showed that the capacitance probe can be used successfully to monitor the biomass and infection process even at higher cell densities.

Animals↗
PubMed1996

A sensitive mutation screening strategy for Fabry disease: detection of nine mutations in the alpha-galactosidase A gene.

Fabry disease is an X-linked recessive lysosomal storage disorder caused by a deficiency of alpha-galactosidase A (alpha-gal; EC 3.2.1.22). In the past, it has been difficult to give an unequivocal diagnosis of carrier status in Fabry disease because of the overlap between normal and heterozygote enzyme levels. To facilitate rapid and accurate carrier and hemizygote detection, a mutation detection strategy was devised to determine the lesion in our Fabry disease patients. The seven alpha-gal exons and adjacent intron boundaries from a representative member of each kindred were PCR amplified and analysed for the presence of sequence alterations by single-stranded conformation polymorphism (SSCP) analysis followed by PCR sequencing. Here we report the use of this strategy in the detection and analysis of the causative mutations in 9 patients with classic severe Fabry disease. Three deletions of 1-, 2-, and 3-bp (987delC, 717delAA, and delta E358), five amino acid substitutions (C52R, G128E, P205T, M284T, and N298K) and a mutation that affects the initiating methionine (M1I) were found in these patients. Counting a previously reported mutation, this strategy has now successfully detected all the Fabry disease mutations present in the 10 kindreds that have been analysed.

Adult↗
PubMed1998

A new informative Alw 26 I polymorphism in exon 10 of the human low density lipoprotein receptor gene. Application to prenatal diagnosis. Mutations in brief no. 145. Online.

Using DNA sequencing of the coding and exon flanking regions of the low density lipoprotein receptor (LDLR) gene we identified an Alw26 I site in exon 10 by a transition G142A. The alleles are represented by one uncut fragment (A1=108bp) or two fragments (A2=82bp and 26 pb). Two other fragments (72bp and 16bp) were systematically found within the amplified product. The alleles were detected in 157 unrelated French Caucasians with A1 frequency = 0.58 and A2=0.42. The observed heterozygoty was 44.5%. Homozygous familial hypercholesterolemie (FH) has a severe clinical picture leading to death during childhood. Because it is very informative, the present polymorphism was very useful as genetic marker for clinical diagnois and counseling as we described in linkage analysis at the LDLR locus for prenatal diagnosis in a fetus who could inherit two LDLR mutant alleles from FH heterozygote parents.

DNA-Cytosine Methylases↗
PubMed1999

Six novel beta-galactosidase gene mutations in Brazilian patients with GM1-gangliosidosis.

GM1-gangliosidosis is a lysosomal storage disease caused by a deficiency of acid beta-galactosidase. Three clinical forms are recognized-infantile, juvenile, and adult-based on age of onset and severity of the symptoms. We have performed molecular analysis of a large cohort of GM1 patients (19 Brazilian and one Uruguayan), using nonradioactive single-strand conformation polymorphism (SSCP) and restriction enzyme analysis of genomic DNA. Six novel mutations (R121S, V240M, D491N, 638-641insT, 895-896insC, 1622-1627insG) and two previously described point mutations (R59H, R208C) were identified. Together they accounted for 90% of the disease alleles of the patients. Two mutations, 1622-1627insG and R59H, were present in 18 of 20 patients. In addition, four polymorphisms (L10P, L12L, R521C, S532G) were identified. All cases reported are infantile GM1 gangliosidosis. This report constitutes the most comprehensive molecular study to date of this disorder in infantile patients. Since GM1-gangliosidosis is the most common lysosomal storage disorder in Southern Brazil, molecular diagnosis will be important for genetic counseling, carrier detection and prenatal diagnosis in index families.

Brazil↗
PubMed1999

Rapid colorimetric assay of urinary beta-galactosidase and N-acetyl-beta-D-glucosaminidase with Cobas Mire Auto-analyzer.

A rapid and simple colorimetric method for the assay of urinary beta-galactosidase (GAL) and N-acetyl-beta-D-glucosaminidase (NAG) using 4-nitrophenyl-glycosides as the substrates and a Cobas Mire Auto-analyzer is described. Optimal conditions for this method including substrate concentration, ionic strength, pH and incubation time were characterized. Endogenous substances in urine did not interfere with the assays. Incubation time could be shortened to 10 minutes. A small volume of an alkaline buffer to terminate the enzyme reaction achieved better sensitivity and accuracy. Buffer-substrate solutions were stable for at least one week at 4 degrees C. Normal values of the two urinary enzymes are also reported.

Acetylglucosaminidase↗
PubMedDate not supplied

Effects of the composition of bacteriological growth media on a chemiluminometric assay of beta-galactosidase in Escherichia coli.

The effects of the composition of bacteriological growth media on the light output in a chemiluminometric assay of beta-galactosidase in Escherichia coli using 1,2-dioxetane substrates has been studied. In this assay a basic conflict exists between conditions that promote optimal bacterial growth and those conducive to maximal chemiluminescence. Common medium ingredients such as yeast or beef extract, protein hydrolysates and lactose suppress light emission and/or lead to high backgrounds. Quenching of light emission is probably partly due to light absorption by medium ingredients such as oxgall, and partly to interference with the reaction triggering the chemiluminescent process. Elevated backgrounds are caused by the presence of high concentrations of protein hydrolysates, which interact with the alkali in the accelerator solution. Only two purposely developed media, i.e. ILM and Colicult are shown to reconcile the requirements of growth support with that of optimal luminescent properties.

Bacteriological Techniques↗
PubMedDate not supplied

Chemiluminescent assay of beta-D-galactosidase based on indole luminescence.

We developed a novel chemiluminescent assay of beta-D-galactosidase (beta-gal) based on the chemiluminescence of indole. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) was used as a substrate for beta-gal and also as a light emitter. X-gal was hydrolysed by beta-gal to liberate free indoxyl, followed by oxidation to indigo dye, and simultaneously produces hydrogen peroxide (H2O2). H2O2 reacts with the residual X-gal in the presence of horseradish peroxidase (HRP) to emit light. The measurable range of beta-gal obtained by this method was 6 x 10(-14) mol/L to 6 x 10(-11) mol/L; the detection limit was 3 amol/assay. This chemiluminescent assay could be applied to an enzyme immunoassay of thyroxine using beta-gal as the enzyme label.

Galactosides↗
PubMed2001

Electrophoretically mediated microanalysis of beta-galactosidase on microchips.

Electrophoretically mediated microanalysis (EMMA) is a method of accomplishing chemical analyses, typically in an open-tubular capillary, due to the difference in the electrophoretic mobility between the particular reagents. This work reports on combining this technique onto microfabricated systems. Two methods of this technique were applied, constant potential and zero potential EMMA onto chips. A dosage response curve was run using this constant potential mode that resulted in a linear response over three orders of substrate concentration magnitude. The chemical system used here is beta-galactosidase (beta-Gal) as the enzyme and fluorescein mono-beta-D-galactopyranoside (FMG) as the substrate. The zero potential mode was used to amplify product turnover using various incubation times. Using this technique and a 10 min incubation, approximately 40000 enzyme molecules could be detected. The zero potential mode is also used in conjunction with an internal standard to show how one can quantitate using this method. The power and ease of utility of this technique is described.

Electrophoresis, Capillary↗
PubMed1995

The 67 kDa spliced variant of beta-galactosidase serves as a reusable protective chaperone for tropoelastin.

Numerous cell types express the 67 kDa galactolectin related to the alternatively spliced variant of beta-galactosidase. This 67 kDa protein, while present on cell surfaces, mediates cell contacts with elastin, laminin and collagen type IV. In elastin-producing tissues, the 67 kDa protein also co-localizes with intracellular tropoelastin and mature elastic fibres. We have established that this elastin binding protein (EBP) serves as a molecular chaperone for tropoelastin. The EBP binds this highly hydrophobic and unglycosylated ligand intracellularly, protecting it from intracellular self aggregation and premature proteolytic degradation, and mediates its orderly assembly upon the microfibrillar scaffold. While some of this protein is incorporated as a permanent component of elastic fibres, most of the EBP, after extracellular dissociation from its ligand, recycles back to the intracellular endosomal compartment and re-associates with the newly synthesized tropoelastin. We suggest that recycling of this reusable shuttle protein is imperative for the effective extracellular deposition of insoluble elastin.

Alternative Splicing↗