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At least 217 records · Page 12Linked to original sources

PubMed1987

Endocytotic internalization of alpha-2-macroglobulin: alpha-galactosidase conjugate by cultured fibroblasts derived from Fabry hemizygote.

Endocytotic internalization of alpha-galactosidase by cultured fibroblasts derived from a patient with Fabry's disease was achieved via receptor-mediated endocytosis of alpha-2-macroglobulin (alpha-2-M). alpha-galactosidase of coffee beans was conjugated to alpha-2-M when the latter was treated with trypsin. Internalization of the conjugate resulted in an increase of alpha-galactosidase activity in the crude cell extracts. The observed internalization was blocked by the presence of bacitracin, an inhibitor of binding between alpha-2-M and its receptor on the cell surface. When the cells were incubated at 4 degrees C with the conjugate, internalization was also inhibited. The alpha-galactosidase activity in the cells was saturated when the concentration of the conjugate in the medium was 40 micrograms/ml. Since non-conjugated alpha-galactosidase was not effectively internalized, the observed internalization of the conjugate was mediated by recognition of alpha-2-M by its receptor. The effective internalization of alpha-galactosidase described in this paper has a potential use in the enzyme replacement therapy of Fabry's disease.

Cells, Cultured↗

PubMed1990

Relationship of the multiple forms of human alpha-D-galactosidase and alpha-D-fucosidase in the normal and in Fabry's disease.

Activities and multiple forms of alpha-D-galactosidase of human kidney and liver in the normal and in Fabry's disease were comparatively studied using alpha-D-galactoside and alpha-D-fucoside as substrates. By isoelectric focusing alpha-D-galactosidase was shown to exist in multiple forms, one of which possesses both alpha-D-galactosidase and alpha-D-fucosidase activity. In Fabry's disease, caused by a deficiency of alpha-D-galactosidase A, we found only one form of alpha-D-galactosidase, which corresponded to form B (alpha-N-acetylgalactosaminidase) and was also able to split alpha-D-fucoside. Thus, in Fabry's disease the alpha-D-fucosidase profile was virtually unchanged, as compared with the normal. The results obtained indicate that the alpha-D-fucosidase activity is due to the action of alpha-D-galactosidase B, encoded for by an autosomal gene of chromosome 22. We suppose these data could be confirmed by revealing the significant reduction of the alpha-D-fucosidase activity in patients with alpha-N-acetylgalactosaminidase deficiency.

Chemical Phenomena↗

PubMed1989

Grafting genetically modified cells into the rat brain: characteristics of E. coli beta-galactosidase as a reporter gene.

The utility of grafting genetically modified cells to the mammalian brain was examined using the E. coli beta-galactosidase gene (lacZ) as a reporter gene in retroviral infection. Following implantation of the infected cells to the brain, lacZ continued to be expressed in vivo and could be detected easily with enzyme histochemistry. However, beta-galactosidase-positive cells were also observed in control grafts which had not been infected with the virus. This false-positive staining was found to be endogenous lysosomal activity associated with macrophage infiltration presumably induced by the damage associated with grafting. The E. coli gene product was distinguished from cellular lysosomal beta-galactosidase by using immunohistochemical staining with an antibody specific for E. coli beta-galactosidase. With this antibody, retrovirus-infected cells could be distinguished in the brain, and no false positives were observed in non-infected cells. We conclude that E. coli beta-galactosidase is a useful reporter gene for determining the fate of implanted cells to the brain if appropriate caution is taken to distinguish it from cellular beta-galactosidase by immunocytochemical procedures.

Animals↗

PubMed1979

A dimer--dimer binding region in beta-galactosidase.

alpha Complementation in beta-galactosidase is the restoration of enzyme activity by addition of the alpha donor CNBr2, from amino acid residues 3--92 of the polypeptide, to inactive M15 protein from the lacZ deletion mutant strain M15. M15 protein lacks residues 11--41 and is a dimer; the active complex, like native beta-galactosidase, is tetrameric [Langley, K. E., & Zabin, I. (1976) Biochemistry 15, 4866--4875]. A dimer--dimer binding region in beta-galactosidase has been identified by proteolytic and immunologic studies of alpha-complementation. Proteolytic experiments were carried out with trypsin. Treatment of native beta-galactosidase with trypsin, followed by reaction of the mixture with cyanogen bromide, yields intact CNBr2 as measured by its ability to complement M15 protein. Active CNBr2 is not obtained when urea-denatured beta-galactosidase is treated in the same way. Therefore the segment corresponding to CNBr2 is apparently buried within the folded protein. Immunologic experiments were carried out with antibodies against CNBr2, tryptic peptide T8 (residues 60--140), and CNBr3 (residues 93--187). Anti-CNBr2 and anti-T8 bind to M15 protein but not to beta-galactosidase, indicating that this area is exposed in the dimer. Anti CNBr2, but not anti-T8 or anti-CNBr3, inhibits the formation of alpha-complemented enzyme. These results indicate that an early part of the sequence, within the segment corresponding to CNBr2, is involved in dimer--dimer interaction.

Antibodies↗

PubMed1977

The separation and characterization of marmoset kidney beta-D-galactosidase and beta-D-glucosidase.

beta-D-Galactosidase and beta-D-glucosidase activities were determined in homogenates of marmoset kidney by using the appropriate 4-methylumbelliferyl glycoside, beta-D-Galactosidase activity was separated into two main components by ion-exchange chromatography on DEAE-cellulose, starch-gel electrophoresis, isoelectric focusing and gel filtration on Sephadex G-200. One form designated A had a pI of 5.1, was loosely bound to DEAE-cellulose at pH7.0, remained near the origin on starch-gel electrophoresis at pH 7.0 and had an apparent molecular weight of 160000. The second beta-D-galactosidase component, designated B, was associated with the total beta-D-glucosidase activity, had a pI of 4.3, was firmly bound to DEAE-cellulose, migrated rapidly towards the anode on starch-gel electrophoresis and had an apparent molecular weight of 50000. The optimum pH values of beta-D-galactosidase A and B were 4.5 and 6.0 respectively. beta-D-Galactosidase A was activated by 0.1 M-NaC1 but the activity of the B form was inhibited by 1 M-NaC1 at pH 4.5. beta-D-galactosidase had a bimodal distribution, the A form being recovered in the lysosomal fraction whereas the B form was present in the soluble fraction, as was the major portion of the beta-D-glucosidase activity. The lysosomal and soluble forms were further characterized by DEAE-cellulose chromatography.

Animals↗

PubMed1983

Dog and human acid beta-D-galactosidases are structurally similar.

The purification of dog liver acid beta-galactosidase is described. The dog enzyme migrated as a single major band on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, with a molecular weight of 60000. Antiserum raised against purified human liver acid beta-galactosidase cross-reacted with beta-galactosidase from dog liver, but not with those from cat liver or Escherichia coli. Tryptic peptide maps of the dog and human acid beta-galactosidases indicate that 21 of the 24 peptides observed were homologous; a similar result was obtained after chymotryptic peptide mapping. We conclude that dog and human acid beta-galactosidases are structurally similar, and that canine GM1 gangliosidosis (acid beta-galactosidase deficiency) is an excellent model for the same disease in man.

Animals↗

PubMed1990

Structure of the lysosomal neuraminidase-beta-galactosidase-carboxypeptidase multienzymic complex.

Lysosomal neuraminidase (sialidase; EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23), together with a carboxypeptidase, the so-called 'protective protein', were co-purified from the human placenta by affinity chromatography on a concanavalin A-Sepharose column followed by a thiogalactoside-agarose affinity column for beta-galactosidase. Analysis of the purified material by gel-filtration h.p.l.c. revealed three distinct molecular forms, all with high beta-galactosidase specific activity, but only the largest one expressed neuraminidase activity. Rechromatography of each individual species separately indicated that all three are in fact part of an equilibrium system (the neuraminidase-beta-galactosidase-carboxypeptidase complex or NGC-complex) and that these species undergo slow conversion into one another through dissociation and association of protomeric components. Each species was sufficiently stable for the determination of their hydrodynamic properties by gel-filtration h.p.l.c. and sedimentation velocity. The largest species had an apparent sedimentation coefficient S20.w, of 18.8 S and a Stokes' radius of 8.5 nm, giving a molecular mass of 679 kDa and a fractional ratio, f/f min, of 1.47. The latter value indicates that the macromolecule is asymmetric or highly hydrated. This large species is composed of four types of polypeptide chains of molecular mass 66 kDa (neuraminidase), 63 kDa (beta-galactosidase), 32 kDa and 20 kDa (carboxypeptidase heterodimer). The 32 kDa and 20 kDa protomers are linked together by a disulphide bridge. Glycopeptidase F digestion of the NGC-complex transformed the diffuse 66-63 kDa band on the SDS gel into two close but sharp bands at 58 and 56 kDa. The two smaller species which were separated on the h.p.l.c. column correspond to tetrameric and dimeric forms of the 66-63 kDa protomers and express exclusively beta-galactosidase activity. Treatment of the NGC-complex with increasing concentrations of guanidinium hydrochloride up to 1.5 M also resulted in dissociation of the complex into the same smaller species mentioned above plus two protomers of molecular mass around 60 and 50 kDa. A model of the largest molecular species as a hexamer of the 66-63 kDa protomers associated to five carboxypeptidase heterodimers (32 kDa and 20 kDa) is proposed

Carboxypeptidases↗

PubMed1990

Active-site-directed inactivation of Aspergillus oryzae beta-galactosidase with beta-D-galactopyranosylmethyl-p-nitrophenyltriazene.

beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene (beta-GalMNT), a specific inhibitor of beta-galactosidase, was isolated as crystals by HPLC and its chemical and physicochemical characteristics were examined. Aspergillus oryzae beta-galactosidase was inactivated by the compound. We studied the inhibition mechanism in detail. The inhibitor was hydrolyzed by the enzyme to p-nitroaniline and an active intermediate (beta-galactopyranosylmethyl carbonium or beta-galactopyranosylmethyldiazonium), which inactivated the enzyme. The efficiency of inactivation of the enzyme (the ratio of moles of inactivated enzyme to moles of beta-GalMNT hydrolyzed by the enzyme) was 3%; the efficiency of Escherichia coli beta-galactosidase was 49%. In spite of the low efficiency, the rate of inactivation of A. oryzae enzyme was not very different from that of the E. coli enzyme, because the former hydrolyzed beta-GalMNT faster than the latter did. A. oryzae beta-galactosidase was also inactivated by p-chlorophenyl, p-tolyl, and m-nitrophenyl derivatives of beta-galactopyranosylmethyltriazene. However, E. coli beta-galactosidase was not inactivated by these triazene derivatives. The results showed that the inactivation of A. oryzae and E. coli beta-galactosidases by beta-GalMNT was an enzyme-activated and active-site-directed irreversible inactivation. The possibility of inactivation by intermediates produced nonenzymatically was ruled out for E. coli, but not for the A. oryzae enzyme.

Aspergillus↗

PubMed1989

Enzyme replacement for lactose malabsorption using a beta-D-galactosidase.

We evaluated 10 healthy symptomatic lactose malabsorbers for effect of an oral beta-D-galactosidase derived from Aspergillus oryzae (Lactrase, Kremers Urban Company, Milwaukee, WI, U.S.A.) on symptom and breath hydrogen response to challenge with 50 g lactose. Basally and at 30-min intervals for 8 h after lactose challenge, end-alveolar breath samples were collected and analyzed for hydrogen using gas chromatography. Symptoms were scored at 30 min and hourly for 8 h, rating bloating, cramps, nausea, pain, diarrhea, and flatulence. Four challenges were performed on 4 separate days with at least 3 days between challenges. The first two challenges served as baselines. Just before ingestion of 50 g powdered lactose dissolved in 200 ml water, beta-D-galactosidase capsules were given orally as a 250-mg dose for the third challenge and a 500-mg dose for challenge 4. Hydrogen excretion, quantified by using a trapezoidal method for computing area under the discontinuous curve of breath hydrogen concentration, was decreased in subjects receiving beta-D-galactosidase (base-line I, 346.0 ppm/h; baseline II, 367.2 ppm/h; 250-mg galactosidase 208.2 ppm/h; 500-mg galactosidase, 178.0 ppm/h; p less than or equal to 0.05). Other analyzed parameters of H2 excretion were also decreased. Analysis of symptom response scores showed a dose-related decrease for bloating and flatus (p less than or equal to 0.05) and no statistical difference in the other assessed symptoms. We conclude that beta-D-galactosidase from Aspergillus oryzae, when given just before ingestion of lactose by lactose malabsorbers, can produce a dose-dependent reduction (statistically significant for the 500-mg dose) in breath hydrogen excretion, bloating, and flatus.

Adult↗

PubMed1985

Human placental neuraminidase. Activation, stabilization and association with beta-galactosidase and its protective protein.

Supernatant of homogenized human placenta hardly contains lysosomal neuraminidase activity. It is, however, possible to generate remarkably high activity by concentration of a partially purified glycoprotein fraction. This activity is labile to dilution, but can be stabilized by incubation at 37 degrees C and acid pH. Using beta-galactosidase specific affinity chromatography and immunotitration, we show that the activated and stabilized human lysosomal neuraminidase exists in a complex with beta-galactosidase. Sucrose density gradient centrifugation experiments demonstrate that the neuraminidase activity is exclusively present in a high density multimeric form of beta-galactosidase. The formation of multimeric forms of beta-galactosidase is known to require a 32000-Mr 'protective' protein. Monospecific antibodies against this 'protective' protein were purified from a conventional antiserum containing a mixture of antibodies against the 64000-Mr beta-galactosidase protein and against the 32000-Mr 'protective' protein, using a nitrocellulose blot immunoaffinity purification procedure. Immunotitration experiments with these antibodies show that the 32000-Mr 'protective' protein is present both in association with the beta-galactosidase multimer and with the high-density multimeric form together with neuraminidase. Our data further suggest that association of the 32000-Mr 'protective' protein and another yet unidentified subunit is essential for the catalytic activity of lysosomal neuraminidase. These results explain the absence of neuraminidase activity in the autosomal recessive human lysosomal storage disorder galactosialidosis, where the 32000-Mr 'protective' protein is known to be absent.

Carboxypeptidases↗

PubMed1987

Coagglutination and enzyme capture tests for detection of Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase.

Polyclonal antibodies to Escherichia coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase were used in coagglutination tests for identification of these three enzymes in cell lysates. Enzyme capture assays were also developed for the detection of E. coli beta-galactosidase and beta-glucuronidase. The enzymes were released by using a gentle lysis procedure that did not interfere with antibody-enzyme interactions. All three enzymes were detected in 93% (51 of 55) of the E. coli strains tested by coagglutination; two of the three enzymes were identified in the remaining 7%. Of 42 non-E. coli tested by coagglutination, only four nonspecifically agglutinated either two or three of the anti-enzyme conjugates. Thirty-two (76%) non-E. coli isolates were negative by coagglutination for all three enzymes. The enzyme capture assay detected the presence of beta-galactosidase in seven of eight and beta-glucuronidase in all eight strains of E. coli tested. Some strains of beta-galactosidase-positive Citrobacter freundii and Enterobacter cloacae were also positive by the enzyme capture assay, indicating that the antibodies were not entirely specific for E. coli beta-galactosidase; however, five other gas-positive non-E. coli isolates were negative by the enzyme capture assay. The coagglutination tests and enzyme capture assays were rapid and sensitive methods for the detection of E. coli beta-galactosidase, beta-glucuronidase, and glutamate decarboxylase.

Agglutination Tests↗

PubMed1978

Regulated expression by readthrough translation from a plasmid-encoded beta-galactosidase.

We have characterized expression of beta-galactosidase from a plasmid cloning vehicle, pBGP120, which carries most of the lacZ gene and contains a single EcoRI site near the end of lacZ. In addition, we have examined expression of heterologous DNA inserted at the position of the EcoRI site. The EcoRI site was shown to be within the sequence coding for beta-galactosidase and its precise location and phase were deduced. Insertion of heterologous EcoRI-generated DNA fragments altered the molecular weight of the plasmid-encoded beta-galactosidase polypeptide. Those insertions that were in the correct phase were expressed at a high level as a fused protein. The different forms of beta-galactosidase polypeptides produced by various hybrid plasmids were all stable proteins. The level of expression of the plasmid-encoded beta-galactosidase was several times higher than maximal expression of chromosome-encoded beta-galactosidase, suggesting that expression is proportional to gene copy number. The expression of the plasmid lacZ gene was controlled by cyclic AMP. When grown in a cya strain (DG74), expression was dependent on exogenous cyclic AMP. Although in normal strains there was insufficient lac repressor to inactivate all copies of the plasmid, repressor regulation was restored when the plasmid was grown in a strain (M96) that overproduces the lac repressor.

Cyclic AMP↗

PubMed1984

Mutation prlF1 relieves the lethality associated with export of beta-galactosidase hybrid proteins in Escherichia coli.

The 42-1 lamB-lacZ gene fusion confers a conditionally lethal, export-dependent phenotype known as maltose sensitivity. A maltose-resistant mutant showing decreased beta-galactosidase activity of the hybrid protein, designated prlF1 (protein localization), was unlinked to the lamB-lacZ fusion. This mutation mapped at 70 min on the Escherichia coli linkage map and conferred maltose resistance, a 30-fold reduction in beta-galactosidase activity, and a 30% decrease in cellular growth rate at 30 degrees C that was independent of the presence of a gene fusion. prlF1 also decreased the beta-galactosidase activity and relieved the maltose sensitivity conferred by fusions of lacZ to the gene specifying the periplasmic maltose-binding protein, malE. The decrease in beta-galactosidase activity, however, was specific for exported hybrid proteins. When export of the hybrid protein was blocked by a signal sequence mutation, prlF1 decreased the beta-galactosidase activity only 2.5-fold. Similarly, prlF1 did not affect the beta-galactosidase activity of fusions of lacZ to a gene specifying a nonexported protein, malK.

Bacteriophage lambda↗

PubMed1986

Cloning and expression of the phospho-beta-galactosidase gene of Staphylococcus aureus in Escherichia coli.

The phospho-beta-galactosidase gene of Staphylococcus aureus was cloned in Escherichia coli. This was done by first isolating a staphylococcal transposon Tn551-induced mutant which rendered phospho-beta-galactosidase synthesis partially constitutive because of an insertion nearby this lac structural gene. This allowed selection in E. coli of chimeric plasmids which expressed the erythromycin resistance determinant of Tn551. A 26-kilobase (kb) BamHI insert in plasmid pBR322 was isolated which encoded phospho-beta-galactosidase, as determined by phospho-beta-galactosidase activity measurements. Maxicell experiments showed the presence of 56-, 13.5-, and 31-kilodalton proteins encoded by the staphylococcal DNA. The presence of the 56-kilodalton protein correlated with phospho-beta-galactosidase activity and corresponded in molecular weight to the reported value for the purified enzyme. The nature of the other proteins is unknown. Phospho-beta-galactosidase was apparently expressed in E. coli by a promoter contained within a 2.1-kb EcoRI chromosomal DNA fragment. This fragment, when inserted into a chloramphenicol acetyl transferase promoter detection plasmid, was transcriptionally active in both E. coli and Bacillus subtilis but was much more active in the latter host.

Acetyltransferases↗

PubMed1989

Genetic studies on the inability of beta-galactosidase to be translocated across the Escherichia coli cytoplasmic membrane.

When a signal sequence is attached to beta-galactosidase, the normally cytoplasmic protein is unable to fully traverse the cytoplasmic membrane. We used a genetic approach to study those features of beta-galactosidase responsible for the block in translocation. By using both in vivo and in vitro techniques, fragments of beta-galactosidase were interposed between a signal sequence and alkaline phosphatase. The alkaline phosphatase acts as a sensor for any blocking effects of beta-galactosidase on export. From these studies, we show that multiple regions of beta-galactosidase contribute to its failure to be translocated. These results are most easily interpreted if the folding of beta-galactosidase or of domains of it is responsible for the block in export. In addition, in certain constructs, positively charged amino acids directly following the signal sequence interfered with export.

Alkaline Phosphatase↗

PubMedDate not supplied

Screening for highly active plasmid promoters via fusion to beta-galactosidase gene.

A plasmid containing promoter-deleted inactive beta-galactosidase gene [1] was used to select promoters of the pEP 121 plasmid [2]. Colonies of cells harboring reactivated beta-galactosidase gene were identified by their red color on McConkey plates. The quantitative amounts of beta-galactosidase produced in each clone were estimated by assaying enzyme activity and by measuring the specific beta-galactosidase protein following fractionation of total cells' proteins on polyacrylamide gel. A wide range of enzyme activities was observed. The most active promoter isolated was shown to promote beta-galactosidase production more efficiently, compared with the original beta-galactosidase promoter, amounting to 20% of all cell proteins. Such highly active promoters may be utilized in the future, to promote expression of cloned genes in bacteria.

Cloning, Molecular↗

PubMed1985

Hydrolysis of galactosylceramide is catalyzed by two genetically distinct acid beta-galactosidases.

Two genetically distinct acid beta-galactosidases are apparently involved in the hydrolysis of galactosylceramide in fibroblasts. These beta-galactosidases were activated by different bile salts. The classical galactosylceramidase (galactosylceramidase I, EC 3.2.1.46) was activated by sodium taurocholate, while the other galactosylceramidase (galactosylceramidase II) was activated by sodium cholate. The former was genetically lacking in globoid cell leukodystrophy (GLD) and the latter in GM1 gangliosidosis. Galactosylceramidase II cross-reacted with antibody raised against purified GM1 ganglioside beta-galactosidase (EC 3.2.1.23) from the human placenta. The purified beta-galactosidase had galactosylceramidase II activity, which was competitively inhibited by GM1 ganglioside. Thus, galactosylceramidase II seems to be identical to GM1 ganglioside beta-galactosidase and lactosylceramidase II. Galactosylceramidase II had a very low affinity for galactosylsphingosine. In the galactosylceramide-loading tests using fibroblasts from patients with GLD and GM1 gangliosidosis, both cell lines hydrolyzed the incorporated galactosylceramide, with lower rates than control fibroblasts but higher than the fibroblasts from patients with I-cell disease, in which both galactosylceramidase I and II were deficient. These results indicate that galactosylceramide is hydrolyzed by two genetically distinct beta-galactosidases and explain well that galactosylsphingosine but not galactosylceramide accumulates in the brain of patients with GLD.

Cell Line↗

PubMed1984

B-galactosidase activity in different toxigenic and nontoxigenic strains of Vibrio cholerae: effect of glucose.

B-galactosidase activity in presence and absence of inducer lactose was examined in eight different strains of both toxigenic and nontoxigenic Vibrio cholerae (V. cholerae). In lactose (0.25 gm%) containing cultures of toxigenic strains, B-galactosidase activity was higher in stationary culture than that of logarithmic. In nontoxigenic strains, B-galactosidase activity was only 1/6th of the toxigenic strains and remained unaltered throughout growth period. While wild strains (toxigenic V. cholerae) showed B-galactosidase activity at reduced rate when grown in the medium containing both lactose and glucose, the rate of activity of B-galactosidase in nontoxigenic strains remained unchanged whether the cells were grown in lactose or lactose plus glucose. The respressive effect of higher concentration of glucose could be reversed by the addition of exogenous cyclic AMP. Low level of B-galactosidase activity in nontoxigenic strains was due to low level of intracellular cyclic AMP, compared to toxigenic strain. Furthermore, induction of enzyme activity occurred in nontoxigenic strains of V. cholerae when cyclic AMP was added exogenously.

Cholera Toxin↗