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At least 1,603 records · Page 89Linked to original sources
PubMed1978

Functional properties of beta-galactosidase from mutant strain 13 PO of Escherichia coli.

The functional properties of CZP protein, a mutant deriving from wild-type beta-galactosidase (beta-D-galactoside galactohydrolase; EC 3.2.1.23) by a point mutation, were investigated. A large decrease of the specificity, as evaluated by the kcat/Km ratio, was observed, principally originated by a weaker binding of the substrates. The catalytic constants, whose values are strongly affected by the presence of divalent cations, were smaller or larger for mutant enzyme than for wild-type enzyme, depending upon the experimental conditions. Analysis of the kinetic pathway indicates, with some substrates, a change in the limiting step for the mutant enzyme compared to the wild type. Because the k'3 step is rate limiting for hydrolysis of p-nitrophenyl-beta-D-galactoside by the mutant enzyme in the absence of Mg2+ and its value is relatively small, it is possible to observe a burst of p-nitrophenol during hydrolysis. This provides conclusive evidence for the occurrence of a two-step mechanism, with a sequential release of the products.

Cations, Divalent↗
PubMed1980

L factor that is required for beta-galactosidase synthesis is the nusA gene product involved in transcription termination.

The DNA-dependent in vitro synthesis of Escherichia coli beta-galactosidase requires the presence of a soluble protein referred to as L factor [Kung, H., Spears, C. & Weissbach, H. (1975) J. Biol. Chem. 250, 1556-1562]. In the present study, comparison of physical, immunological, and biological properties shows that L factor is the product of the E. coli nusA gene. The nusA gene product is known to interact with bacteriophage lambda N gene protein and to prevent premature termination of transcription from the early lambda promoters. Our results suggest that premature transcription termination in the lac operon of E. coli may also be overcome by the nusA protein.

Bacterial Proteins↗
PubMed1981

Antibody-mediated activation of genetically defective Escherichia coli beta-galactosidases by monoclonal antibodies produced by somatic cell hybrids.

Six hybridomas producing monoclonal antibodies against Escherichia coli beta-galactosidase (beta-D-galactoside galoctohydrolase, EC 3.2.1.23) have been derived from two separate somatic cell fusions. Three of these antibodies can activate defective enzymes produced by strains of E. coli carrying Z-gene point mutations. In antigen excess, one monoclonal antibody shows similar enzyme binding and mutant-activating capacity. Characteristically, the former reaction has a 200-fold higher equilibrium constant. These data provide direct evidence that the enzyme-activation reaction is a single-hit event in which one antibody site favors the correct conformation of one active center of the enzyme. Because each "activating" hybridoma is able to activate several but not all point mutant enzymes tested, it appears that the correction of the genetic defect is produced by binding key sites of the protein three-dimensional structure rather than the sites affected by the mutation.

Animals↗
PubMed1984

In vivo formation of gene fusions encoding hybrid beta-galactosidase proteins in one step with a transposable Mu-lac transducing phage.

A Mu-lac bacteriophage transposon, MudII301 (Ap, lac), was constructed to form hybrid protein gene fusions. When it integrates into structural genes in the appropriate direction and reading phase, transcription and translation from outside gene controlling regions can proceed across 116 nucleotides from the right end of Mu into lacZ codons to form hybrid proteins that are enzymatically active for beta-galactosidase. Integration can be obtained either by infection to form lysogens or by transposition during growth of a lysogen. The size of the hybrid protein product either corresponds to or, in the cases of translation restart or protein degradation, is a minimal estimate of the distance of the Mu insertion from the translation initiation site of the gene. Hybrid proteins formed by insertions in randomly selected genes and in the araB and A genes were examined by polyacrylamide gel electrophoresis.

Bacteriophage mu↗
PubMed1985

Fabry disease: isolation of a cDNA clone encoding human alpha-galactosidase A.

Fabry disease is an X-linked inborn error of metabolism resulting from the deficient activity of the lysosomal hydrolase, alpha-galactosidase A (alpha-Gal A; alpha-D-galactoside galactohydrolase, EC 3.2.1.22). To investigate the structure, organization, and expression of alpha-Gal A, as well as the nature of mutations in Fabry disease, a clone encoding human alpha-Gal A was isolated from a lambda gt11 human liver cDNA expression library. To facilitate screening, an improved affinity purification procedure was used to obtain sufficient homogeneous enzyme for production of monospecific antibodies and for amino-terminal and peptide microsequencing. On the basis of an amino-terminal sequence of 24 residues, two sets of oligonucleotide mixtures were synthesized corresponding to adjacent, but not overlapping, amino acid sequences. In addition, an oligonucleotide mixture was synthesized based on a sequence derived from an alpha-Gal A internal tryptic peptide isolated by reversed-phase HPLC. Four positive clones were initially identified by antibody screening of 1.4 X 10(7) plaques. Of these, only one clone (designated lambda AG18) demonstrated both antibody binding specificity by competition studies using homogeneous enzyme and specific hybridization to synthetic oligonucleotide mixtures corresponding to amino-terminal and internal amino acid sequences. Nucleotide sequencing of the 5' end of the 1250-base-pair EcoRI insert of clone lambda AG18 revealed an exact correspondence between the predicted and known amino-terminal amino acid sequence. The insert of clone lambda AG18 appears to contain the full-length coding region of the processed, enzymatically active alpha-Gal A, as well as sequences coding for five amino acids of the amino-terminal propeptide, which is posttranslationally cleaved during enzyme maturation.

Amino Acid Sequence↗
PubMed1986

Human alpha-galactosidase A: nucleotide sequence of a cDNA clone encoding the mature enzyme.

The complete nucleotide sequence has been determined for a lambda gt11 cDNA clone (lambda AG18) containing the full-length coding region for the mature lysosomal form of human alpha-galactosidase A (alpha-Gal A; EC 3.2.1.22). The lambda AG18 insert contained a 1226-base-pair sequence with an open reading frame encoding 398 amino acids of the mature polypeptide (predicted Mr = 45,356) and the last 5 amino acids of the propeptide sequence. The poly(A) signals AATACA and ATTAAA occurred 28 and 11 nucleotides prior to the TAA stop codon, respectively. There was no 3' untranslated region as the poly(A) sequence immediately followed the TAA termination codon; a second independently cloned cDNA confirmed this finding. The predicted amino acid sequence was colinear with 86 nonoverlapping residues (22% of the mature subunit) determined by microsequencing amino-terminal, tryptic, and cyanogen bromide peptides of the purified mature enzyme. Four potential N-glycosylation sites were identified, all of which occurred at predicted beta turns in hydrophilic regions of secondary structure. RNA transfer hybridization analysis of HeLa poly(A)+ RNA demonstrated a single 1.45-kilobase band whose signal was decreased by prior immunoabsorption of polysomes with monospecific alpha-Gal A antibodies. Searches of nucleic acid and protein data bases did not reveal significant homology even with the limited sequences available for mammalian lysosomal enzymes.

Amino Acid Sequence↗
PubMed1990

Infection of cultured central nervous system neurons with a defective herpes simplex virus 1 vector results in stable expression of Escherichia coli beta-galactosidase.

We have developed a defective herpes simplex virus (HSV) vector system that permits the introduction of virtually any gene into mammalian central nervous system neurons. The prototype vector, pHSVlac, contains a transcription unit that places the Escherichia coli lacZ gene under the control of the HSV-1 immediate early 4/5 promoter. pHSVlac was propagated using the HSV-1 temperature-sensitive mutant ts K as helper virus. Infection of rat neurons in primary culture derived from various regions throughout the central nervous system, including spinal cord, cerebellum, thalamus, basal ganglia, hippocampus, occipital cortex, temporal cortex, and frontal cortex, resulted in stable expression of high levels of beta-galactosidase for at least 2 weeks, without cell damage. Since other genes can be expressed from pHSVlac, HSV-1 vectors may prove useful for delivery of genes into central nervous system neurons for studies on nervous system physiology or to perform gene therapy for neurological conditions.

Animals↗
PubMed1985

A simple method for the conjugation of affinity-purified Fab' to horseradish peroxidase and beta-D-galactosidase from Escherichia coli.

A simple method was described for the conjugation of affinity-purified Fab' to horseradish peroxidase and beta-D-galactosidase from Escherichia coli. IgG was subjected to successive processes of pepsin digestion, reduction with 2-mercaptoethylamine, affinity-purification and reaction with maleimide groups introduced into the enzymes. In the present method, gel filtration was required only once to separate the conjugate from unconjugated components in the final step, while gel filtration had to be repeated 3-4 times in the previous methods. The conjugate preparations obtained by the present method contained less nonspecific conjugate and gave a lower background by immunoenzymometric assay technique than those obtained by the previous method.

Binding Sites↗
PubMed1978

Suppression of induced beta-galactosidase synthesis by cysteamine and its reversion by gamma-irradiation in the presence of ascorbate.

The induced synthesis of beta-galactosidase in E. coli was found to be inhibited by cysteamine. This inhibitory effect of the SH compound was antagonized by the addition of ascorbate followed by gamma-irradiation with relatively low doses. The cAMP level which, it has been suggested, plays a role in the radioprotective action of cysteamine, is stabilized by ascorbate against changes induced by irradiation.

Ascorbic Acid↗
PubMed1985

Repertoires of T cells directed against a large protein antigen, beta-galactosidase. II. Only certain T helper or T suppressor cells are relevant in particular regulatory interactions.

11 cyanogen bromide (CB) peptides, comprising 70% of the large protein, Escherichia coli beta-galactosidase (GZ), were studied for their ability to induce T suppressor (Ts) cells capable of strongly suppressing the in vitro anti-fluorescein (FITC) response to GZ-FITC. Only CB-2 (amino acid residues 3-92) and CB-3 (residues 93-187) were found to bear such Ts-inducing epitopes. In examining the specificity of T helper cell (Th) targets susceptible to CB-2 and CB-3-specific Ts, it appeared that only nearly Th targets could be suppressed. Thus, CB-10-primed Th were not suppressed by either Ts; even CB-3-primed Ts did not suppress CB-2-specific Th, although CB-2-specific Ts were effective. Furthermore, analysis of the suppression pattern revealed a hierarchical use of potential epitopes on native GZ in triggering functional regulatory T cells. A dominant Th epitope near the amino terminus of GZ tops a hierarchy of potential Th, most of which are never engaged. The dominant determinant seems to exist on the peptide CB-2-3 (residues 3-187), and presumably is destroyed by its cleavage at Met 92; the Th cells that it induces are suppressible by each of the Ts-inducing peptides. In the GZ system, where the native antigen is quite large, the interactions between Th and Ts are highly circumscribed. This may be attributable to the topology of antigen fragments produced during processing; any relevant fragment must bear at least a Ts- and Th-reactive determinant to permit intercellular regulation. A final implication of these results is that, not only does the existence of a Th-inducing determinant depend on its being an appropriate distance from a B cell epitope, but the existence of Ts-inducing determinants likewise depends on the existence of a neighboring Th-B cell association.

Animals↗
PubMed1987

Site-specific recombination directed by single-stranded crossover linkers: specific deletion of the amino-terminal region of the beta-galactosidase gene in pUC plasmids.

The "duplex crossover linker" technique was simplified and used to delete the beta-galactosidase (beta-Gal)-coding sequence upstream from the multiple restriction sites in pUC plasmids. A single-stranded crossover linker, with a homology-searching sequence as short as 5 bases, was initially ligated to a linearized plasmid. Inside Escherichia coli, the plasmid was circularized by intramolecular, homologous recombination between the (5'-or 3'-) protruding homology-searching sequence and a targeted region in the opposite terminus. As a consequence, sequences beyond the point of integration were deleted. Specific deletion of sequences up to 1472 bp was demonstrated. The single-stranded linkers apparently avoided generation of undesirable mutants associated with the usage of duplex linkers. A mechanism has been proposed for the intramolecular recombination directed by the crossover linkers. It principally involves either 3'- or 5'-exonucleolytic breakdown of the homologous terminus of the plasmid, circularization by spontaneous pairing of the exposed complementary strands, and subsequent degradation of any redundant sequence.

Base Sequence↗
PubMed1985

Leukocyte interferon conjugated to beta-D-galactosidase for receptor studies: a preliminary report.

In this report, we describe a simple procedure for labeling leukocyte interferon (IFN) with the enzyme beta-D-galactosidase (beta-gal), through a low-molecular-weight linking agent. The labeling procedure did not appear to alter the properties of the enzyme, the antiviral activity of the recombinant DNA-derived human clone A leukocyte IFN (rIFN alpha A), or the ability of the IFN to bind to Daudi cells. The binding of the putative IFN-enzyme complex to Daudi cells could be blocked by excess unlabeled IFN or by monoclonal antibody to IFN receptors. Our preliminary results indicate that enzyme labeling of IFN can result in a biologically active agent that has potential for use in the quantitation of circulating IFN and specific receptor proteins. In addition, this labeling procedure may be potentially useful with other immunomodulatory proteins.

Antibodies, Monoclonal↗
PubMed1987

Lactose digestion by yogurt beta-galactosidase: influence of pH and microbial cell integrity.

Lactase-deficient subjects more effectively digest lactose in yogurt than lactose in other dairy products, apparently due to yogurt microbial beta-galactosidase (beta-gal) which is active in the GI tract. We evaluated the effects of buffering capacity of yogurt, gastric pH, and microbial cell disruption on beta-gal activity and lactose digestion. Three times more acid was required to acidify yogurt than to acidify milk. Yogurt beta-gal was stable at pH 4.0 but inactivated at lower pH. When yogurt was sonicated to disrupt microbial cell structure, only 20% activity remained after incubation at pH 4.0 for 60 min. In vivo gastric pH remained greater than 2.7 for 3 h after ingestion of yogurt. Acidified milk alone or with disrupted yogurt microorganisms caused twice as much lactose malabsorption as did acidified milk containing intact yogurt microorganisms. The results provide a possible explanation for the survival of beta-gal activity from yogurt in the GI tract.

Adult↗
PubMed1980

Inhibition of DNA-directed beta-galactosidase synthesis in a cell-free system by dimethyl sulfate and N-methyl-N-nitrosourea.

Methylation of a genome DNA lambda h80dlacps by N-methyl-Nitrosourea (MNU, a potent carcinogen) and dimethyl sulfate (DMS, a weak carcinogen) results in loss of its template activity in directing beta-galactosidase (beta G) synthesis in vitro, the degree of inhibition in template activity is proportional to the extent of methylation which is in turn related to the concentration of the methylating agents during 10 minutes incubation at 37 degrees C. When these methylating agents are added to the complete synthesizing system, beta G synthesis is also impaired. Maximum inhibition occurs when the chemicals are added at the initiation of this coupled transcription-translation assay. Inhibition gradually decreases at later times of addition until ten minutes after initiation when no inhibition is observed. This suggests that the early stages in mRNA synthesis are most sensitive to these agents. Preincubation of DNA with MNU for 10 min at 37 degrees C prior to the addition of other assay components (including S30 cell lysate) results in greater inhibition than preincubation of the S30 preparation with MNU prior to DNA and cofactors addition. The opposite result is obtained with DMS suggesting that while DNA is the more sensitive component of the system for MNU, S30 is the more sensitive component for DMS.

Alkylating Agents↗
PubMed1988

Effects of amino acid substitutions at the active site in Escherichia coli beta-galactosidase.

Forty-nine amino acid substitutions were made at four positions in the Escherichia coli enzyme beta-galactosidase; three of the four targeted amino acids are thought to be part of the active site. Many of the substitutions were made by converting the appropriate codon in lacZ to an amber codon, and using one of 12 suppressor strains to introduce the replacement amino acid. Glu-461 and Tyr-503 were replaced, independently, with 13 amino acids. All 26 of the strains containing mutant enzymes are Lac-. Enzyme activity is reduced to less than 10% of wild type by substitutions at Glu-461 and to less than 1% of wild type by substitutions at Tyr-503. Many of the mutant enzymes have less than 0.1% wild-type activity. His-464 and Met-3 were replaced with 11 and 12 amino acids, respectively. Strains containing any one of these mutant proteins are Lac+. The results support previous evidence that Glu-461 and Tyr-503 are essential for catalysis, and suggest that His-464 is not part of the active site. Site-directed mutagenesis was facilitated by construction of an f1 bacteriophage containing the complete lacZ gene on a single EcoRI fragment.

Amino Acids↗
PubMed1987

An attenuated aroA Salmonella typhimurium vaccine elicits humoral and cellular immunity to cloned beta-galactosidase in mice.

beta-Galactosidase (GZ) is an intracellular protein that is frequently used to express cloned antigens as fusion proteins in Escherichia coli. Salmonella typhimurium strain SL3261, an attenuated aroA vaccine strain, was used as a carrier for the plasmid pXY411, which directs the expression of GZ in salmonellae (which do not normally produce this protein). The resulting strain--SL3261(pXY411)--expressed GZ as an intracellular antigen. The plasmid was stable in vitro and in vivo and did not significantly alter the behavior of strain SL3261 in mice. Animals intravenously vaccinated with this construct developed circulating antibodies to GZ, as measured by ELISA, and delayed hypersensitivity to the antigen injected in the footpad. These results indicate that attenuated salmonellae may be expected to elicit both humoral and cellular responses to intracellular cloned antigens.

Animals↗
PubMed1988

The use of beta-galactosidase as a marker gene to define the regulatory sequences of the herpes simplex virus type 1 glycoprotein C gene in recombinant herpesviruses.

The expression of Herpes Simplex Virus 1 (HSV-1) glycoprotein C (gC), a well defined herpesvirus late gene, was studied by linking the promoter-regulatory region of this gene to the coding sequences for the bacterial enzyme, beta-galactosidase (beta-gal). A chimeric gene, containing the beta-gal gene under the control of gC sequences from -1350 to +30 relative to the mRNA start site, was inserted by homologous recombination into the thymidine kinase (TK) locus of the HSV-1 genome. Selection of the TK- recombinant virus by plaque assay was facilitated by addition of a beta-gal indicator to the agarose overlay. Recombinant virus containing the gC promoter-beta-gal chimeric gene faithfully expressed beta-gal as a viral late gene, as shown by the absence of beta-gal expression when viral DNA replication was inhibited with phosphonoacetic acid. In contrast, the inhibition of viral DNA replication had no effect on the expression of beta-gal when the beta-gal gene was under the control of the early HSV-1 TK promoter in a separate recombinant virus. Analysis of recombinant viruses containing 5' to 3' deletions in the gC regulatory region revealed no apparent difference in beta-gal expression as deletions extended from -1350 to -109 base-pairs (bp) before the RNA start site, demonstrating that sequences between -109 and +30 are sufficient for regulated gC expression in the viral genome. Analysis of the mRNA made by these recombinant viruses confirmed the results of the beta-gal assays, and demonstrated that the transcriptional start sites of the gC promoter-beta-gal chimeric genes were the same as the start site of the gC gene.

Animals↗
PubMed1980

pH-dependent association-dissociation of GM1-beta-galactosidase purified from porcine spleen.

A beta-galactosidase [EC 3.1.23] catalyzing the hydrolysis of GM1-ganglioside was purified from porcine spleen to a homogeneous form. By applying a hydrophobic chromatography procedure as the first purification step, the enzyme could be purified through subsequent purification steps as a dissociated form. The purified enzyme was a monomer with an apparent molecular weight of 70,000-74,000 at neutral pH and associated to a dimer with an apparent molecular weight of 158,000-160,000 at acidic pH, near optimal for its activity (pH 4.6). It had specific activities of 1,820 mumol/mg/h towards GM1 with an apparent Km of 3.18 X 10(-5) M, 1,880 mumol/mg/h towards lactosylceramide with an apparent Km of 1.99 X 10(-4) M, and 1,340 mumol/mg/h towards p-nitrophenyl-beta-galactopyranoside (pNp-beta galactoside) with an apparent Km of 2,14 X 10(-4) M. Kinetic studies suggested that common catalytic site(s) cleaved the two natural substrates mentioned above.

Animals↗