Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Monoclonal antibodies were raised against two soluble, galactose-binding lectins from cells of Dictyostelium discoideum, discoidin I and II. These antibodies reacted not only with both discoidins, but also with a plasma membrane glycoprotein of aggregation competent cells, called contact site A, and with two carbohydrate-binding proteins of E. coli, beta-galactosidase and lac repressor. The possibility that the antibody recognizes a structure common to different carbohydrate-binding proteins is discussed. The two carbohydrate-binding proteins of E. coli share with discoidin I the sequence -Ser-X-X-Ile-His(Pro)-Pro(His)-Leu-Thr- which might be responsible for the cross-reactivity.
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PMID:Monoclonal antibody against cytoplasmic lectins of Dictyostelium discoideum: cross-reactivity with a membrane glycoprotein, contact site A, and with E. coli beta-galactosidase and lac repressor. 620 16

The bacteriophage Mu d1(Apr lac cts62 ) obtained from an Escherichia coli double lysogen carrying the defective Mu d1 phage and a Mu-P1 hybrid phage was utilized as a vector for phage mutagenesis in Erwinia carotovora subsp. carotovora. Among ampicillin-resistant transductants. 1.4% were auxotrophs. The synthesis of beta-galactosidase was derepressed upon starvation for histidine in two different his-lac fusion strains.
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PMID:Mutagenesis of Erwinia carotovora subsp. carotovora with bacteriophage Mu d1 (Apr lac cts62): construction of his-lac gene fusions. 623 63

Enzymatic activity was investigated in metal-binding proteins from rat epidermal cells. Tris-HCl buffer soluble and KSCN solubilized proteins were extracted stepwise from granular and cornified cells of 2-day old rat epidermis. Each extract was separately applied to a Cu2+ or Zn2+ chelate Sepharose 6B column and the proteins were eluted with buffers of different pHs and finally with EDTA solution. Metal chelate-binding proteins were found in both soluble and solubilized proteins but there was a larger amount in the latter. Affinity of the proteins to bind with Cu2+ chelate was greater than that with Zn2+ chelate. In Tris-HCl buffer extract, histidase activity was detected in Cu2+ chelate-binding proteins, but not in Zn2+ chelate-binding proteins. Acid phosphatase, cysteine proteinase, dipeptidase, cathepsin D, beta-galactosidase, gelatin hydrolase, and superoxide dismutase did not bind to metal chelates although these enzymes, except acid phosphatase, were inhibited by Cu2+, but not by Zn2+. In contrast, KSCN solubilized metal chelate-binding proteins showed plasminogen activator, acid phosphatase, and gelatin and casein hydrolases while histone hydrolase did not bind to either chelate column. Since metal-binding proteins in rat epidermal cells have been shown previously to be histidine- and cysteine-rich proteins concentrated in keratohyalin granules, interaction of metals and the structural proteins with certain enzymes may be involved in the regulation of epidermal cell functions.
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PMID:Enzymatic activity of metal-binding proteins in epidermal cells. 653 44

The characteristics of the sialidase (N-acetyl-alpha-neuraminidase) of human leukocytes, fibroblasts and amniotic fluid cell cultures were determined with a radioactive assay method utilizing neuramin-[3H]lactitol as the enzyme substrate. Fibroblast cultures from patients with the inherited sialidase deficiency diseases including mucolipidosis I, sialidosis I and sialidosis II, juvenile type have less than 10% of normal sialidase activity using either this substrate, 2-(3'-methoxyphenyl)-N-acetyl-alpha-neuraminic acid, or 2'-(4-methylumbelliferyl)-N-acetyl-alpha-neuraminic acid. The total sialic acid content of fibroblasts and leukocytes from mucolipidosis I and sialidosis I patients is greatly elevated; this parameter is useful in establishing a diagnosis of sialidase deficiency. The sialic acid content of sialidosis II, juvenile type, with coexistent sialidase and beta-galactosidase deficiencies, is only slightly elevated above normal levels. A patient with mucolipidosis I has 16% of normal neuramin-[3H]lactitol sialidase activity in his peripheral leukocytes. His parents were clearly distinguished from the normal range using leukocyte enzyme levels and a maternal aunt was identified as a possible carrier. The presence of this enzyme in amniotic fluid cell cultures, both fibroblastic and mixed cell type, makes possible the prenatal detection of these diseases. A pregnancy from a family at risk for having a child with mucolipidosis I was monitored by amniocentesis and subsequent sialidase measurement of the amniotic fluid cell cultures.
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PMID:Mucolipidosis I: studies of sialidase activity and a prenatal diagnosis. 722 21

Plasmids containing the ebgAo and ebgAa genes of Escherichia coli under the control of the lac repressor and promoter have been constructed and inserted into Salmonella typhimurium CH3. This system expresses the large subunit of the ebgo and ebga beta-galactosidase in high yield (20-60% of total protein). The large subunits have been purified to homogeneity. As isolated they are tetramers of significant catalytic activity; the N-terminal amino acid residue is Met, but it is not formylated. The kcat. values for a series of aryl galactosides were 6-200-fold reduced from the corresponding values for the holoenzymes. kcat/Km Values for glycosides of acidic aglycones, though, were unchanged, whilst kcat./Km values for galactosides of less acidic aglycones showed a modest (up to 10-fold) decrease. The kcat. values for glycosides of acidic aglycones hydrolysed by ebgo and ebga large subunits were essentially invariant with aglycone pK, suggesting that hydrolysis of the galactosyl-enzyme intermediate had become rate-determining for these substrates. Rate-determining hydrolysis of the glycosyl-enzyme intermediate was confirmed by pre-steady-state measurements and nucleophilic competition with methanol. Absence of the small subunit was thus estimated to cause a 200-fold decrease in degalactosylation rate for ebgo and a 20-fold one for ebga. beta 1g(V/K) values of -0.57 +/- 0.08 for ebgo and -0.54 +/- 0.08 for ebga isolated subunits were significantly more negative than for holoenzymes. It is suggested that the small subunit is associated with the optimal positioning of the electrophilic Mg2+ ions in these enzymes. Use of PCR in the construction of the plasmid also inadvertently led to the production of psi ebgo large subunit in which there was a PCR-introduced Leu9-->His change. Values of kcat. for aryl galactosides, calculated on the assumption that the psi ebgo large subunit, like the ebgo and ebga large subunits, was 100% active as isolated, were about an order of magnitude lower than for true ebgo large subunit, whilst Km values were similar. The very significant kinetic effect of this inadvertant site-undirected mutagenesis indicates that quite large kinetic effects of amino-acid replacements in enzymes may have no obvious mechanistic significance.
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PMID:Catalysis by the large subunit of the second beta-galactosidase of Escherichia coli in the absence of the small subunit. 749 25

beta-galactosidase (Escherichia coli) with a His substituted for Glu-461 retained about 10% of its normal activity in the absence of divalent metals but was inactivated rather than activated by Mg2+, Mn2+, Zn2+, Ni2+, Cu2+, and Co2+. Since Zn2+, Ni2+, Cu2+, and Co2+ do not interact with wild type beta-galactosidase while Mg2+ and Mn2+ activate and Ca2+ binds but has no effect on wild type beta-galactosidase activity, the substituted enzyme has very different divalent metal interactions. A much larger amount of Mg2+ than of the other divalent metal ions was needed to inactivate the substituted enzyme at pH 7 (half-maximal activity was at 12.5 mM Mg2+ while the half-maximal activities with the other metals were at micromolar levels) compared to the amount of Mg2+ needed to activate the wild type enzyme. The inactivation of E461H-beta-galactosidase caused by Mg2+ took about 20 min. Reactivation by removal of the divalent metal took about 60 min. Interaction with Mg2+ was about 10(7)-fold stronger at pH 9 than at pH 7, and inactivation occurred in less than 2 min at higher pH values. "Galactosylation" (k2, cleavage of the glycosidic bond) seemed to be rate-limiting for E461H-beta-galactosidase at pH values above 6 with both o-nitrophenyl beta-D-galactopyranoside and p-nitrophenyl beta-D-galactopyranoside in both the presence and absence of Mg2+. Mg2+ caused decreases (about 50-fold) of the k2 values of E461H-beta-galactosidase (apparent pKa was about 6.8).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:E461H-beta-galactosidase (Escherichia coli): altered divalent metal specificity and slow but reversible metal inactivation. 757 31

Morquio B disease was found in a 15-year-old Japanese boy who presented with progressive generalized skeletal dysplasia without neurological manifestations. Mild keratan sulfaturia was found, and beta-galactosidase was deficient in fibroblasts. Gene analysis revealed two mutant alleles, 83Tyr-->His (Y83H) and 482Arg-->Cys (R482C). The former expressed a low enzyme activity (2-5% of normal), and the latter expressed no detectable enzyme activity.
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PMID:Clinical and molecular analysis of a Japanese boy with Morquio B disease. 758 49

An E. coli expression clone coding for human proinsulin, which was fused to NH2-terminal beta-galactosidase, was engineered for the separation from host proteins by introducing peptide devices, and for the sequential removal of the fused polypeptide by cyanogen bromide in front of the NH2 terminal residue (methionine) of the human proinsulin gene. Short synthetic genes encoding oligopeptide residues including (Glu)n, (His)n, (Trp)n, and (Ser)n (n = 10 or 11), which have certain characteristic physical properties such as metal-affinity, polarity, hydrophobicity, and hydrophilicity, respectively, were inserted at the junction region of the gene fusion. Interestingly, it was found that among the oligopeptides, the oligohistidine residue as an affinity-tag has greatly facilitated the procedures for FPI purification, particularly in the manner of selective metal-affinity precipitation. The chelating peptide covering the NH2-terminal beta-galactosidase portion could then be removed simply after purification to generate a protein with the natural amino acid sequence of proinsulin by cyanogen bromide.
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PMID:Metal affinity engineering of proinsulin carrying genetically attached (His)10-X-Met affinity tail and removal of the tag by cyanogen bromide. 776 85

The RNA polymerase gene of human coronavirus (HCV) 229E encodes a large polyprotein that contains domains with motifs characteristic of both papain-like cysteine proteinases and proteinases with homology to the 3C proteinase of picornaviruses. In this study, we have, first, expressed the putative HCV 229E 3C-like proteinase domain as part of a beta-galactosidase fusion protein in Escherichia coli and have shown that the expressed protein has proteolytic activity. The substitution of one amino acid within the predicted proteinase domain (His-3006-->Asp-3006) abolishes, or at least significantly reduces, this activity. Amino-terminal sequence analysis of a purified, 34-kDa cleavage product shows that the bacterial fusion protein is cleaved at the dipeptide Gln-2965-Ala-2966, which is the predicted amino-terminal end of the putative 3C-like proteinase domain. Second, we have confirmed the proteolytic activity of a bacterially expressed polypeptide with the amino acid sequence of the predicted HCV 229E 3C-like proteinase by trans cleavage of an in vitro translated polypeptide encoded within open reading frame 1b of the RNA polymerase gene. Finally, using fusion protein-specific antiserum, we have identified a 34-kDa, 3C-like proteinase polypeptide in HCV 229E-infected MRC-5 cells. This polypeptide can be detected as early as 3 to 5 h postinfection but is present in the infected cell in very low amounts. These data contribute to the characterization of the 3C-like proteinase activity of HCV 229E.
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PMID:Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity. 776 94

Site directed mutagenesis was used to replace His-418 of beta-galactosidase with Phe (H418F) or Glu (H418E). Kinetic analysis revealed that H418F beta-galactosidase was not significantly affected by the presence of Mg2+ whereas H418E beta-galactosidase retained its sensitivity to Mg2+. H418F had a kcat similar to that of Mg(2+)-free wild type beta-galactosidase. Its pH profile was shifted 1.0 pH unit lower on the alkaline side as compared to wild type beta-galactosidase (with Mg2+). This was similar to the shifting of the wild type beta-galactosidase pH profile when Mg2+ was absent. H418E beta-galactosidase was inactivated (rather than activated) by Mg2+ binding. Equilibrium dialysis studies indicated that H418E and wild type beta-galactosidase bind Mg2+ tightly whereas H418F does not. The results indicate that His-418 is probably a ligand to Mg2+.
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PMID:Site directed substitutions suggest that His-418 of beta-galactosidase (E. coli) is a ligand to Mg2+. 800 24


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