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)

A method is described that allows the expression of a stable human proinsulin product in Escherichia coli as encoded by either a fused or an unfused gene construction. In the fused system, the human proinsulin coding sequence is joined to the 3' side of a fragment containing the lac promoter and the coding sequence for a small part of the NH2 terminus of beta-galactosidase. In the unfused system, the proinsulin coding sequence is linked directly to a fragment containing the Tac promoter followed by a bacterial Shine-Dalgarno sequence. In both systems, the human proinsulin product is too unstable to be detected by NaDodSO4/polyacrylamide gel electrophoresis or even pulse-chase analysis. However, when multiple copies of the proinsulin coding sequence are tandemly linked such that the resultant protein product contains multiple copies of the proinsulin domain, the stability of the product is markedly increased in both the fused and the unfused expression systems. In the unfused system, three tandemly linked proinsulin polypeptide domains are required for stabilization, whereas two proinsulin domains plus the bacterial leader protein enhance stability in the fused system. The polypeptide product of a multiple copy proinsulin gene can be cleaved into single proinsulin units by cyanogen bromide treatment.
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PMID:Multiple joined genes prevent product degradation in Escherichia coli. 637 48

A cloned synthetic DNA fragment coding for 27-desamidosecretin (DAS), which differs from natural porcine secretin by the lack of the amide group at the carboxyl-terminal valine residue, was fused to the beta-galactosidase gene (lacZ) at the EcoRI site near the 3'-terminus of the gene. The fusion gene was efficiently expressed in Escherichia coli, yielding 1.15 X 10(6) fused-protein molecules per cell. After the treatment of the fused protein with cyanogen bromide, DAS was purified by a combination of ion-exchange column chromatography and reverse-phase high-performance liquid chromatography (HPLC). The structure of bacterial DAS was confirmed by tryptic mapping and amino acid composition analyses. The bacterial DAS was not amidated at its carboxyl-terminal valine residue. Nevertheless, it stimulated pancreatic secretion in rats as does authentic porcine secretin, indicating that the carboxyl-terminal amide is not essential to secretin activity.
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PMID:Overproduction of a gastrointestinal hormone, secretin, in Escherichia coli cells and its chemical characterization. 638 4

beta-Galactosidase from T. cornutus was resolved into two activity peaks by gel filtration column chromatography. The pH optima of the two peaks designated P1 and P2, were 5.5 and 3.0, respectively, when p-nitrophenyl-beta-D-galactopyranoside was used as the substrate. The molecular weights of P1 and P2 were 700,000 +/- 70,000 and 78,000 +/- 7800, respectively, when estimated by gel filtration chromatography. The activities of both forms of the enzymes are stimulated by anions such as Cl-, Br- and NO-3. While the activity of P1 was stimulated by low anion concentrations, P2 requires 700 times higher anion concentration for similar enhancement of activity. P1, the high molecular weight form hydrolyzes mainly galactose from small molecular weight beta-galactosides, such as p-nitrophenyl-beta-D-galactopyranoside, 4-methylumbelliferyl-beta-D-galactopyranoside, lactose, lactosylceramide and 3-O-beta-D-galactopyranosyl-D-arabinose, whereas P2, the low molecular weight form cleaves, in addition, all the beta-galactosides tested, including 2-hexadecanoylamino-4-nitrophenyl-beta-D-galactopyranoside, GM1-ganglioside, asialo-GM1-ganglioside, asialo fetuin, alpha 1-acid glycoproteins and the tryptic peptides of the glycoproteins. The optimal conditions for the hydrolysis of the terminal galactose from GM1-ganglioside which does not occur in gastropods, such as T. cornutus, was found to require 40 mM NaCl and 1 mM sodium taurodeoxycholate at pH 3.0 in 50 mM sodium citrate buffer, conditions similar to those by mammalian beta-galactosidase.
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PMID:A beta-galactosidase isoenzyme from Turbo cornutus with substrate specificity toward GM1-ganglioside and glycoproteins. 641 42

Structural studies are reported on seven hybrid proteins produced by gene fusions that contain a "foreign" amino acid sequence substituting for part of the NH2-terminal region of the beta-galactosidase polypeptide. All of these hybrid proteins retain beta-galactosidase enzyme activity. A simple and rapid purification scheme for the hybrid beta-galactosidase is described, involving ammonium sulfate fractionation, DEAE-Bio-Gel, and Bio-Gel A-1.5 chromatography. The proteins are tetramers and have activity equivalent to that of wild type enzyme. Their amino acid sequences were determined by isolation and sequence determination of the cyanogen bromide peptide containing the joining site. The subunit sizes vary from 1009 to 1355 residues compared to 1023 for wild type. Up to 26 amino acid residues at the NH2 terminus of beta-galactosidase can be substituted by the new sequence. The nature of the new sequence apparently has no influence on stability or activity of the hybrid, but those hybrids with more of the beta-galactosidase sequence deleted are less stable to heat or urea treatment and tend to dissociate to dimeric form. All hybrids are less stable to heat and urea than wild type. Antipeptide antibodies raised against peptides derived from the NH2-terminal region of wild type beta-galactosidase were found to bind to the hybrid proteins, although they do not bind to the normal enzyme. These results indicate that the quaternary structure is disturbed but not disrupted by substitution of the different sequence, and these results help to localize one of the intersubunit contact regions in beta-galactosidase.
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PMID:Purification, structure, and properties of hybrid beta-galactosidase proteins. 641 39

[3H] Conduritol C cis-epoxide (1,2-anhydro-epi-inositol, I) was synthesized as an active-site-directed inhibitor for lacZ beta-galactosidase from Escherichia coli. A considerable kinetic isotope effect was noted in the reduction by [3H]NaBH4 of the p-benzoquinone-derived precursor for I. Complete loss of beta-galactosidase activity occurred on incorporation of 4 mol I/mol beta-galactosidase tetramer. The inhibitor was very labile in the denatured enzyme at pH greater than 8, implying the formation of an ester bond between I and a carboxylate at the active site. The radioactive material released from the labeled enzyme was identified as allo-inositol. The stereochemistry of the expoxide reaction (trans-diaxial ring opening) is thus the same as for beta-glucosidases with the corresponding epoxides. The binding site for I was identified as Glu-461 by the isolation and partial sequence analysis of a radioactive octapeptide from the cyanogen bromide and pepsin fragments of the labeled enzyme. A failure to determine the N-terminal amino acid of the labeled peptide is ascribed to the great reactivity of the esterified gamma-carboxyl group of its N-terminal Glu-461 which causes rapid cyclisation of this residue to pyroglutamate, even under weakly basic conditions. The participation of the carboxylate of Glu-461 in catalysis is discussed.
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PMID:Identification of an essential carboxylate group at the active site of lacZ beta-galactosidase from Escherichia coli. 642 Jan 54

The cyanogen bromide fragment CB67-129 of human prethrombin 1, corresponding to residues 54-116 of the thrombin B chain, is a potent chemotaxin for human peripheral blood monocytes and the murine macrophage like cell line, J774. Both of these cell types have been shown to respond chemotactically to alpha-thrombin and iPr2P-alpha-thrombin. Effective concentrations for stimulating directed cell movement with the fragment vary from 10(-11) to 10(-7) M. Moreover, CB67-129 and its parent protein compete for the same chemotactic receptor site. Fragment CB67-129, representing residues 54-116 of the human thrombin B chain sequence, contains a nine-residue insertion ("loop B") that is absent in homologous sequences derived from the closely related proteases chymotrypsin and trypsin. Unlike iPr2P-alpha-thrombin, iPr2P derivatives of these latter enzymes possess little or no chemotactic activity, suggesting a relationship between the insertion sequence and thrombin chemotactic activity. The loop B sequence is unique insofar as it contains all of the carbohydrate moieties known to reside in alpha-thrombin. However, chemotactic activity is only minimally reduced subsequent to hydrolysis by both neuraminidase and beta-galactosidase, indicating that receptor recognition and stimulated cell movement are mainly a function of structure of the cyanogen bromide derived fragment rather than of asparagine-linked carbohydrates.
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PMID:Localization of a chemotactic domain in human thrombin. 670 77

The complex between lactase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) and phlorizin hydrolase (glycosyl-N-acylsphingosine glycohydrolase, EC 3.2.1.62) has been purified from the proximal and distal regions of the small intestine of suckling rats. The two enzymes behaved differently on DEAE-cellulose ion-exchange chromatography and during electrophoresis in the presence and absence of sodium dodecyl sulphate (SDS), but they have very similar cyanoge bromide cleavage patterns. Kinetic studies on the proximal and distal enzymes showed the same pH optimum of 6.0 and the same heat stability at 45 degrees C, but a small difference in Km. Treatment of both enzymes with fucosidase, mannosidase or N-acetylhexosaminidase did not affect enzymic activity or electrophoretic mobility. Neuraminidase digestion abolished the electrophoretic differences and gave two active enzymes with similar isoelectric points.
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PMID:Isolation and characterization of the proximal and distal forms of lactase-phlorizin hydrolase from the small intestine of the suckling rat. 677 1

Studies on beta-galactosidase alpha-complementation are reviewed. The isolation and structure of two beta-galactosidase fragments that form an enzymically active complex are described. One of these is a cyanogen bromide peptide from whole beta-galactosidase; the other is a dimeric protein from a lacZ deletion mutant of Escherichia coli. The mechanism most likely involves an initial binding of two cyanogen bromide peptides to the dimer, followed by formation of a tetramer, and finally a slow conformational change of the complex to a native-like enzyme. The overall reaction is essentially irreversible. A region of the polypeptide chain involved in dimer-dimer contact must be supplied by the cyanogen bromide peptide. alpha-Complemented enzyme contains overlapping sequences. Proteolytic experiments were carried out to determine the origin of the functionally important segment. The effect on alpha-complementation of amino acid substitutions at four positions in the polypeptide chain was investigated. The implications of these results for beta-galactosidase structure and for proteins in general are discussed.
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PMID:beta-Galactosidase alpha-complementation. A model of protein-protein interaction. 681 52

A gene has been constructed which codes for an analog of human proinsulin in which the normal 35-amino acid connecting peptide is replaced by a "mini-C" peptide of six amino acids (Arg-Arg-Gly-Ser-Lys-Arg). The gene, composed of oligonucleotide fragments synthesized by the triester method, was cloned and expressed as a beta-galactosidase hybrid protein. The proinsulin analog was separated from beta-galactosidase by cyanogen bromide cleavage and purified. Controlled disulfide exchange in the S-sulfonate of the analog generated a molecule having high-pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) behavior consistent with a proinsulin-like structure.
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PMID:Expression in Escherichia coli of a chemically synthesized gene for a "mini-C" analog of human proinsulin. 704 95

The feasibility of using permeabilized whole cells as a source of intracellular enzymes instead of isolated expensive enzymes for the estimation of biomolecules has been studied. Alcohol dehydrogenase (ADH), glucose-6-phosphate dehydrogenase (G6PDH), hexokinase (HK), and beta-galactosidase (beta-GAL) activities of cetyltrimethylammonium bromide (CTAB)-permeabilized whole yeast cells were employed to estimate ethyl alcohol, glucose, and lactose. The method using permeabilized cells was comparable to that of isolated enzymes and was applicable for the estimation of these analytes in complex samples such as blood, milk, and fermented samples. The usefulness of permeabilized cells as a single source of more than one enzyme required for coupled enzyme assays was demonstrated.
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PMID:Detergent permeabilized yeast cells as the source of intracellular enzymes for estimation of biomolecules. 776 9


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