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)

We have introduced the firefly luciferase gene of Photinus pyralis into the vaccinia virus genome. This gene is expressed in a coordinate fashion during virus infection. Luminescence produced by the action of luciferase [Photinus-luciferin:oxygen 4-oxidoreductase(decarboxylating, ATP-hydrolyzing), EC 1.13.12.7] was easily detectable in infected cells in culture as well as in cells of tissues of infected mice. The limits of detection were about one infected cell in a background of a million noninfected cells. The luciferase assay was about 1000-fold more sensitive than that of beta-galactosidase. Our findings show that the luciferase assay can be conveniently used to follow viral gene expression and virus dissemination both in cell cultures and in tissues of infected animals.
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PMID:Expression of the firefly luciferase gene in vaccinia virus: a highly sensitive gene marker to follow virus dissemination in tissues of infected animals. 342 54

Cell-free fusion between endocytic vesicles has been obtained in a sensitive assay based on the avidin-biotin binding reaction. Chinese hamster ovary cells are allowed to endocytose either avidin-linked beta-galactosidase or biotinylated IgG. Postnuclear supernatant extracts prepared from these cells are incubated at 37 degrees C in the presence of an ATP-regenerating system. Fusion between vesicles from the two extracts permits the avidin-biotin association to occur, so that the amount of avidin-enzyme-biotinylated IgG complex produced is proportional to the amount of fusion between vesicles. The amount of complex formed is measured, after detergent lysis of the vesicles, by an ELISA technique, using a fluorogenic substrate for beta-galactosidase. The fusion process requires ATP hydrolysis and specific cytosolic proteins. The vesicles that fuse appear to be endocytic vesicles populated by the endocytosed proteins within 5 min of their internalization at 37 degrees C. Ionophores and weak bases do not inhibit fusion, suggesting that a pH gradient across the vesicle membrane is not crucial for the fusion process.
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PMID:Fusion between endocytic vesicles in a cell-free system. 346 57

Plasmid genes redirect some components of cellular metabolism into synthesis of plasmid gene products and additional plasmids. The stoichiometric and kinetic implications of these host-plasmid interactions have been investigated theoretically and experimentally. Using known pathway energetics, maximum theoretical yield factors based on ATP, glucose, and O2 have been estimated for recombinant Escherichia coli and compared with corresponding estimates for host cells alone, indicating major changes in carbon and energetic stoichiometry in recombinant cells in cases of high cloned gene expression. The influence of the number of plasmids in recombinant E. coli has been experimentally characterized using a series of pMB1 derivatives stably propagated at copy numbers from 12 to 408. Recombinant cell growth rate declines monotonically as plasmid content increases as does efficiency of plasmid gene expression. A detailed metabolically structured single-cell model for E. coli has successfully simulated these trends. Interrelationships among number of plasmids per cell, induction of expression of a plasmid gene, and recombinant population growth rate have been experimentally delineated for Saccharomyces cerevisiae containing plasmid pLGSD5 and derivatives in which the 2-micron origin of replication has been replaced by a cloned ARS1 sequence or its deletion fragments. The CEN4 centromere sequence has been included in some of these plasmids to provide more regular segregation. Specific growth rate of these recombinant yeasts exhibits a maximum as a function of plasmid content, an effect attributed to the interplay between beneficial effects of the plasmid in selective medium and parasitic effects on metabolism at larger plasmid content or with more plasmid gene expression activity. The yeast strains investigated exhibit substantial segregational instability that was characterized using a rapid-flow cytometry measurement based upon single-cell deletion of E. coli beta-galactosidase activity in recombinant cells.
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PMID:Studies of host-plasmid interactions in recombinant microorganisms. 352 97

The lon gene product in Escherichia coli is an ATP-dependent protease (La) that plays an important role in the breakdown of abnormal proteins and certain normal polypeptides. Since transcription of the lon gene rises as part of the heat-shock response, we studied the physiological effects of increased levels of protease La. In cells carrying additional copies of the lon gene under the control of the lac or tac promoter, induction of the protease resulted in a rapid cessation of cell growth and in a loss of viability at stationary phase. Similarly, cells carrying a multicopy plasmid encoding the lon gene contained 2-5-fold more protease La and grew much more slowly than did control cells. In such cells, insertion sequences appeared spontaneously in the lon gene on the plasmid and prevented the excess protease production and allowed more rapid growth. The cells with increased content of protease La (due to the lon plasmid or induction of the lon gene) exhibited severalfold higher rates of degradation of abnormal proteins containing amino acid analogs and of incomplete polypeptides containing puromycin. Also, a beta-galactosidase fusion protein with enzymatic activity was relatively stable in control cells but unstable in the cells with high protease La content. In these cells, the overall degradation of normal proteins increased 2-fold, and certain cellular polypeptides appeared particularly sensitive to proteolysis. Thus, rates of protein degradation in vivo are limited in part by the cellular content of the ATP-dependent protease, and increases in transcription of the lon gene enhance proteolysis and can be deleterious to the cell.
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PMID:An increased content of protease La, the lon gene product, increases protein degradation and blocks growth in Escherichia coli. 354 9

Activation of proteolysis by ATP was studied in lysates of crude and purified lysosomal preparations from liver and kidney at acid pH. In the crude system, from kidney, it was found that ATP activates proteolysis over a concentration range of 0.1-2 mM. Up to 4-fold activation was observed. GTP and CTP also activated proteolysis, but to a lesser extent. Proteolysis was inhibited by vanadate and molybdate. Fractionation of the kidney lysosomes on Percoll gradients produced two fractions containing lysosomal marker enzymes. Most of the acid phosphatase and the acid pyrophosphatase were found in the lighter band, while most of the beta-galactosidase and cathepsin activity was found in a more dense band. Proteolysis by lysates of both fractions was activated by ATP and inhibited by vanadate and molybdate. In the dense band proteolysis was also nearly totally blocked by pepstatin, and was enhanced by an inhibitor of pyrophosphatases, sodium fluoride. ATP also activates proteolysis in crude lysosomes from liver, but upon fractionation of this tissue it was found that all the lysosomal enzyme markers are present in the dense fraction obtained from the Percoll gradient. Again, proteolysis by lysates of the purified fractions was activated by ATP and inhibited by vanadate and molybdate. These data indicate that ATP can activate proteolysis at acid pH in a lysosomal milieu containing enzymes which also catalyze its breakdown. In the kidney there may be two lysosomal compartments which separate the enzymes catalyzing ATP breakdown from the proteolytic enzymes, but this is not essential for ATP activation as shown by the data from the liver and the crude lysosomal fractions.
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PMID:ATP activation of protein degradation by extracts of crude and purified lysosomal preparations. 385 74

1. The intermediary metabolism of two strains of Escherichia coli has been examined. One strain (Q22) exhibits acute transient repression of beta-galactosidase synthesis when glucose is supplied to cells growing on glycerol; the other strain (W3110) does not. The two strains do not differ genetically in their lac operons. 2. Strain Q22 uses about twice as much glucose as strain W3110 per unit of cell mass produced. 3. Pentose phosphate-cycle activity in the presence of glucose is much stronger in strain Q22 than in strain W3110. 4. In strain Q22 the pool sizes of glucose 6-phosphate, 6-phosphogluconate, fructose 1,6-diphosphate and NADPH increase when glucose is added to cells growing on glycerol, and beta-galactosidase synthesis is severely inhibited. After about 1hr. the synthesis of beta-galactosidase is partly resumed, and the pool sizes of the four compounds fall. ATP, NADH and several other phosphorylated compounds show no concentration changes. 5. These concentration changes do not occur in strain W3110, in which beta-galactosidase synthesis is only rather weakly repressed by glucose. 6. It is suggested that repression of enzyme synthesis by glucose requires the rapid operation of the pentose phosphate cycle, and is mediated by one of the four substances whose concentration rises and later falls in strain Q22. A definite choice of effector from among these four possibilities cannot at present be made.
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PMID:Pool sizes of metabolic intermediates and their relation to glucose repression of beta-galactosidase synthesis in Escherichia coli. 438 55

Yersinia pestis strain KIM requires plasmid pCD1 for expression of the low calcium response, plague virulence antigen V, and virulence. We constructed Mu d1(Ap lac) insertion mutants of this plasmid which were unable to express the low calcium response. The insertions mapped to a 17-kilobase region of the plasmid. By determining the orientation of the insertions and examining beta-galactosidase production from the Mu d1 lac genes, we determined that this region contains three units of transcription, one of which is transcribed in a direction opposite the direction of transcription of the other two. Transcription of at least two of these units was induced significantly at 37 degrees C compared with 26 degrees C. Ca2+ (2.5 mM) and ATP (18 mM) had no significant effect on the level of expression of the Mu d1 lac genes of these mutants. All insertions in the region strongly reduced production of the V antigen. Insertions from each unit of transcription also reduced virulence in mice.
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PMID:Genetic analysis of the low calcium response in Yersinia pestis mu d1(Ap lac) insertion mutants. 609 9

The H+/ATP stoichiometry of the H+-ATPase was investigated in Escherichia coli cells growing under anaerobic conditions at pH 6 and 7. The protonmotive force was determined from the intracellular accumulation of benzoate and tetraphenylphosphonium ions, as well as the accumulation of lactose in this lac operon inducible, but beta-galactosidase negative strain. The phosphorylation potential was calculated from the cellular concentrations of ATP, ADP and inorganic phosphate. By comparing the phosphorylation potential and the proton motive force under these steady state conditions, the H+/ATP stoichiometry was determined to be 3, similar to the value previously found in the same cells growing under aerobic conditions.
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PMID:Stoichiometry of the H+-ATPase of Escherichia coli cells during anaerobic growth. 621 95

The H+/ATP stoichiometry of the proton-translocating ATPase was investigated in growing and nongrowing, respiring cells of Escherichia coli. The protonmotive force, delta p, was determined by measuring the transmembrane chemical gradient of protons, delta pH, from the cellular accumulation of benzoate anions, and the electrical gradient, delta psi, from the accumulation of the lipophilic cation tetraphenylphosphonium (TPP+). The accumulation of lactose was also used to calculate the delta p in this lactose operon constitutive beta-galactosidase negative mutant. The phosphorylation potential, delta GP', was determined by measuring the cellular concentration of ATP, ADP, and inorganic phosphate. According to chemiosmotic principles, at steady state the phosphorylation potential is in thermodynamic equilibrium with the protonmotive force, and thus the ratio delta p/delta GP' can be used to determine the H+/ATP ratio. Respiring E. coli cells, in mid-exponential phase of growth or incubated in buffer, at external pHs from 6.25 to 8.25 had a constant delta GP' of about 500 mV. The H+/ATP ratio was found to be 3 when the delta p value derived from lactose accumulation levels was used. However, when the delta p values derived from delta pH and delta psi were used in the calculations, the H+/ATP ratio varied from about 2.5 at external pH 6.25 to about 4 at pH 8.25. Arguments are presented for the hypothesis that the delta psi values obtained from the TPP+ measurements are likely to be inaccurate and that a value of 3 H+/ATP, independent of the external pH, is likely to be the valid stoichiometry.
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PMID:Stoichiometry of the H+-ATPase of growing and resting, aerobic Escherichia coli. 629 45

The gene coding for the yeast mitochondrial F1-ATPase beta subunit (ATP2) has been fused to the Escherichia coli lacZ gene. The chimeric ATP2-lacZ gene codes for a hybrid protein consisting of some 350 amino acids of the F1-ATPase beta subunit at its amino terminus and a large enzymatically active portion of the lacZ gene product, beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), at its carboxyl terminus. The beta-subunit-beta-galactosidase hybrid protein is expressed in both E. coli and yeast. In yeast, this hybrid molecule is targeted to the mitochondrion and is protected in isolated mitochondria from added protease under conditions in which an outer membrane enzymatic marker is digested. Yeast cells carrying the ATP2-lacZ gene fusion on plasmid p beta Z1 are unable to grow on a nonfermentable carbon source. Upon loss of the p beta Z1 plasmid, growth of the cured host strain on the nonfermentable substrate is restored. In the presence of the beta-subunit-beta-galactosidase hybrid protein, the energy-transducing capacity of the mitochondrial membrane as measured by the 32Pi-ATP exchange reaction is only 9% of that measured in the absence of the gene fusion product. The results indicate that it is the presence of the beta-subunit-beta-galactosidase hybrid protein within mitochondria that interferes with function(s) essential for respiratory growth. These observations open up the prospect of genetic characterization of the signals and cellular machinery responsible for mitochondrial protein delivery.
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PMID:Intracellular targeting and import of an F1-ATPase beta-subunit-beta-galactosidase hybrid protein into yeast mitochondria. 633 Jul 27


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