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)

Escherichia coli B infected with T4 phage ghosts at 10 mM Mg2+ regains its protein synthesizing activity upon addition of ATP, GTP, and their generator to approximately 2% of the intact exponentially growing cells. In contrast to amino acid incorporation by intact cells, this system is sensitive to EDTA or low Mg2+. On the other hand, this system, differing from the regular cell-free system, does not respond to addition of soluble protein and ribonuclease. The ghost-infected cells were able to synthesize beta-galactosidase upon addition of the inducer isopropyl thiogalactoside. The initial rate of the induction was 2.6% of intact cells. For this induction, the addition of cyclic AMP, amino acids, ATP, GTP, UTP, CTP, and their generator was necessary. The induction of beta-galactosidase in these ghost-infected cells was very sensitive to the addition of EDTA, CaCl2, sulfhydryl blocking reagent, rifampin and chloramphenicol but insensitive to DNA synthesis inhibitors such as nalidixic acid and DNase.
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PMID:Protein synthesis in bacteriophage ghost-infected cells. 17 55

Escherichia coli Q13 was infected with bacteriophage Q beta and subjected to energy source shift-down (from glucose-minimal to succinate-minimal medium) 20 min after infection. Production of progeny phage was about fourfold slower in down-shifted cultures than in the cultures in glucose medium. Shift-down did not affect the rate of phage RNA replication, as measured by the rate of incorporation of [14C]uracil in the presence of rifampin, with appropriate correction for the reduced entry of exogenous uracil into the UTP pool. Phage coat protein synthesis was three- to sixfold slower in down-shifted cells than in exponentially growing cells, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The polypeptide chain propagation rate in infected cells was unaffected by the down-shift. Thus, the reduced production of progeny phage in down-shifted cells appears to result from control of phage protein synthesis at the level of initiation of translation. The reduction in the rate of Q beta coat protein synthesis is comparable to the previously described reduction in the rate of synthesis of total E. coli protein and of beta-galactosidase, implying that the mechanism which inhibits translation in down-shifted cells is neither messenger specific nor specific for 5' proximal cistrons. The intracellular ATP pool size was nearly constant after shift-down; general energy depletion is thus not a predominant factor. The GTP pool, by contrast, declined by about 40%. Also, ppGpp did not accumulate in down-shifted, infected cells in the presence of rifampin, indicating that ppGpp is not the primary effector of this translational inhibition.
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PMID:Control of protein synthesis in Escherichia coli: control of bacteriophage Q beta coat protein synthesis after energy source shift-down. 38 18

Varicella-zoster virus (VZV) ORF 47 lies in the unique long region of the VZV genome. Sequence homology studies have demonstrated that gene 47 possessed conserved protein kinase motifs. In this study, we investigated the properties of the ORF 47 product. First, a rabbit antiserum was raised against a protein generated from the fusion of the most antigenic ORF 47 domain with Escherichia coli beta-galactosidase. The high-titer antiserum reacted specifically with ORF 47 polypeptides translated in vitro. When incubated with VZV-infected cell lysate, the antiserum immunoprecipitated a phosphoprotein of M(r) 54,000, a size comparable with the predicted molecular mass. The precipitated viral protein was phosphorylated in a protein kinase assay; subsequent phosphoamino acid analysis indicated that the phosphotransferase associated with the ORF 47 protein was a serine protein kinase. Synthesis of the ORF 47 product in VZV-infected cell culture increased in the first and second days and plateaued after the third day of infection. The protein kinase activity associated with VZV ORF 47 had several distinctive biochemical properties: (i) its phosphotransferase activity was enhanced more by manganese than by magnesium, (ii) it utilized both ATP and GTP as donors of phosphate, and (iii) it phosphorylated both acidic and basic substrates. In summary, this report lends support to the computer homology data which predicted that VZV ORF 47 would encode a serine protein kinase.
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PMID:Serine protein kinase associated with varicella-zoster virus ORF 47. 132 39

The bovine dopamine D2 receptor was purified by wheat-germ-agglutinin-Sepharose chromatography and affinity chromatography, using the D2-receptor-specific agonist N-0434. Purification yields a preparation with a major protein band of 95 kDa. In order to ascertain the identity of this protein, polyclonal antibodies against the dopamine D2 receptor have been raised using synthetic peptides based on the predicted amino acid sequence of the cloned D2 receptor. For the initial screening of these antibodies, three fusion proteins consisting of beta-galactosidase and receptor fragments were constructed. One antiserum reacted strongly with the corresponding D2 receptor fusion protein, both on Western blots and in immunoprecipitation experiments. In each case, recognition was inhibited by competition with free peptide. On Western blots of partially purified receptor preparations from bovine striatum, the antiserum specifically recognized a 95-kDa glycoprotein. From similar preparations, the antiserum precipitated a substantial proportion of active D2 receptor, as determined by a decrease in [3H]spiperone binding in the supernatant. Active receptor could be released from the immunoprecipitate by addition of free peptide. Immunocytochemical analysis of cells transiently transfected with DNA coding for the D2 receptor showed specific staining of transfected cells. The antibody raised against a sequence in the third intracellular loop is able to shift the affinity of the receptor for dopamine from high to low, indicating that the antiserum may be interfering with receptor-GTP-binding-protein interactions.
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PMID:An anti-peptide antibody that recognizes the dopamine D2 receptor from bovine striatum. 135 Feb 46

Pharmacological and biochemical characteristics of the partially purified gamma-aminobutyric acid (GABA)B receptor using baclofen affinity column chromatography have been examined. The Scatchard analysis of [3H]GABA binding to the purified GABAB receptor showed a linear relationship and the KD and Bmax values were 60 nM and 118 pmol/mg of protein, respectively. Although GTP and Mg2+ did not affect on the GABAB receptor binding, Ca2+ significantly increased [3H]GABA binding to the purified GABAB receptor in a dose-dependent manner and showed its maximum effect at 2 mM. The enhancement of the binding by Ca2+ was found to be due to the increase of Bmax by the Scatchard analysis. The treatments with pronase and trypsin significantly decreased the binding of [3H]GABA, but phospholipase A2 had no significant effect on the binding. In addition, treatment with glycosidases such as glycopeptidase A and beta-galactosidase significantly decreased the binding of [3H]GABA to the purified GABAB receptor. These results suggest that purification of the solubilized GABAB receptor by the affinity column chromatography may result in the functional uncoupling of GABAB receptor with GTP-binding protein. Furthermore, the present results suggest that cerebral GABAB receptor may be a glycoprotein and membrane phospholipids susceptible to phospholipase A2 treatment may not be involved in the exhibition of the binding activity.
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PMID:Pharmacological and biochemical characteristics of partially purified GABAB receptor. 166 62

We initiated a genetic reversion analysis at the HIS4 locus to identify components of the translation initiation complex that are important for ribosomal recognition of an initiator codon. Three unlinked suppressor loci, suil, sui2, and SUI3, that restore expression of both HIS4 and HIS4-lacZ in the absence of an AUG initiator codon were identified. In previous studies, it was demonstrated that the sui2 and SUI3 genes encode mutated forms of the alpha and beta subunits, respectively, of eukaryotic translation initiation factor 2 (eIF-2). In this report, we describe the molecular and biochemical characterizations of the sui1 suppressor locus. The DNA sequence of the SUI1+ gene shows that it encodes a protein of 108 amino acids with a calculated Mr of 12,300. The sui1 suppressor genes all contain single base pair changes that alter a single amino acid within this 108-amino-acid sequence. sui1 suppressor strains that are temperature sensitive for growth on enriched medium have altered polysome profiles at the restrictive temperature typical of those caused by alteration of a protein that functions during the translation initiation process. Gene disruption experiments showed that the SUI1+ gene encodes an essential protein, and antibodies directed against the SUI1+ coding region identified a protein with the predicted Mr in a ribosomal salt wash fraction. As observed for sui2 and SUI3 suppression events, protein sequence analysis of His4-beta-galactosidase fusion proteins produced by sui1 suppression events indicated that a UUG codon is used as the site of translation initiation in the absence of an AUG start codon in HIS4. Changing the penultimate proline codon 3' to UUG at his4 to a Phe codon (UUC) blocks aminopeptidase cleavage of the amino-terminal amino acid of the His4-beta-galactosidase protein, as noted by the appearance of Met in the first cycle of the Edman degradation reaction. The appearance of Met in the first cycle, as noted, in either a sui1 or a SUI3 suppressor strain showed that the mechanism of suppression is the same for both suppressor genes and allows the initiator tRNA to mismatch base pair with the UUG codon. This suggests that the Sui1 gene product performs a function similar to that of the beta subunit of eIF-2 as encoded by the SUI3 gene. However, the Sui1 gene product does not appear to be a required subunit of eIF-2 on the basis of purification schemes designed to identify the GTP-dependent binding activity of eIF-2 for the initiator tRNA. In addition, suppressor mutations in the sui1 gene, in contrast to suppressor mutations in the sui2 or SUI3 gene, do not alter the GTP-dependent binding activity of the eIF-2. The simplest interpretation of these studies is that the sui1 suppressor gene defines an additional factor that functions in concert with eIF-2 to enable tRNAiMet to establish ribosomal recognition of an AUG initiator codon.
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PMID:The suil suppressor locus in Saccharomyces cerevisiae encodes a translation factor that functions during tRNA(iMet) recognition of the start codon. 172 2

A gene from Rhizobium meliloti coding for an adenylate cyclase was sequenced, and the deduced protein sequence was compared with those of other known adenylate cyclases. No similarity could be detected with the procaryotic counterparts. However, striking similarity was found with the catalytic region of Saccharomyces cerevisiae adenylate cyclase, the cytoplasmic domains of bovine adenylate cyclase, and two mammalian guanylate cyclases. The gene was fused to the enteric beta-galactosidase, and the chimeric protein was purified by affinity chromatography. This fusion protein was found to direct the synthesis of cyclic AMP in vitro. This activity was strongly inhibited by the presence of GTP, but no cyclic GMP synthesis could be detected in conditions permitting cyclic AMP synthesis.
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PMID:Rhizobium meliloti adenylate cyclase is related to eucaryotic adenylate and guanylate cyclases. 197 May 65

The product of the CDC25 gene of Saccharomyces cerevisiae, in its capacity as an activator of the RAS/cyclic AMP pathway, is required for initiation of the cell cycle. In this report, we provide an identification of Cdc25p, the product of the CDC25 gene, and evidence that it promotes exchange of guanine nucleotides bound to Ras in vitro. Extracts of strains containing high levels of Cdc25p catalyze both removal of GDP from and the concurrent binding of GTP to Ras. This same activity is also obtained with an immunopurified Cdc25p-beta-galactosidase fusion protein, suggesting that Cdc25p participates directly in the exchange reaction. This biochemical activity is consistent with previous genetic analysis of CDC25 function.
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PMID:The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras. 201 69

1. Metabolically stable analogues of GTP, e.g. guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta,gamma-imido]triphosphate (pp[NH]pG), enhance the extent of Ca2(+)-dependent secretion of beta-N-acetylglucosaminidase and beta-galactosidase from electropermeabilised human platelets in the presence of less than 5 microM Ca2+. A similar effect is observed on addition either of 1,2-dioctanoin or of GTP in in the presence or absence of thrombin. 2. In the presence of higher Ca2+ concentrations the extent of enhancement of lysosomal secretion declines and little, or no, enhancement is observed at a [Ca2+] of 30-40 microM. Addition of leupeptin or antipain prevents this decrease in lysosomal secretion and enhances the extent of Ca2(+)-dependent lysosomal secretion obtained in the presence or absence of guanine nucleotides, thrombin or 1,2-dioctanoin. 3. The concentration of GTP[S] or pp[NH]pG required to obtain half-maximal enhancement of lysosomal secretion is dependent on [Ca2+] for secretion of 5-hydroxytryptamine, beta-N-acetylglucosaminidase and beta-galactosidase. At two fixed [Ca2+] the median effective concentration (EC50) values for GTP[S] and pp[NH]pG which characterise enhancement of 5-hydroxytryptamine secretion are significantly different from those characterising enhancement of the secretion of beta-N-acetylglucosaminidase and beta-galactosidase. 4. In the presence of a saturating concentration of GTP[S] marked 5-hydroxytryptamine and beta-N-acetylglucosaminidase secretion is observed at nanomolar [Ca2+] and these responses show little dependence on [Ca2+] over the attainable range. Secretion of beta-N-acetylglucosaminidase is also induced at nanomolar Ca2+ concentrations by addition of activators of protein kinase C. 5. Guanosine 5'-[beta-thio]diphosphate inhibits enhancement of beta-N-acetylglucosaminidase secretion induced by GTP[S] but has no effect on secretion of this enzyme induced by Ca2+ when added alone. 6. Our data provide some support for a model in which addition of metabolically stable guanine nucleotides enhances Ca2(+)-dependent platelet lysosomal secretion by activating a guanine-nucleotide-binding protein (GE) located close to the exocytotic site. However, not all the data are consistent with this postulate.
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PMID:Guanine nucleotides and Ca2(+)-dependent lysosomal secretion in electropermeabilised human platelets. 211 63

A cell-free assay to monitor receptor-mediated endocytic processes has been developed that uses biotinylated transferrin and avidin-linked beta-galactosidase as receptor-associated and fluid-phase probes, respectively (Wessling-Resnick, M., and Braell, W. A. (1990) J. Biol. Chem. 265, 690-699). The fusion of vesicles from heterologous sources can be detected in this assay: endocytic vesicles from K562 cells (a human cell line) will fuse with vesicles from Chinese hamster ovary cells. Fusion between endocytic vesicles is inhibited upon treatment with N-ethylmaleimide but can be restored by the addition of untreated cytosol from either cell type. The in vitro fusion reaction is also inhibited by the nonhydrolyzable nucleotide analogs guanosine 5'-(3-thiotriphosphate) (GTP gamma S) and adenosine 5'-(3-thiotriphosphate) (ATP gamma S). Other nonhydrolyzable guanine nucleotides are found to inhibit the in vitro reaction in the following order of potency: GTP gamma S greater than 5'-guanylyl imidodiphosphate (GTP-PNP) greater than alpha,beta-methylene GTP (GTP-PCP). The inhibitory effects of the nonhydrolyzable analogs of GTP and ATP are not additive. Moreover, excess GTP relieves the inhibition by GTP gamma S more than it relieves the inhibition by ATP gamma S, while excess ATP preferentially alleviates ATP gamma S (not GTP gamma S) inhibition. These properties suggest that the two nucleotides exert their effects at distinct points in the fusion process. Although micromolar levels of excess Ca2+ also inhibit vesicle fusion, the inhibition exerted by GTP gamma S appears to proceed via a pathway independent of the divalent cation. The GTP gamma S-sensitive step in endocytic vesicle fusion is found to occur at a mechanistic stage prior to and distinct from the N-ethylmaleimide-sensitive step of the reaction. This situation permits the accumulation of a membrane vesicle intermediate in the presence of GTP gamma S; subsequent incubation of these vesicles with cytosol and GTP restores their fusion competence. Characteristics of in vitro endocytic vesicle fusion suggest that similarities exist with steps of the fusion mechanism involved with membrane traffic events of the secretory pathway.
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PMID:Characterization of the mechanism of endocytic vesicle fusion in vitro. 212 Feb 6


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