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

---

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

Expression of the soluble (SH) and membrane-bound (MBH) hydrogenases in the facultatively lithoautotrophic bacterium Alcaligenes eutrophus is dependent on the transcriptional activator HoxA and the alternative sigma factor sigma 54. Deletion analysis revealed that a region 170 bp upstream of the transcriptional start of the SH operon is necessary for high-level promoter activity. Mobility shift assays with DNA fragments containing the SH upstream region and purified beta-galactosidase-HoxA fusion protein isolated from Escherichia coli or authentic HoxA isolated by immunoaffinity chromatography from A. eutrophus failed to detect specific binding. In contrast, A. eutrophus extracts enriched for HoxA by heparin-Sepharose chromatography and ammonium sulfate fractionation produced a weak but discrete shift in the mobility of the target DNA. This effect was not observed with comparable extracts prepared from hoxA mutants. A similar experiment using antibodies against HoxA confirmed that HoxA was responsible for the observed mobility shift. Extracts prepared from a temperature-tolerant mutant of A. eutrophus gave a stronger retardation than did those from the wild type. Unlike the wild type, the hox(Tr) mutant is able to grow with hydrogen at temperatures above 33 degrees C because of a mutation in the regulatory gene hoxA. In this paper, we show that a single amino acid substitution (Gly-468-->Val) in the C-terminal part of HoxA is responsible for temperature tolerance. The SH upstream region also contains sequence motifs resembling the E. coli integration host factor (IHF) binding site, and purified E. coli IHF protein shifted the corresponding indicator fragment.
...
PMID:Temperature tolerance of hydrogenase expression in Alcaligenes eutrophus is conferred by a single amino acid exchange in the transcriptional activator HoxA. 773 Feb 67

The heterogeneous nuclear RNP (hnRNP) A1 protein is one of the major pre-mRNA/mRNA binding proteins in eukaryotic cells and one of the most abundant proteins in the nucleus. It is localized to the nucleoplasm and it also shuttles between the nucleus and the cytoplasm. The amino acid sequence of A1 contains two RNP motif RNA-binding domains (RBDs) at the amino terminus and a glycine-rich domain at the carboxyl terminus. This configuration, designated 2x RBD-Gly, is representative of perhaps the largest family of hnRNP proteins. Unlike most nuclear proteins characterized so far, A1 (and most 2x RBD-Gly proteins) does not contain a recognizable nuclear localization signal (NLS). We have found that a segment of ca. 40 amino acids near the carboxyl end of the protein (designated M9) is necessary and sufficient for nuclear localization; attaching this segment to the bacterial protein beta-galactosidase or to pyruvate kinase completely localized these otherwise cytoplasmic proteins to the nucleus. The RBDs and another RNA binding motif found in the glycine-rich domain, the RGG box, are not required for A1 nuclear localization. M9 is a novel type of nuclear localization domain as it does not contain sequences similar to classical basic-type NLS. Interestingly, sequences similar to M9 are found in other nuclear RNA-binding proteins including hnRNP A2.
...
PMID:A nuclear localization domain in the hnRNP A1 protein. 773 Mar 95

The tolerance of bacteriophage lambda morphogenesis for C-terminal additions to the tail tube major protein subunit (the V gene product; gpV) has been investigated. A second modified copy of the lambda V gene, either within a novel phage vector itself or plasmid-borne, was expressed during phage growth. High-level substitution of wild-type gpV by modified gpV bearing a basic C-terminal peptide sequence (RRASV; a target site for cAMP-dependent protein kinase) was possible using multiple repeats of a serine-glycine (SGGG) linker sequence. Highly purified phage bearing copies of gpV-RRASV could be efficiently phosphorylated by the appropriate protein kinase, and the incorporated label was shown to migrate exclusively at the expected size in protein gels. A large tetrameric protein (beta-galactosidase) could be incorporated into active virions in at least one copy, again using a Ser-Gly linker. These studies suggest that with a suitable spacing linker and controlled levels of expression, it is likely that a wide range of protein or peptide substitutents can be fused with gpV at its C terminus and assembled as component subunits of the tail tube.
...
PMID:Assembly of functional bacteriophage lambda virions incorporating C-terminal peptide or protein fusions with the major tail protein. 775 19

The lac I gene of Escherichia coli encodes the lactose repressor. We isolated temperature-sensitive mutants of the lac I gene by in vitro mutagenesis with hydroxylamine. The new mutation sites were determined, and replacement of a single amino acid had respectively occurred at amino acid positions 241 (Ala-->Thr), 265 (Gly-->Asp) and 300 (Ser-->Asn). These mutation sites were located in the core region of the lac repressor protein. Temperature-dependent expression of beta-galactosidase was observed in the strains having these mutant lac I genes. By using these temperature-sensitive lac I genes, we developed a thermo-inducible expression system for a foreign gene under the control of the lac promoter. A recombinant fusion protein, consisting of a derivative of E. coli beta-galactosidase and the human calcitonin precursor peptide, was efficiently produced by using this system.
...
PMID:Thermo-inducible expression of a recombinant fusion protein by Escherichia coli lac repressor mutants. 776 12

The cDNA encoding sea bass (Dicentrarchus labrax) prolactin (sbPRL) was obtained by reverse transcription-polymerase chain reaction (RT/PCR) from pituitary RNA with degenerate primers designed on the basis of the cDNAs of the two PRLs (tPRL188 and tPRL177) from the tilapia, Oreochromis niloticus. The sbPRL cDNA encodes a preprotein of 212 amino acids composed of a putative signal peptide of 24 residues and a mature protein of 188 amino acids that is the homologue of tiPRL188. The cDNA coding for the mature protein was cloned into the pAX4a+ expression vector and expressed efficiently in Escherichia coli as a beta-galactosidase-fusion protein. To split the fusion protein, a sequence encoding the hexapeptide, (Asn-Gly)3, that contains three Asn-Gly hydroxylamine-cleavable bonds, had been previously introduced by PCR upstream of the sbPRL cDNA. N-terminal sequencing confirmed that the cleaved product corresponded to sbPRL. An antiserum raised against the recombinant hormone detected by immunoblotting a single band in sea bass pituitaries and two bands in tilapia pituitaries, suggesting the occurrence of a single PRL form in sea bass.
...
PMID:The prolactin of European sea bass (Dicentrarchus labrax L.): cloning of cDNA and efficient expression in Escherichia coli. 780 37

Substitutions of Gly-794 (beta-galactosidase) with Asp, Asn, Glu, and Lys caused decreased binding of substrates and inhibition by substrate analogs, while inhibition by planar and positively charged galactose analogs increased relative to the binding of substrates and the inhibition by substrate analogs. There was a correlation of the relative inhibition with the size of the substituted residue but no relationship to the presence or absence of a negative charge, and as the relative inhibition by the planar and positively charged galactose analogs increased, k3 (hydrolysis; degalactosylation) and kcat/Km (catalytic efficiency) values decreased. The k2 values (glycolytic cleavage; galactosylation) mainly increased for poor substrates (p-nitrophenyl beta-galactoside and lactose) but decreased for o-nitrophenyl beta-galactoside (a good substrate). Enzymes substituted with Asp or Asn were inhibited to a similar extent by planar and positively charged inhibitors and had similar effects on catalysis, while inhibition and catalytic effects on the enzyme substituted by Glu were quite different. If the negative charge was important, the Asp- and Glu-substituted enzymes should have been inhibited to a similar extent, while the Asn-substituted enzyme should have caused a different degree of inhibition. The enzyme substituted with a Lys at position 794 bound substrates and inhibitors very poorly, but the relative inhibition and the catalysis still correlated to size. Alterations of the size of the residue at position 794 cause modifications in the binding interactions and affected activity.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Substitutions for Gly-794 show that binding interactions are important determinants of the catalytic action of beta-galactosidase (Escherichia coli). 789 71

We describe four new mutations in the beta-galactosidase gene. These are the first mutations causing infantile and juvenile GM1-gangliosidosis to be described in American patients. Cell lines from two patients with juvenile and from six patients with infantile GM1-gangliosidosis were analyzed. Northern blot analysis showed the acid beta-galactosidase message to be of normal size and quantity in two juvenile and four infantile cases and of normal size but reduced quantity in two infantile cases. The mutations are distinct from the Japanese mutations. All are point mutations leading to amino acid substitutions: Lys577-->Arg, Arg590-->His, and Glu632-->Gly. The fourth mutation, Arg208-->Cys, accounts for 10 of 16 possible alleles. Two infantile cases from Puerto Rico of Spanish ancestry are homozygous for this mutation, suggesting that this allele may have come to South America and North America via Puerto Rico. That these mutations cause clinical disease was confirmed by marked reduction in catalytic activity of the mutant proteins in the Cos-1 cell expression system.
...
PMID:Mutations in acid beta-galactosidase cause GM1-gangliosidosis in American patients. 821 16

Recombinant rat brain inositol 1,4,5-triphosphate [Ins(1,4,5)P3] 3-kinase was expressed in Escherichia coli as a beta-galactosidase fusion product. It could be adsorbed onto calmodulin-Sepharose and eluted in Ca(2+)-free medium as a 48-kDa protein. Purification could be achieved in a single step. Molecular evidence for a calmodulin-binding domain on Ins(1,4,5)P3 3-kinase can be shown by the following approaches. (a) Inhibition of Ca2+/calmodulin stimulation by a synthetic peptide based on a candidate calmodulin-binding domain. The inhibition was mimicked by a well-characterized peptide derived from the sequence of smooth muscle myosin light-chain kinase calmodulin-binding site. (b) The construction of two mutants by site-directed mutagenesis of Trp165 to Gly or Arg. Both mutants displayed kinase activity but were no longer Ca2+/calmodulin sensitive, supporting, therefore, the role of Trp165 in calmodulin binding.
...
PMID:Interaction of calmodulin with a putative calmodulin-binding domain of inositol 1,4,5-triphosphate 3-kinase. Effects of synthetic peptides and site-directed mutagenesis of Trp165. 839 Mar 54

The kinetics of hydrolysis of a series of synthetic substrates by two experimentally evolved forms ('evolvants'), ebgabcd and ebgabcde, of the second beta-galactosidase of Escherichia coli have been measured. The ebgabcd enzyme differs from the wild-type (ebgo) enzyme by Asp92-->Asn (a) and Trp977-->Cys (b) changes in the large subunit, as well as two changes hitherto considered to have no kinetic effect, Ser979-->Gly in the large subunit (c) and Glu122-->Gly in the small subunit (d). The enzyme ebgabcde contains in addition a Glu93-->Lys change in the large subunit (e). Comparison of ebgabcd with ebgab [Elliott, K, Sinnott, Smith, Bommuswamy, Guo, Hall and Zhang (1992) Biochem. J. 282, 155-164] indicates that the c and d changes in fact accelerate the hydrolysis of the glycosyl-enzyme intermediate by a factor of 2.5, and also decrease the charge on the aglycone oxygen atom at the first transition state; the charge on the glycone, however, is unaltered [see K, Konstantinidis, Sinnott and Hall (1993) Biochem. J. 291, 15-17]. The e mutation causes a fall in the degalactosylation rate of about a factor of 3, and its occurrence only together with c and d mutations [Hall, Betts and Wootton (1989) Genetics 123, 635-648] suggests that degalactosylation of a hypothetical ebgabe enzyme would be so slow that the enzyme would have no biological advantage over the ancestral ebgab. The transfer products from galactosyl-ebgabcd and galactosyl-ebgabcde to high concentrations to glucose have been measured; the predominant product is allolactose, but significant quantities of lactose are also formed; however, at apparent kinetic saturation of the galactosyl-enzyme, hydrolysis rather than transfer is the preponderant pathway. A knowledge of the rates of enzyme-catalysed exchange of 18O from [1-18O]galactose to water permits the construction of the free-energy profiles for hydrolysis of lactose by begabcd and ebgabcde. As with the other evolvants, changes in the profile away from the rate-determining transition state are essentially random, and there is no correlation between the changes in the free energies of intermediates and of their flanking transition states. We consider the aggregate of our kinetic data on the ebg system to be telling experimental support for the theoretical objections of Pettersson [Pettersson (1992) Eur. J. Biochem. 206, 289-295 and previous papers] to the Albery-Knowles theory of the evolution of enzyme kinetic activity.
...
PMID:Catalytic consequences of experimental evolution: catalysis by a 'third-generation' evolvant of the second beta-galactosidase of Escherichia coli, ebgabcde, and by ebgabcd, a 'second-generation' evolvant containing two supposedly 'kinetically silent' mutations. 855 46

The primary and atomic structures of the porin protein from Rhodobacter (Rb.) capsulatus strain 37b4 were determined several years ago by peptide sequencing and X-ray crystallography. In this work the gene encoding this porin (named porCa) was cloned and sequenced. The porin open reading frame encodes 320 amino acids-a mature protein of 300 residues (molecular mass 31 552 kDa) and a presequence of 20 amino acids. Our deduced amino-acid sequence was directly confirmed by purifying the porin protein from the same bacterial strain and sequencing the amino terminus as well as several peptides derived from trypsin digestion. However, comparison of this deduced amino-acid sequence with the published primary structure of this porin, nominally from the same strain (but cultivated for ca. 30 years in a different laboratory) reveals seven differences in the amino-acid sequence at the following positions in the mature protein (published/present): 59 (Gly/Ala), 123 (Tyr/Asn), 135 Ser/Thr), 189 (Ile/Val), 196 (Asn/His), 231 (Ala/Thr) and 238 (Ser/deleted). Surprisingly, analysis of the positioning of these mutations revealed that they are located exclusively on transmembrane strands, with two of them deeply buried within the structure. These mutations may in fact have only marginal influence on porin structure and function. Northern blot analysis revealed that porCa encodes an RNA transcript of 1070 nucleotides. No differential response in the abundance or size of this mRNA was seen upon growth under phototrophic/anaerobic vs. chemotrophic/aerobic conditions, under high or low osmotic pressure. Primer extension experiments revealed a transcription start site 73 bases upstream from the ATG translation start, juxtaposed to the identified putative promoter region. Fusion of lacZ with this putative promoter region (using a 288-bp upstream region) revealed similar promoter activity in beta-galactosidase assays under both physiological conditions tested, again suggesting that this gene is constitutively expressed. The molecular genetic characterization described in this work opens the way for structure-function studies by site-directed mutagenesis.
...
PMID:Molecular characterization and organization of porin from Rhodobacter capsulatus strain 37B4. 899 88


<< Previous 1 2 3 4 5 6 7 Next >>

---