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 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.
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PMID:Mutations in acid beta-galactosidase cause GM1-gangliosidosis in American patients. 821 16

Rhodobacter capsulatus is a Gram-negative photosynthetic bacterium that requires c-type cytochromes for photosynthetic electron transport. Our studies demonstrate that the gene helX is required for the biogenesis of c-type cytochromes in R. capsulatus. A helX chromosomal deletion mutant cannot grow photosynthetically, due to a deficiency of all c-type cytochromes. The predicted amino acid sequence of the helX gene product (176 residues) is related to that of thioredoxin and shares active-site homology with protein disulfide isomerase. Cytochrome c2-alkaline phosphatase gene fusions are used to show that HelX is not required for the transcription, translation, or secretion of apocytochrome c2. HelX-alkaline phosphatase and HelX-beta-galactosidase gene fusions are used to demonstrate that HelX is a periplasmic protein, which is consistent with the presence of a typical signal sequence in HelX. Based on these results, we propose HelX functions as a periplasmic disulfide oxidoreductase that is essential for cytochromes c biogenesis. This role is in accordance with the observation that both heme and the cysteines of apocytochromes c (Cys-Xaa-Yaa-Cys-His) must be in the reduced state for covalent linkage between the two moieties to occur.
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PMID:Cytochromes c biogenesis in a photosynthetic bacterium requires a periplasmic thioredoxin-like protein. 838 15

The 3', 5' cyclic adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to substitute leucine, glutamine, or aspartate for glutamate 72; and lysine, histidine, leucine, isoleucine, or glutamine for arginine 82. Substitutions were made in wild-type CRP and in a CRP*, or cAMP-independent, form of the protein to assess the effects of the amino acid substitutions on CRP structure. Cells containing the binding pocket residue-substituted forms of CRP were characterized through beta-galactosidase activity and by measurement of cAMP binding activity. This study confirms a role for both glutamate 72 and arginine 82 in cAMP binding and activation of CRP. Glutamine or leucine substitution of glutamate 72 produced forms of CRP having low affinity for the cAMP and unresponsive to the nucleotide. Aspartate substituted for glutamate 72 produced a low affinity cAMP-responsive form of CRP. CRP has a stringent requirement for the positioning of the position 72 glutamate carboxyl group within the cyclic nucleotide binding pocket. Results of this study also indicate that there are differences in the binding requirements of cAMP and cGMP, a competitive inhibitor of cAMP binding to CRP.
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PMID:Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 72 and 82 of the cyclic nucleotide binding pocket. 838 97

The rat monoclonal antibody, mAb 12C11, reacts with numerous proteins from mature asexual stages of Plasmodium falciparum. The largest is 315 kDa and is designated PfEMP3. A lambda gt11 expression library, generated from genomic DNA of Malayan Camp strain parasites, was screened with mAb 12C11. One positive clone, lambda 12.1.3, contained a 1.4-kb fragment in frame with the beta-galactosidase gene of lambda gt11. The deduced 455-amino acid sequence is a novel, highly charged sequence encoding two 15-amino acid repeats at the N-terminus followed by 27 repeats of 13 amino acids. The last 59 C-terminal residues are non-repetitive. Two in-frame stop codons at the 3' end of the DNA suggests that this DNA fragment encodes the C-terminus of the protein. Southern blotting with the cloned fragment identified two copies of this fragment per haploid genome in knob-positive, parasitized erythrocytes (K+PE). Both DNA fragments are absent from K - PE. Northern blotting of trophozoite-stage PE total RNA revealed mRNAs of 10, 4.4 and 2 kb in K+PE, but no hybridization with K - PE. Immune sera were elicited against the lambda 12.1.3 beta-galactosidase fusion protein and peptides generated from the predicted lambda 12.1.3 amino acid sequence. These sera and mAb 12C11 reacted specifically with PfEMP3 in Western blots of mature K+PE but not with K - PE. Rat and mouse sera against the recombinant protein produced an immunofluorescence pattern in fixed mature K+PE almost identical to the pattern produced by a monoclonal antibody against the knob-associated protein, Histidine Rich Protein 1. The same antibodies were immunofluorescence negative with fixed K - PE. Mouse antibodies against the recombinant protein reacted on immunoelectron microscopy with the erythrocyte membrane of K+PE, labeling knobs as well as the membrane between knobs. In contrast, a mAb against Histidine Rich Protein 1 reacted only under the electron dense material of knobs. We conclude that the lambda 12.1.3 clone encodes the C-terminal portion of the 315 kD PfEMP3 antigen and that PfEMP3 may be involved in knob formation or other perturbations of the erythrocyte membrane.
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PMID:Cloning and characterization of a Plasmodium falciparum gene encoding a novel high-molecular weight host membrane-associated protein, PfEMP3. 851 84

beta-Galactosidases with substitutions for His-540 were only poorly reactive with galactosyl substrates. However, the activity with substrates that were like galactose but did not have a C6 hydroxyl group was not decreased much as a result of such substitutions. The loss of transition state stabilization for galactosyl substrates as a result of substitution was between -15.4 and -22.8 kJ/mol but only between +0.34 and -6.5 for substrates that were identical to galactose but lacked the C6 hydroxyl. These findings indicate that an important function of His-540 is to aid in the stabilization of the transition state by forming a stable interaction with the C6 hydroxyl group. This suggestion was strengthened by the results of competitive inhibition studies showing that L-arabinolactone (a transition state analog inhibitor of beta-galactosidase without a C6 hydroxymethyl group) was bound as well by the substituted enzymes as by wild type, whereas transition state analog inhibitors that contain C6 hydroxyls (L-ribose and D-galactonolactone) were bound much more poorly by the substituted enzymes than by the wild type enzyme. Substrate analog inhibitor studies showed that His-540 was also important for binding interactions with the C6 hydroxyl group of the ground (substrate) state. The activation by Mg2+ was the same for the substituted enzymes as for the wild type, and equilibrium dialysis showed that H540F-beta-galactosidase bound Mg2+ as well as did normal beta-galactosidase. The k2 and Ks values seem to have the same pH interactions as wild type enzyme, whereas the k3 interactions are affected differently by pH in the substituted enzymes than in the wild type enzyme. The rate of the "degalactosylation" reaction was affected more by substitutions for His-540 than was the rate of the "galactosylation" reaction. All three substituted beta-galactosidases were less stable to heat than was wild type, but H540N-beta-galactosidase was somewhat more stable than the other two substituted enzymes. There were some differences in activity and inhibitory properties that resulted from the different substitutions.
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PMID:The beta-galactosidase (Escherichia coli) reaction is partly facilitated by interactions of His-540 with the C6 hydroxyl of galactose. 866 37

The potential genotoxicity of dihydroabikoviromycin was assessed in bacterial and sister-chromatid exchange (SCE) test systems. Direct cytotoxicity was also assayed using bioluminescence methods to screen for differences in cell viability among different tumour cell lines following exposure to the drug. Differential killing tests with Escherichia coli WP2 and its repair-deficient derivative CM871 indicated that a functional DNA repair system was protective against the action of dihydroabikoviromycin, implying that this compound causes some form of DNA damage and is almost certainly therefore genotoxic. Dose-dependent reversion from His- to His+ with dihydroabikoviromycin was observed in the Ames test with Salmonella typhimurium TA100, but not in frameshift tester strain TA98. Dihydroabikoviromycin also induced the sfiA gene, as indicated by beta-galactosidase induction in an SOS-chromotest with E. coli PQ37 strain. A dose-related increase in SCEs by dihydroabikoviromycin was observed in CHO cells. Growth of tumour cells was also suppressed by dihydroabikoviromycin at a dose of 10 micrograms/ml.
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PMID:Genotoxicity of dihydroabikoviromycin, a secondary metabolite of Streptomyces anulatus. 869 21

The Drosophila abnormal wing discs (awd) gene encodes the subunit of a protein that has nucleoside diphosphate kinase (NDP kinase) activity. Null mutations of the awd gene cause lethality after puparium formation. Larvae homozygous for such mutations have small imaginal discs, lymph glands, and brain lobes. Neither the imaginal discs nor the ovaries from such null mutant larvae are capable of further growth or normal differentiation when transplanted into suitable host larvae. This null mutant phenotype can be entirely rescued by one copy of a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA. Tissue-specific expression of AWD protein from this rescue transgene is identical to tissue-specific expression of beta-galactosidase from a reporter transgene that has the same regulatory region fused to the bacterial lac Z gene. However, this rescue transgene or reporter transgene expression pattern is only a subset of the endogenous pattern of expression detected by either in situ hybridization or immunohistochemistry. This suggests that awd is normally expressed in some tissues where it is not required. The null mutant phenotype cannot be rescued at all by a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA with a mutation that eliminates NDP kinase activity by replacement of the active site histidine with alanine. This suggests that the enzymatic activity of the AWD protein is necessary for its biological function. The human genes nm23-H1 and nm23-H2 encode NDP kinase A and B subunits, respectively. The protein subunit encoded by either human nm23 gene is 78% identical to that encoded by the Drosophila awd gene. Transgenes that have the 750-bp awd upstream regulatory DNA fused to human nm23-H2 cDNA but not to nm23-H1 cDNA can rescue the imaginal disc phenotype and the zygotic lethality caused by homozygosis for an awd null mutation as efficiently as an awd transgene. However, rescue of female sterility requires twice as much nm23-H2 expression as awd expression. This implies that the enzymatic activity of the AWD protein is not sufficient for its biological function. The biological function may require nonconserved residues of the AWD protein that allow it to interact with other proteins.
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PMID:The enzymatic activity of Drosophila AWD/NDP kinase is necessary but not sufficient for its biological function. 880 30

The Drosophila abnormal wing discs (awd) gene encodes the subunit of a protein that has nucleoside diphosphate kinase (NDP kinase) activity. Null mutations of the awd gene cause lethality after puparium formation. Larvae homozygous for such mutations have small imaginal discs, lymph glands, and brain lobes. Neither the imaginal discs nor the ovaries from such null mutant larvae are capable of further growth or normal differentiation when transplanted into suitable host larvae. This null mutant phenotype can be entirely rescued by one copy of a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA. Tissue-specific expression of AWD protein from this rescue transgene is identical to tissue-specific expression of beta-galactosidase from a reporter transgene that has the same regulatory region fused to the bacterial lac Z gene. However, this rescue transgene or reporter transgene expression pattern is only a subset of the endogenous pattern of expression detected by either in situ hybridization or immunohistochemistry. This suggests that awd is normally expressed in some tissues where it is not required. The null mutant phenotype cannot be rescued at all by a transgene that has 750 bp of awd upstream regulatory DNA fused to a full-length awd cDNA with a mutation that eliminates NDP kinase activity by replacement of the active site histidine with alanine. This suggests that the enzymatic activity of the AWD protein is necessary for its biological function. The human genes nm23-H1 and nm23-H2 encode NDP kinase A and B subunits, respectively. The protein subunit encoded by either human nm23 gene is 78% identical to that encoded by the Drosophila awd gene. Transgenes that have the 750-bp awd upstream regulatory DNA fused to human nm23-H2 cDNA but not to nm23-H1 cDNA can rescue the imaginal disc phenotype and the zygotic lethality caused by homozygosis for an awd null mutation as efficiently as an awd transgene. However, rescue of female sterility requires twice as much nm23-H2 expression as awd expression. This implies that the enzymatic activity of the AWD protein is not sufficient for its biological function. The biological function may require nonconserved residues of the AWD protein that allow it to interact with other proteins.
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PMID:The Enzymatic Activity of Drosophila AWD/NDP Kinase Is Necessary but Not Sufficient for Its Biological Function 881 47

Rat myoblast primary cultures were tested as a model for proinsulin synthesis and processing and unregulated insulin delivery for insulin-dependent diabetes mellitus (IDDM) gene therapy. Three human proinsulin cDNA constructs containing genetically engineered furin endoprotease cleavage sites between the B-chain and C-peptide (IFur) and between the C-peptide and A-chain (IIFur) and/or containing a histidine B10 to aspartic acid point mutation were subcloned into a mammalian expression vector (pCMV) containing the cytomegalovirus (CMV) promoter. The altered cleavage sites enable the insulin to be processed by the ubiquitous endoprotease furin. The histidine B10 to aspartic acid mutation creates a more stable form of insulin leading to an increase in insulin accumulation. Myoblasts transfected with a proinsulin cDNA construct mutated at all three sites (pCMV.IFur.IIFur.B10), a construct with only the furin sites (pCMV.IFur.IIFur), and a construct containing only the mutation at the B10 position (pCMV.B10) accumulated 852 +/- 16, 150 +/- 13, and 883 +/- 39 microU (pro)insulin/ml, respectively, in the culture medium during a 48-hr incubation. (Pro)insulin was detected in the culture medium within 2 hr post-transfection. Significant (pro)insulin release continued for 1 week and gradually diminished over a month. Approximately 50% of the proinsulin released from rat myoblasts transfected with pCMV.IFur.IIFur.B10 was completely processed into mature insulin based on densitometric analysis of autoradiographs of gels containing immunoprecipitated 35S-Cys-labeled (pro)insulin. However, only a trace of the proinsulin encoded by pCMV.B10 was processed. In an isolated rat adipocyte [14C]glucose oxidation assay, insulin released from myoblasts transfected with pCMV.IFur.IIFur.B10 was active biologically, displaying more biological activity than normal human insulin. Plasmid expression was studied by transfecting myoblasts with the beta-galactosidase (beta-Gal) gene in pCMV, allowing them to divide and fuse into multinucleated myotubes, followed by staining for beta-Gal. Approximately 80% of myotubes expressed beta-Gal. The results indicate that proinsulin encoded by genetically modified proinsulin cDNA is processed into mature insulin, which is secreted at high levels, making myoblasts a viable target cell for gene therapy of IDDM.
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PMID:Synthesis and processing of genetically modified human proinsulin by rat myoblast primary cultures. 882 70

Although single bacterial recombinant antigens have been used successfully to stimulate individual T-cell clones and elicit recall responses in peripheral lymphocytes, the broader use of molecular cloning systems for the identification of autoantigens recognised by the cellular arm of the immune system has met with only limited success. In a systematic approach to address this problem, a series of bacterial expression vectors were examined for their potential use as cloning vectors to elicit a proliferative response in vitro from a non-obese diabetic (NOD) mouse T-cell clone which recognises the immunodominant ovalbumin epitope (aa 323-339). The use of the vector pRSET, which produces a hexa-histidine tagged fusion protein, was confounded by non-specific responses to bacterial protein contaminants. pGEX, which generates a glutathione-S-transferase hybrid, avoided this problem but suffered from the disadvantage that a universally applicable purification procedure for the hybrid antigen could not be easily developed. A practical screening protocol was developed using the pUEX expression system (beta-galactosidase hybrid) and purification based upon electroelution of the hybrid protein from purified inclusion bodies subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). This system can be used to screen expression libraries for the detection of T-cell epitopes provided that the T-cell clones give low background responses to irrelevant pUEX recombinant proteins. Low abundance antigens may be obtained using this system in combination with subtractive hybridisation to construct cDNA libraries enriched in the target antigen.
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PMID:Development of a procedure for the direct cloning of T-cell epitopes using bacterial expression systems. 884 44


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