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: UNIPROT:Q07644 (polypeptide)
72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The complete amino acid sequence of fibrolase, a fibrinolytic enzyme from southern copperhead (Agkistrodon contortrix contortrix) venom, has been determined. This is the first report of the sequence of a direct-acting, nonhemorrhagic fibrinolytic enzyme found in snake venom. The majority of the sequence was established by automated Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. The amino-terminus is blocked by a cyclized glutamine (pyroglutamic acid) residue, and the sequence of this region of the molecule was determined by mass spectrometry. Fibrolase is composed of 203 residues in a single polypeptide chain with a molecular weight of 22,891, as determined by the sequence. Its sequence is homologous to the sequence of the hemorrhagic toxin Ht-d of Crotalus atrox venom and with the sequences of two metalloproteinases from Trimeresurus flavoviridis venom. Microheterogeneity in the sequence was found at both the amino-terminus and at residues 189 and 192. All six cysteine residues in fibrolase are involved in disulfide bonds. A disulfide bond between cysteine-118 and cysteine-198 has been established and bonds between cysteines-158/165 and between cysteines-160/192 are inferred from the homology to Ht-d. Secondary structure prediction reveals a very low percentage of alpha-helix (4%), but much greater beta-structure (39.5%). Analysis of the sequence reveals the absence of asparagine-linked glycosylation sites defined by the consensus sequence: asparagine-X-serine/threonine.
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PMID:Amino acid sequence of fibrolase, a direct-acting fibrinolytic enzyme from Agkistrodon contortrix contortrix venom. 130 58

The proposal that the active site vacuole of NAD(+)-S-lactate dehydrogenase is unable to accommodate any imbalance in electrostatic charge was tested by genetically manipulating the cDNA coding for human muscle lactate dehydrogenase to make a protein with an aspartic acid introduced at position 140 instead of the wild-type asparagine. The Asn 140-Asp mutant enzyme has the same kcat as the wild type (Asn 140) at low pH (4.5), and at higher pH the Km for pyruvate increases 10-fold for each unit increase in pH up to pH 9. We conclude that the anion of Asp 140 is completely inactive and that it binds pyruvate with a Km that is over 1,000 times that of the Km of the neutral, protonated aspartic-140. Experimental results and molecular modeling studies indicate the pKa of the active site histidine-195 in the enzyme-NADH complex is raised to greater than 10 by the presence of the anion at position 140. Energy minimization and molecular dynamics studies over 36 ps suggest that the anion at position 140 promotes the opening of and the entry of mobile solvent beneath the polypeptide loop (98-110), which normally seals off the internal active site vacuole from external bulk solvent.
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PMID:Charge balance in the alpha-hydroxyacid dehydrogenase vacuole: an acid test. 130 74

We describe a genomic clone encoding the human 5-HT1B receptor. This apparently intronless gene encodes a 390 amino acid polypeptide homologous to the rat 5-HT1B serotonin receptor, with which it shares 93% amino acid sequence identity. Remarkably, [3H]5-hydroxytryptamine binding studies with transfected HeLa cells show that the human 5-HT1B receptor has a pharmacological profile that is markedly different from that of the corresponding rat receptor. Instead, human 5-HT1B drug specificity is highly similar to that of the human 5-HT1D receptor, with which it shares 59% amino acid sequence identity. The human 5-HT1B receptor, like the 5-HT1D receptor, can couple to Gi proteins. The presence of the threonine355 in the human receptor rather than an asparagine, as found in the corresponding rat gene product, may explain much of the marked pharmacological difference between the human and rat 5-HT1B receptors.
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PMID:Molecular cloning and functional characterization of a human 5-HT1B serotonin receptor: a homologue of the rat 5-HT1B receptor with 5-HT1D-like pharmacological specificity. 131 31

The three-dimensional structure of the enzyme myeloperoxidase has been determined by X-ray crystallography to 3 A resolution. Two heavy atom derivatives were used to phase an initial multiple isomorphous replacement map that was subsequently improved by solvent flattening and non-crystallographic symmetry averaging. Crystallographic refinement gave a final model with an R-factor of 0.257. The root-mean-square deviations from ideality for bond lengths and angles were 0.011 A and 3.8 degrees. Two, apparently identical, halves of the molecule are related by local dyad and covalently linked by a single disulfide bridge. Each half-molecule consists of two polypeptide chains of 108 and 466 amino acid residues, a heme prosthetic group, a bound calcium ion and at least three sites of asparagine-linked glycosylation. There are six additional intra-chain disulfide bonds, five in the large polypeptide and one in the small. A central core region that includes the heme binding site is composed of five alpha-helices. Regions of the larger polypeptide surrounding this core are organized into locally folded domains in which the secondary structure is predominantly alpha-helical with very little organized beta-sheet. A proximal ligand to the heme iron atom has been identified as histidine 336, which is in turn hydrogen-bonded to asparagine 421. On the distal side of the heme, histidine 95 and arginine 239 are likely to participate directly in the catalytic mechanism, in a manner analogous to the distal histidine and arginine of the non-homologous enzyme cytochrome c peroxidase. The site of the covalent linkage to the heme has been tentatively identified as glutamate 242, although the chemical nature of the link remains uncertain. The calcium binding site has been located in a loop comprising residues 168 to 174 together with aspartate 96. Myeloperoxidase is a member of a family of homologous mammalian peroxidases that includes thyroid peroxidase, eosinophil peroxidase and lactoperoxidase. The heme environment, defined by our model for myeloperoxidase, appears to be highly conserved in these four mammalian peroxidases. Furthermore, the conservation of all 12 cysteine residues involved in the six intra-chain disulfide bonds and the calcium binding loop suggests that the three-dimensional structures of members of this gene family are likely to be quite similar.
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PMID:X-ray crystal structure of canine myeloperoxidase at 3 A resolution. 132 Jan 28

To identify amino acid residues that influence the assembly or stability of the manganese cluster in photosystem II, we have generated site-directed mutations in the D1 polypeptide of the cyanobacterium, Synechocystis sp. PCC 6803. Indirect evidence has suggested that the D1 polypeptide provides some of the ligands that are required for metal binding. Mutations at position 170 of D1 were selected for characterization, since an aspartate to asparagine mutation (DN170D1) at this position completely abolishes photoautotrophic growth, while retention of a carboxylic acid at this position (aspartate to glutamate, DE170D1) supports photoautotrophic growth. Photosystem II particles were purified from control, DE170D1, and DN170D1 cells by a procedure that retains high rates of oxygen evolution activity in control particles [Noren, G.H., Boerner, R.J., & Barry, B.A. (1991) Biochemistry 30, 3943-3950]. Spectroscopic analysis shows that the tyrosine radical, Z+, which normally oxidizes the manganese cluster, is rapidly reduced in the DE170D1 mutant, but not in the DN170D1 mutant. A possible explanation of this block or dramatic decrease in the rate of electron transfer between the manganese cluster and tyrosine Z is an alteration in the properties of the metal center. Quantitation of manganese in these particles is consistent with aspartate 170 influencing the stability or assembly of the manganese cluster, since the aspartate to asparagine mutation results in a decrease in the manganese content per reaction center. Photosystem II particles from DN170D1 show a 60% decrease in the amount of specifically bound manganese per reaction center, when compared to control particles. Also, we observe a 70% decrease in the amount of specifically bound manganese per reaction center in partially purified DN170D1 particles and at least an 80% decrease in the amount of hydroxylamine-reducible manganese in DN170D1 thylakoid membranes. Single-turnover fluorescence assays and steady-state EPR measurements demonstrate that the remaining, endogenous manganese does not rapidly reduce tyrosine Z+ in the DN170D1 mutant. Additional evidence that aspartate 170 influences the assembly or stability of the metal site comes from analysis of the DE170D1 mutant. Although this mutant assembles a functional manganese cluster, as assessed by oxygen evolution and spectroscopic assays, the properties of the manganese site are perturbed.
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PMID:Evidence from directed mutagenesis that aspartate 170 of the D1 polypeptide influences the assembly and/or stability of the manganese cluster in the photosynthetic water-splitting complex. 132 68

HPV types associated with genital disease are termed "high-risk" or "low-risk" viruses according to their prevalence in cancers. Two viral genes, E6 and E7, are invariably expressed in cervical carcinomas. The E7 gene product has been found to bind the retinoblastoma tumor suppressor protein and to be phosphorylated by casein kinase II. Although present in both high- and low-risk E7 proteins, these activities are diminished in the low-risk HPV-6 E7 polypeptide. To better understand the oncogenic potential of the HPV-6 E7 protein, we replaced four of its amino acids with HPV-16 E7 residues present in the analogous region of the N-terminal half of the protein. Replacement of the arginine at position 4 of the HPV-6 E7 protein with an aspartate present in HPV-16 E7 slowed the mobility of the protein when expressed in vivo. Replacement of the glycine at position 22 with an aspartate resulted in higher affinity for retinoblastoma protein binding. Replacement of valine residues at positions 30 and 37 with asparagine and aspartate, respectively, resulted in higher levels of casein kinase II phosphorylation. The substitution at position 22 was the only mutation that exhibited increased transforming activity, suggesting a correlation between the HPV E7 protein affinity for the retinoblastoma tumor suppressor protein and its ability to transform established cells. Our results show that subtle changes in sequence may result in marked differences in biological activity of HPV oncogenes.
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PMID:Single amino acid substitutions in "low-risk" human papillomavirus (HPV) type 6 E7 protein enhance features characteristic of the "high-risk" HPV E7 oncoproteins. 132 43

Human platelet arylsulphatase A (ASA) exhibits a multiple banding pattern when examined by PAGE. The isoform pattern (IVa) of the enzyme obtained from normal subjects differs from variants (IIIa, IIIb, IVb) which are primarily found in alcoholic patients. Alkaline phosphatase and endo-N-acetylglucosaminidase H treatments, as well as ion-exchange chromatography, demonstrate that the isoforms of ASA arise because of charge heterogeneity caused by phosphoglycan moieties. The isoform with the slowest mobility in PAGE lacks phosphate substituents. Based upon mannose-6-phosphate-receptor affinity chromatography it can be concluded that most, if not all, of the enzyme-linked phosphate is in the form of 6-O-phospho-D-mannosyl units. Lectin affinity chromatography and peptide-N-glycosidase F treatment followed by SDS/PAGE and Western-blot analysis indicate that normal platelet ASA contains two oligomannose and/or hybrid glycan moieties of which one, or both, possess a 6-O-L-fucosyl substituent on the asparagine-linked N-acetylglucosamine residue. Comparative analysis indicates that the variant IIIa and IIIb types of ASA differ from the IVa ASA with regard to the level of glycan phosphorylation and glycan structure, as well as the polypeptide structure.
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PMID:The structural basis for the electrophoretic isoforms of normal and variant human platelet arylsulphatase A. 135 79

Mevalonic aciduria is the first proposed inherited disorder of the cholesterol/isoprene biosynthetic pathway in humans, and it is presumed to be caused by a mutation in the gene coding for mevalonate kinase. To elucidate the molecular basis of this inherited disorder, a 2.0-kilobase human mevalonate kinase cDNA clone was isolated and sequenced. The 1188-base pair open reading frame coded for a 396-amino acid polypeptide with a deduced M(r) of 42,450. The predicted protein sequence displayed similarity to those of galactokinase and the yeast RAR1 protein, indicating that they may belong to a common gene family. Southern hybridization studies demonstrated that the mevalonate kinase gene is located on human chromosome 12 and is a single copy gene. No major rearrangements were detected in the mevalonic aciduria subject. The relative size (2 kilobases) and amounts of human mevalonate kinase mRNA were not changed in mevalonic aciduria fibroblasts. Approximately half of the mevalonic aciduria cDNA clones encoding mevalonate kinase contained a single base substitution (A to C) in the coding region at nucleotide 902 that changed an asparagine residue to a threonine residue. The presence of this missense mutation was confirmed by polymerase chain reaction amplification and allele-specific hybridization of the genomic DNAs from the proband and the proband's father and brother. Similar analysis failed to detect this mutation in the proband's mother, seven normal subjects, or four additional mevalonic aciduria subjects, indicating that the mutation does not represent a common gene polymorphism. Functional analysis of the defect by transient expression confirmed that the mutation produced an enzyme with diminished activity. Our data suggest that the index case is a compound heterozygote for a mutation in the mevalonate kinase gene.
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PMID:Molecular cloning of human mevalonate kinase and identification of a missense mutation in the genetic disease mevalonic aciduria. 137 80

A human yolk sac tumor cell line, TG1, which was established from a testicular yolk sac tumor, was found to replicate continuously in a chemically defined medium supplemented with Na2SeO3 (ISRPMI). TG1 produced several plasma proteins and growth factors: albumin, alpha-fetoprotein (AFP), ferritin, carcinoembryonic antigen, beta-2-microglobulin, polyamine, neuron specific enolase, tissue polypeptide antigen, transferrin (Tf), epidermal growth factor, and platelet derived growth factor. By analysis of lentil lectin (LcHA)-affinity electrophoresis, to examine the microheterogeneity of carbohydrate chains of synthetic glycoproteins, TG1 cells cultured with ISRPMI produced only LcHA reactive Tf and AFP based on core fucose attached to asparagine-linked N-acetylglucosamine residues instead of LcHA-nonreactive Tf and AFP produced by TG1 cells cultured with fetal bovine serum (FBS)-containing medium. alpha 1-6 Fucosyltransferase activity was significantly greater in the TG1 cells cultured with ISRPMI (39.9 +/- 1.5 pmol.h-1.mg-1 protein) than cultured with FBS-containing media (18.2 +/- 1.2 pmol.h-1.mg-1 protein). These results have indicated that the selective increase of alpha 1-6 fucosyltransferase occurred when the cells were cultured with the FBS-free synthetic media.
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PMID:Growth of a human yolk sac tumor cell line with yolk sac-derived functions in selenium-supplemented chemically defined synthetic medium. 137 30

The gene PHO5 coding for one of the repressible acid phosphatases of the yeast Saccharomyces cerevisiae has been expressed at high efficiency in the baby hamster kidney (BHK) cell line. The expression vector was constructed from PHO5 driven by the human beta-actin promoter and was transfected into BHK cells by the calcium phosphate method. The recombinant APase (r-APase) which was secreted in active form from the cells was estimated by SDS/polyacrylamide gel electrophoresis to have molecular mass M(r) = 62,000, indicating substitution of the polypeptide moiety by 2-3 asparagine-linked glycans. Analysis by sequential lectin affinity chromatography of glycopeptides obtained from r-APase with Pronase showed that the glycans are predominantly of the 2.2.4 triantennary and tetraantennary complex-type. These data suggest that the extensive glycosylation of yeast APase, which contains eight polymannose substituents, is not essential for secretion and expression of enzymatic activity of the transfected gene product.
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PMID:Expression, glycosylation and secretion of yeast acid phosphatase in hamster BHK cells. 139 64


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