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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Complete primary structure of an extracellular low molecular mass ribonuclease of Bacillus thuringiensis was determined using Edman degradation and mass-spectrometry analysis of individual peptides obtained after hydrolysis of the protein by cyanogen bromide and staphylococcal protease. The peptides were isolated and purified by HPLC and denaturing PAGE. The enzyme consists of 109 amino acid residues (Asp 8, Asn 6, Thr 6, Ser 10, Glu 3, Gln 1, Pro 3, Gly 9, Ala 12, Val 7, Ile 7, Leu 7, Tyr 7, Phe 4, His 1, Arg 10, Trp 3 and Lys 5) and has a molecular weight of 12182 Da. A single difference was detected between primary structures of the enzyme and an extracellular ribonuclease of B. intermedius.
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PMID:[Complete primary structure of Bacillus thuringiensis extracellular ribonuclease]. 825 Sep 78

Systematic replacement of the amino acid residues in Escherichia coli ribonuclease HI with those in the thermophilic counterpart has revealed that two mutations, His62-->Pro (H62P) and Lys95-->Gly (K95G), increased the thermostability of the protein. These single-site mutant proteins, together with the mutant proteins His62-->Ala (H62A), Lys95-->Asn (K95N) and Lys95-->Ala (K95A), were crystallized and their structures were determined at 1.8 A resolution. The crystal structures of these mutant proteins reveal that only the local structure around each mutation site is essential for the increase in thermostability. For each mutant protein, the stabilization mechanism is considered to be as follows: (i) H62P is stabilized because of a decrease in the entropy of the unfolded state, without a change in the native backbone structure; (ii) K95G is stabilized since the strain caused by the left-handed backbone structure in the typical 3:5 type loop is eliminated; and (iii) K95N is slightly stabilized by a hydrogen bond formed between the side-chain N delta-atom of the mutated aspargine residue and the main-chain carbonyl oxygen within the same residue.
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PMID:Structural study of mutants of Escherichia coli ribonuclease HI with enhanced thermostability. 838 58

The crystal structure of Thermus thermophilus RNase H was determined at 2.8 A resolution. The structure was solved by the molecular replacement method, based on the accurately refined structure of Escherichia coli RNase HI, which shows 52% amino acid sequence identity. Crystallographic refinement led to an R-factor of 0.205, with a 0.019 A root-mean-square deviation from ideal bond lengths and 0.048 A from ideal bond angle distances. Structural comparison shows a striking similarity in the overall folding of the thermophilic and mesophilic enzymes. The root-mean-square displacement is 0.95 A between equivalent alpha-carbon atoms from all elements of secondary structure (five alpha-helices and five beta-strands). However, some notable differences, which account for the enhanced thermostability of T. thermophilus RNase H, are observed in loop structures and side-chain conformations. The substitution of Gly for the left-handed helical residue (Lys95) in the E. coli enzyme is proposed to substantially enhance the thermostability, due to the release of steric hindrance caused by the beta-carbon atom. Furthermore, it is likely that the expansion of an aromatic cluster, arising from the replacement of Ile78 in the mesophilic enzyme by Phe, and the increased number of salt-bridges additively contribute to the stability.
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PMID:Crystal structure of ribonuclease H from Thermus thermophilus HB8 refined at 2.8 A resolution. 838 28

The insertion of a Gly residue (designated as Gly-80b) between the C-cap of the alpha II-helix (Gln-80) and the N-cap of the alpha III-helix (Trp-81) in Escherichia coli ribonuclease HI enhances the protein stability by 0.4 kcal/mol in delta G (Kimura, S., Nakamura, H., Hashimoto, T., Oobatake, M., & Kanaya, S. (1992) J. Biol. Chem. 267, 21535-21542). Another mutation within the alpha II-helix, Gly-77-->Ala, reduces the stability by 0.9 kcal/mol. Simultaneous introduction of these mutations enhances the stability by 0.8 kcal/mol, indicating that the effects of these mutations are cooperative and not simply independent. We determined the crystal structures of these three mutant proteins (G80b-, A77-, and A77/G80b-RNase H) to investigate this cooperative mechanism of the protein stabilization. The structures revealed that the inserted Gly-80b assumes a left-handed helical conformation in both the G80b- and the A77/G80b-RNase H. This inserted glycine residue allows the formation of a "paperclip", which is a common motif at the C-termini of alpha-helices. Accompanying the formation of the paperclip motif, two intrahelical hydrogen bonds are formed between the backbone atoms (O78-N80b and O80b-N84). The stabilization caused by the insertion of Gly-80b can be ascribed to the formation of these hydrogen bonds. The Gly-77-->Ala substitution destabilizes the protein due to the deformed packing interactions in the hydrophobic core around Ala-77 and the stress in the wedged indole ring of Trp-81. These effects are alleviated by the insertion of Gly-80b, which relaxes the backbone structure.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cooperative stabilization of Escherichia coli ribonuclease HI by insertion of Gly-80b and Gly-77-->Ala substitution. 839 6

Because the ferric uptake regulator (fur) appears to be an essential gene in Pseudomonas aeruginosa, resistance to manganese was used as an enrichment to isolate strains carrying point mutations in the fur gene in order to assess its role in the co-ordinate expression of siderophores and exotoxin A (ETA). This report describes a detailed molecular and phenotypic characterization of four mutants and one revertant, which carry point mutations in the fur gene. Two parental strains were used in this study. Three mutants were isolated from the widely used strain, PAO1. One of these, CS (cold sensitive), has a mutation in the 5' non-coding region of the fur gene while the two other mutants derived from this parent have mutations resulting in the following deduced changes in Fur: mutant A2, H86-->R; mutant A4, H86-->Y. The other mutant (C6) and its revertant (C6Rv) were derived from PAO6261, a mutant of PAO1 with a deletion in the anr gene (anaerobic regulation of arginine deiminase and nitrate reduction) that controls anaerobic respiration in P. aeruginosa. Fur from the C6 mutant has an A10-->G mutation while in the C6Rv spontaneous revertant the mutant Gly residue has been changed to Ser at this position. All mutants were examined for alterations in the iron-regulated expression of siderophores and ETA. The A2 and A4 mutants expressed higher levels of siderophores in iron-deficient media and in iron-replete media. The CS mutant constitutively expressed siderophores at 25 degrees C. At 42 degrees C siderophore biosynthesis was iron repressed as in the parental strain PAO1. The deletion of anr in PAO6261 had no apparent effect on the iron-mediated regulation of siderophore synthesis, but the C6 mutant derived from this strain produces siderophores constitutively. The iron-regulated production of siderophores by C6Rv was similar to the parental strain PAO6261 and PAO1. Because one of the parental strains used in this study is an Anr mutant, regulation of ETA production was assessed under aerobic and microaerobic conditions. Iron-dependent repression of ETA synthesis in both parental strains and A2 and A4 mutants was found to be 50-100-fold under aerobic and microaerobic conditions, as assayed by quantitative Western dot-blot assays. By contrast in the CS and C6 mutants, while iron-dependent repression os ETA synthesis was similar to both parental strains under aerobic conditions, ETA production in these mutants was constitutive in a microaerobic environment. RNase protection analysis of toxA and regAB transcription in PAO1, PAO6261 and the C6 mutant corroborated the results of quantitative dot-blot assays of ETA. The mutant Fur proteins were purified and examined for their ability to bind to the promoter of a gene (pvdS) that positively regulates the expression of siderophores and ETA. Fur from the A2 and A4 mutants and from the C6Rv revertant was able to bind to the target DNA, but with reduced affinity by comparison to wild-type Fur. Fur from the C6 mutant in DNase I footprint experiments failed to protect the promoter region of the pvdS gene, but it retained some weak binding activity in gel mobility shift assays. The data presented in this study not only furnish some additional insights into the structure-function relationships of Fur, but also afford novel perspectives of virulence factors in P. aeruginosa under environmental conditions that have not previously been considered.
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PMID:Ferric uptake regulator mutants of Pseudomonas aeruginosa with distinct alterations in the iron-dependent repression of exotoxin A and siderophores in aerobic and microaerobic environments. 888 70

The human and murine pregnancy-specific glycoprotein (PSG) gene families encode a large number of closely related proteins which are abundantly expressed in the fetal trophoblast and secreted into the maternal circulation. Although the presence of a well conserved tripeptide sequence His or Arg-Gly-Asp or Glu or Lys (H/RGD/E/K) similar to the RGD motif found in extracellular matrix proteins hints towards a possible interaction with integrin-type receptors, the function of this group of proteins related to the carcinoembryonic antigen family is still unknown. It is also not clear whether the various members of the PSG family exert the same function. Here we describe the cloning of two splice variants of Cea4 (Cea4a, Cea4b), a murine PSG family member, which lacks the RGD-related consensus motif. Cea4a, like most of the other rodent PSG members, is composed of three immunoglobulin (Ig) variable-like domains (N1-N3) and and one Ig constant-like domain (A). In contrast, Cea4b lacks the N2 domain (N1N3A), demonstrating for the first time that PSG isoforms produced by alternative splicing also exist in mice. The mRNAs coding for Cea4a and Cea4b exhibit the same expression kinetics during placental development as found for two other murine PSGs, Cea2 and Cea3, which contain the RGD-like motif. Expression starts after day 12.5 of embryonic development (E12.5) and maximum steady-state levels are reached around E15.5-E17.5 as determined by RNase protection analyses. At E17.5, PSG transcripts can be detected exclusively in the spongiotrophoblast of the placenta. In addition, PCR analyses revealed that Cea2, Cea3, and Cea4 transcripts are also found in RNA from a pool of embryos (E12-E15) but are absent from a number of adult tissues tested (kidney, lung, testis, ovary, liver, brain, thymus, heart, spleen). These results indicate that the various PSG isoforms exert their function(s) at the same time during placental and embryonic development.
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PMID:Coordinate expression of splice variants of the murine pregnancy-specific glycoprotein (PSG) gene family during placental development. 897 44

Radiation-induced formation of 3,4-dihydroxyphenylalanine (DOPA) in Tyr and Tyr-containing peptides and proteins was investigated as a function of X-irradiation dose. Irradiated Tyr (0-30 Gy) and the acid hydrolysates of irradiated peptide and protein (0-240 Gy) were conjugated with dansyl chloride. The dansylated amino acids were analyzed by reversed-phase HPLC using fluorescence detection. Formation of DOPA, determined by integrated peak area, increased with dose. Analysis of the major product from irradiated tripeptide Tyr-Gly-Gly detected Gly and DOPA (2:1). Extension of the model study to irradiated BSA and RNase A showed correlation of DOPA formation with Tyr modification up to 120 Gy. Higher dose induced further transformation of DOPA. The fluorescence signal of dansylated DOPA was linear from 1.5 nmol to 0.5 pmol (correlation coefficient of 0.999, n = 3). The detection limit allows the detection of 1 molecule of DOPA/300 molecules of BSA in 5 micrograms of dansylated hydrolysate. Most standard amino acid analysis techniques are limited to detect normal residues of protein. Protein-bound DOPA has been suggested to have a role in the replenishment of reduced transition metal ion involved free-radical-generating system in vivo. Sensitive analysis of protein-bound DOPA will be useful to study amplification of the radical-damaging event.
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PMID:Radiation-induced formation of 3,4-dihydroxyphenylalanine in tyrosine-containing peptides and proteins as a function of X-irradiation dose. 909 57

Escherichia coli ribonuclease HI has a cavity within the hydrophobic core. Two core residues, Ala52 and Val74, resided at both ends of this cavity. We have constructed a series of single mutant proteins at Ala52, and double mutant proteins, in which Ala52 was replaced by Gly, Val, Ile, Leu, or Phe, and Val74 was replaced by Ala or Leu. All of these mutant proteins, except for A52W, A52R, and A52G/V74A, were overproduced and purified. Measurement of the thermal denaturations of the proteins at pH 3.2 by CD suggests that the cavity is large enough to accommodate three methyl or methylene groups without creating serious strains. A correlation was observed between the protein stability and the hydrophobicity of the substituted residue. As a result, a number of the mutant proteins were more stable than the wild-type protein. The stabilities of the mutant proteins with charged or extremely bulky residues at the cavity were lower than those expected from the hydrophobicities of the substituted residues, suggesting that considerable strains are created at the mutation sites in these mutant proteins. However, examination of the far- and near-UV CD spectra and the enzymatic activities suggest that all of the mutant proteins have structures similar to that of the wild-type protein. These results suggest that the cavity in the hydrophobic core of E. coli RNase HI is conformationally fairly stable. This may be the reason why the cavity-filling mutations effectively increase the thermal stability of this protein.
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PMID:Conformational stabilities of Escherichia coli RNase HI variants with a series of amino acid substitutions at a cavity within the hydrophobic core. 922 39

The human epithelial proteinase inhibitor SKALP/elafin and the porcine sodium-potassium ATPase inhibitor SPAI-2 are two highly homologous proteins that share an NH2-terminal transglutaminase substrate domain and a COOH-terminal whey acidic protein (WAP) domain. Here we describe the bovine and simian orthologs of SKALP/elafin as well as two new bovine family members that are designated Trappin-4 and Trappin-5 on the basis of a new nomenclature that we propose (Trappin = TRansglutaminase substrate and WAP motif-containing ProteIN). Sequence analysis of Trappin-4 and Trappin-5 revealed a domain structure that is very similar to SPAI-2 (Trappin-1) and SKALP/elafin (Trappin-2). The transglutaminase substrate motifs are conserved although the number of repeats varies among species and among family members. The sequence of Trappin-4 and Trappin-5 diverges from Trappin-1 and Trappin-2 at the putative reactive site in the WAP domain. The bovine ortholog of Trappin-2 is expressed in tongue and snout epidermis; Trappin-4 is expressed in trachea, ileum, and tongue; and Trappin-5 is expressed at low levels in trachea, as determined by RNase protection and Northern blot analysis. Based on the analysis of 67 transglutaminase substrate repeats as present in all known Trappin gene family members from four different mammalian species a consensus sequence could be established: Gly-Gln-Asp-Pro-Val-Lys (GQDPVK). Using biotinylated hexapeptide probes we found that the GQDPVK sequence is a very efficient transglutaminase substrate both for guinea pig liver transglutaminase and for epidermal transglutaminase, and it acts as acyl donor as well as acceptor. We propose that the Trappin protein family forms a new group of enzyme inhibitors with various specificities of the WAP domain, which share transglutaminase substrate motifs that can act as an anchoring sequence.
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PMID:Identification and sequence analysis of two new members of the SKALP/elafin and SPAI-2 gene family. Biochemical properties of the transglutaminase substrate motif and suggestions for a new nomenclature. 925 57

1. Inhibition of gastric acid secretion by proton pump inhibitors like omeprazole increases the synthesis and secretion of the pyloric antral hormone gastrin. We report here how omeprazole influences the conversion of the gastrin precursor to its final products, and the abundance of mRNAs encoding proteins associated with progastrin processing in rat antral mucosa. 2. Progastrin processing was studied using a pulse-chase protocol in antral mucosa, incubated in vitro, from rats treated with omeprazole for up to 5 days. Labelled peptides were detected by on-line scintillation counting after immunoprecipitation and HPLC. The mRNAs encoding prohormone-processing enzymes were identified by Northern blot, polymerase chain reaction or ribonuclease protection assay, and their cellular origins identified by immunocytochemistry. 3. Cleavage of [3H]- and [35S]-labelled progastrins at Arg-94-95 or Arg-57-58, and amidation at Phe-92 were not influenced by pretreatment with omeprazole. In contrast, cleavage of G34 (the thirty-four amino acid amidated gastrin) at Lys-74-75 to give G17 (the seventeen amino acid amidated gastrin), and of G34-Gly to G17-Gly (G34 and G17 with COOH-terminal glycine), was increased 3-fold after treatment with omeprazole for either 1 or 5 days. 4. Approximately 20% of newly synthesized amidated and Gly-extended gastrins were secreted within 240 min of the labelling period in omeprazole-treated samples, but secretion of labelled gastrins from control tissue was undetectable over a comparable period. 5. The amidating enzyme, peptidyglycine alpha-amidating mono-oxygenase (PAM), the prohormone convertases PC1/3, PC2, PC5 and the PC2 chaperone 7B2 were localized to rat antral gastrin cells by immunocytochemistry. The relative abundance of mRNA species encoding 7B2, PC5 and PAM were unchanged after treatment with omeprazole for 5 days, whereas gastrin, PC1/3 and PC2 mRNAs are known to increase at this time. 6. The main consequence of increased cleavage at Lys-74-75 is the production of G17 and G17-Gly at the expense of G34 and G34-Gly, respectively. The latter have longer plasma half-lives, and so their increased cleavage may serve to limit the rise in plasma gastrin concentration after inhibition of acid secretion. Changes in the abundance of mRNAs encoding prohormone-processing enzymes cannot account for the rapidity of the changes in cleavage of progastrin at Lys residues after omeprazole.
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PMID:Regulation by gastric acid of the processing of progastrin-derived peptides in rat antral mucosa. 926 20


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