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:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of the conserved Asp134 residue in Escherichia coli ribonuclease HI, which is located at the center of the alpha V helix and lies close to the active site, was analyzed by means of site-directed random mutagenesis. Mutant rnhA genes encoding proteins with ribonuclease H activities were screened by their ability to suppress the ribonuclease-H-dependent, temperature-sensitive growth phenotype of E. coli strain MIC3001. Based on the DNA sequences, nine mutant proteins were predicted to have ribonuclease H activity in vivo. All of these mutant proteins were purified to homogeneity and examined for enzymic activity and protein stability. Among them, only the mutant proteins [D134H]RNase H and [D134N]RNase H were shown to have considerable ribonuclease H activities. Determination of the kinetic parameters revealed that replacement of Asp134 by amino acid residues other than asparagine and histidine dramatically decreased the enzymic activity without seriously affecting the substrate binding. Determination of the CD spectra indicated that none of the mutations seriously affected secondary and tertiary structure. The protein stability was determined from the thermal denaturation curves. All mutant proteins were more stable than the wild-type protein. Such stabilization effects would be a result of a reduction in the negative charge repulsion between Asp134 and the active-site residues, and/or an enhancement of the stability of the alpha V helix. These results strongly suggest that Asp134 does not contribute to the maintenance of the molecular architecture but the carboxyl oxygen at its delta 1 position impacts catalysis.
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PMID:Investigating the role of conserved residue Asp134 in Escherichia coli ribonuclease HI by site-directed random mutagenesis. 812 23

The 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of monosaccharides, maltooligosaccharides, and oligosaccharides enzymatically released from asparagine-linked sites in ribonuclease B and fetuin have been investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Use of the matrix 2,6-dihydroxyacetophenone containing diammonium hydrogen citrate (DHAP/DAHC) resulted in predominance of protonated over sodiated pseudomolecular ions of PMP-derivatized oligosaccharides. By comparison, the matrices alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid resulted in predominantly sodiated pseudomolecular ions. In addition, tendencies for fragmentation of PMP-oligosaccharide derivatives were significantly lower with DHAP/DAHC which enabled meaningful data to be obtained in reflector mode, even for samples with high excipient levels. The relative magnitude of the ion signals for PMP-derivatized maltooligosaccharides and ribonuclease B oligosaccharides correlated well with the oligomer distribution apparent by HPLC. PMP-maltohexose was used as an internal standard to quantitate PMP-oligosaccharides from ribonuclease B and asialofetuin in crude derivatization mixtures. A linear relationship was observed between the ratio of the intensities of pseudomolecualr ions and the amount of glycoprotein derivatized. The limit of detection for the major oligosaccharide of each protein was reached with ca. 3 micrograms of glycoprotein but may be further enhanced by optimization of sample handling. PMP derivatives of sialylated fetuin oligosaccharides were readily detected as protonated pseudomolecular ions by linear mode analyses. By comparison, reflector mode analyses revealed substantially reduced magnitudes of protonated pseudomolecular ions and considerable post-source fragmentation of sialic acid residues. The PMP derivatives of fetuin oligosaccharides were also amenable to exoglycosidase treatment as shown by the mass shifts found upon treatment with sialidase.
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PMID:Oligosaccharide characterization and quantitation using 1-phenyl-3-methyl-5-pyrazolone derivatization and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 917 25

The contribution of hydrogen bonding by peptide groups to the conformational stability of globular proteins was studied. One of the conserved residues in the microbial ribonuclease (RNase) family is an asparagine at position 39 in RNase Sa, 44 in RNase T1, and 58 in RNase Ba (barnase). The amide group of this asparagine is buried and forms two similar intramolecular hydrogen bonds with a neighboring peptide group to anchor a loop on the surface of all three proteins. Thus, it is a good model for the hydrogen bonding of peptide groups. When the conserved asparagine is replaced with alanine, the decrease in the stability of the mutant proteins is 2.2 (Sa), 1.8 (T1), and 2.7 (Ba) kcal/mol. When the conserved asparagine is replaced by aspartate, the stability of the mutant proteins decreases by 1.5 and 1.8 kcal/mol for RNases Sa and T1, respectively, but increases by 0.5 kcal/mol for RNase Ba. When the conserved asparagine was replaced by serine, the stability of the mutant proteins was decreased by 2.3 and 1.7 kcal/mol for RNases Sa and T1, respectively. The structure of the Asn 39 --> Ser mutant of RNase Sa was determined at 1.7 A resolution. There is a significant conformational change near the site of the mutation: (1) the side chain of Ser 39 is oriented differently than that of Asn 39 and forms hydrogen bonds with two conserved water molecules; (2) the peptide bond of Ser 42 changes conformation in the mutant so that the side chain forms three new intramolecular hydrogen bonds with the backbone to replace three hydrogen bonds to water molecules present in the wild-type structure; and (3) the loss of the anchoring hydrogen bonds makes the surface loop more flexible in the mutant than it is in wild-type RNase Sa. The results show that burial and hydrogen bonding of the conserved asparagine make a large contribution to microbial RNase stability and emphasize the importance of structural information in interpreting stability studies of mutant proteins.
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PMID:Contribution of a conserved asparagine to the conformational stability of ribonucleases Sa, Ba, and T1. 981 11

Glucosidase I initiates the processing of asparagine (N-) linked glycoproteins by removing the distal alpha1,2-linked glucosyl residue of the tetradecasaccharide Glc(3)Man(9)GlcNAc(2). The gene encoding this enzyme was isolated and its structural organization and promoter activity determined. The major transcript for glucosidase I on northern blot appeared to be 3.1 kb; Southern blotting and DNA sequencing indicated the size of the gene to be 6.8 kb, comprising four exons separated by three introns. The first exon encodes the cytoplasmic tail and transmembrane domain; the fourth encodes the putative catalytic domain of the enzyme. Exon-intron junctions are flanked by consensus splice donor and acceptor sequences. Transcription initiation sites were mapped by primer extension, ribonuclease protection assay and RT-PCR analysis. Primer extension results showed multiple initiation sites at -150, -156, and -272 bp relative to the translation initiation codon ATG. Sequence analysis of 5' flanking region showed no canonical TATA box, a high GC content, Sp1 and ETF binding sites (typical of a housekeeping gene promoter). Also noteworthy, the promoter region contains several generic STAT factor binding sites, one nearly perfect, and two half GR binding elements. Other cis- acting elements recognized by transcription factors such as AP-2, NF-kappaB, estrogen receptor, and progesterone receptor (PR) were also present in the putative promoter region. To determine the promoter activity, a construct encompassing the region between -2114 to -5 bp of the putative promoter was ligated to the chloramphenicol acetyltransferase (CAT) reporter plasmid and transiently transfected into COS 7 cells. CAT assay results clearly show transcriptional activity of the promoter.
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PMID:Genomic organization and promoter activity of glucosidase I gene. 1040 45

Bovine seminal ribonuclease (BS RNase) displays immunosuppressive and antitumor activities on mammalian cells, whereas bovine pancreatic ribonuclease (RNase A) is not cytotoxic. To learn more about the mechanism of BS RNase cytotoxicity, various mutants and hybrid proteins were prepared. A series of RNase A variants substituted with amino acid residues from BS RNase were prepared. Concerning quaternary structure, a significant impact was achieved in the variant TM (Q28L K31C S32C), which forms a dimer joined covalently by two intersubunit disulfide bonds. This variant is more efficient than RNase A but less active than BS RNase. Introduction of cationic residues at positions 55, 62, and 64 or substitution at positions 111 and 113 enhanced the immunosuppressive activity of RNase A but did not confer its antitumor activity. The substitution at positions 28, 31, 32, 55, 62, 64, 111, and 113 in variant T13 exerted the best immunosuppressive and antitumor effect observed among the round of the RNase A variants. Replacement of the active-site histidine residues H12 and H119 with asparagine led to the loss of both catalytic and biological activities. Five previously prepared hybrid enzymes (SRA 1-5), synthesized by introducing 16 amino acid residues from RNase A into BS RNase, exerted the same immunosuppressive activities as did the wild-type BS RNase. However, the substitution at positions 111, 113, and 115 in variant SRA 5 caused a marked decrease in its antitumor effect, indicating that these residues play an important role in antitumor efficiency. A different mechanism of action of RNases on tumor cells and/or on blastogenic transformed lymphocytes has been assumed.
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PMID:Structural changes to ribonuclease A and their effects on biological activity. 1044 19

Cultivated tomato (Lycopersicon esculentum), a self-compatible species, evolved from self-incompatible (SI) species in the genus Lycopersicon following a breakdown of the self-incompatibility system. In order to elucidate the molecular basis of this breakdown in L. esculentum, we first analysed the stylar proteins with an in-gel assay for ribonuclease activity and 2D-PAGE. No S-RNase protein or its activity was detected in the style of L. esculentum. We then introduced the S6-RNase gene from an SI relative, L. peruvianum, into L. esculentum. However, the styles of transgenic plants expressing S6-RNase at levels comparable to those found in the L. peruvianum style were unable to reject self-pollen and L. peruvianum pollen in an allele-specific manner. This indicated that defect in the S-RNase expression was not the sole reason for the loss of self-incompatibility in tomato. The asparagine-rich HT protein, originally identified from the style of Nicotiana alata, is the other stylar factor involved in self-incompatibility reaction. We cloned and sequenced two distinct genes encoding HT-A and HT-B proteins from L. peruvianum (LpHT-A and LpHT-B) and L. esculentum (LeHT-A and LeHT-B). A frame shift mutation in the coding sequence of LeHT-A and a stop codon in the ORF of LeHT-B were found, and no LeHT-B transcript was detected in the style of L. esculentum. The results suggest that the breakdown of self-incompatibility in cultivated tomato is associated with loss-of-function mutations in both S-RNase and HT genes.
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PMID:Cultivated tomato has defects in both S-RNase and HT genes required for stylar function of self-incompatibility. 1187 75

Tourtellotte, Mark E. (University of Connecticut, Storrs), Harold J. Morowitz, and Phil Kasimer. Defined medium for Mycoplasma laidlawii. J. Bacteriol. 88:11-15. 1964.-A defined medium for the pleuropneumonia-like organism Mycoplasma laidlawii B is described in which absolute requirements for coenzyme A and longchain fatty acids were demonstrated. This organism did not require cholesterol or macromolecules of high molecular weight, but did show a growth requirement for peptides. Optimal growth in the basal medium was obtained in the presence of two purified peptides from crystalline ribonuclease, one of which has the amino acid sequence threonine - threonine - glutamine - alanine - asparagine-lysine, and the other lysine-glutamic acid-threonine-alanine-alanine-alanine-lysine. Continuous, but suboptimal, growth was obtained with the single ribonuclease peptide: lysine-glutamic acid-threonine-alanine-alanine-alanine-lysine.
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PMID:DEFINED MEDIUM FOR MYCOPLASMA LAIDLAWII. 1419 75

Poor protein solubility is a common problem in high-resolution structural studies, formulation of protein pharmaceuticals, and biochemical characterization of proteins. One popular strategy to improve protein solubility is to use site-directed mutagenesis to make hydrophobic to hydrophilic mutations on the protein surface. However, a systematic investigation of the relative contributions of all 20 amino acids to protein solubility has not been done. Here, 20 variants at the completely solvent-exposed position 76 of ribonuclease (RNase) Sa are made to compare the contributions of each amino acid. Stability measurements were also made for these variants, which occur at the i+1 position of a type II beta-turn. Solubility measurements in ammonium sulfate solutions were made at high positive net charge, low net charge, and high negative net charge. Surprisingly, there was a wide range of contributions to protein solubility even among the hydrophilic amino acids. The results suggest that aspartic acid, glutamic acid, and serine contribute significantly more favorably than the other hydrophilic amino acids especially at high net charge. Therefore, to increase protein solubility, asparagine, glutamine, or threonine should be replaced with aspartic acid, glutamic acid or serine.
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PMID:Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa. 1717 28

A simple and rapid "one-pot" methylation method to esterify sialic acids and construct a permanent charge was developed for N-linked glycan analysis, which combined complete nonspecific proteolytic digestion and methylation. A mixture of Asn-glycans prepared from Pronase E digestion of the glycoprotein was passed through a cation-exchange column to convert carboxylic acids to the Na+ form before being methylated with methyl iodide. Derivatives could be easily purified with a hydrophilic affinity chromatography cartridge. Mass spectrometry analysis was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and MALDI-TOF/TOF. The mass spectrometric data indicated that carboxylic acids were methylated in addition to the formation of a quaternary ammonium in the amino group of asparagine residues. Three model glycoproteins, including ribonuclease B, ovalbumin, and transferrin, were employed to demonstrate the merits of this technique. Results showed that the stabilization of sialic acid was achieved in addition to the formation of a permanent charge. Compared to the analysis of underivatized N-glycans, detection sensitivity improved approximately 10-fold. The new technique was further evaluated with glycan profiling of serum transferrin and proved to be a sensitive method for the characterizing protein glycosylation.
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PMID:"One-pot" methylation in glycomics application: esterification of sialic acids and permanent charge construction. 1741 Oct 71

This laboratory has introduced a chemical method for residue-specific protein cleavage and has provided a preliminary assessment of the suitability of microwave accelerated acid cleavage as a proteomic tool. This report is a continuing assessment of the fate of common protein modifications in microwave-accelerated acid cleavage. We have examined the cleavage of ribonuclease A and the related N-linked glycoprotein ribonuclease B, and the O-linked glycoprotein alpha crystallin A chain, using MALDI-TOF and LC-ESI-MS to identify the peptide products. RNase A and B each contain four disulfide bonds, and the addition of a reducing reagent, such as dithiothreitol, was found to be required to achieve efficient acidic proteolysis. The linkage of the glycosidic group to the asparagine side-chain in ribonuclease B was found not to be cleaved by brief microwave treatment in 12.5 % acetic acid. The distribution of the heterogeneous carbohydrate side chain in the glycopeptide products of acid cleavage was compared to that of the glycopeptide products of tryptic digestion. Hydrolysis within the carbohydrate chain itself is minimal under the conditions used. The O-linked side-chain on alpha crystalline A was found to be cleaved during acid cleavage of the protein.
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PMID:Extension of microwave-accelerated residue-specific acid cleavage to proteins with carbohydrate side chains and disulfide linkages. 1995 38


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