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)

The performance of an electrospray (ESI) source was evaluated and compared to that of a coaxial continuous-flow fast-atom bombardment source on a magnetic mass spectrometer using ten different peptides over the mass range of 400 to 3500 Da and using a variety of scanning modes. Results show that sensitivities using the two ionization techniques are similar, with limits-of-detection in the attomole to low femtomole range. In addition, proteins can be routinely detected using ESI at femtomole levels. The observance of the noncovalent complex of RNase A and cytidine 2'-monophosphate yields evidence that these complexes can be studied using instruments that operate at high accelerating voltages.
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PMID:Coaxial continuous-flow fast-atom bombardment vs electrospray ionization: a sensitivity comparison on a magnetic sector instrument. 856 17

S-Ribonucleases (S-RNases) that cosegregate with S-alleles in the styles of solanaceous and rosaceous plants are associated with gametophytic self-incompatibility (GSI). The amino acid sequences of many S-RNases have been derived from cDNA sequences, but the state of half-cystines has not been clarified. We report the locations of the two free cysteine residues and four disulfide bridges of tobacco S6-RNase and of the four disulfide bridge of Japanese pear S4-RNase. The protein was first S-pyridylethylated at a low pH to selectively modify the free cysteines without thiol-disulfide exchange. The S-pyridylethylated protein (PE-protein) was digested with Achromobacter protease I (API) at pH 6.5 then analyzed by liquid chromatography/electrospray-ionization mass spectrometry (LC/ESI-MS). This analysis showed that tobacco S6-RNase has two free cysteine residues, Cys77 and Cys95, and four disulfide bonds at Cys16-Cys21, Cys45-Cys94, Cys153-Cys182 and Cys165-Cys176. Similarly, four disulfide bonds were identified for pear S4-RNase, which bears no free cysteine, at Cys15-Cys22, Cys48-Cys92, Cys156-Cys195 and Cys172-Cys183. The eight cysteines forming these four disulfide bonds are conserved in all the known S-RNases, indicative that these cross-links are important in stabilizing the tertiary structures of the self-incompatibility-associated glycoproteins in the solanaceous and rosaceous families. The LC/ ESI-MS analysis also provided some structural informations regarding sugar chains attached to the S-RNases.
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PMID:Location of cysteine and cystine residues in S-ribonucleases associated with gametophytic self-incompatibility. 902 90

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

S-RNases are the stylar products of the self-incompatibility (S)-locus in solanaceous plants (including Nicotiana alata), and as such, are involved in the prevention of self-pollination. All cDNA sequences of S-RNase products of functional S-alleles contain potential N-glycosylation sites, with one site being conserved in all cases, suggesting that N-glycosylation is important in self-incompatibility. In this study, we report on the structure and localization of the N-glycans on the S7-allele RNase of N. alata. A total of nine N-glycans, belonging to the high-mannose- and xylosylated hybrid-classes, were identified and characterized by a combination of electrospray-ionization mass-spectrometry (ESI-MS), 1H-NMR spectroscopy, and methylation analyses. The glycosylation pattern of individual glycosylation sites was determined by ESI-MS of the glycans released from isolated chymotryptic glycopeptides. All three N-glycosylation sites showed microheterogeneity and each had a unique complement of N-glycans. The N-glycosylation pattern of the S7-RNase is significantly different to those of the S1- and S2-RNases.
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PMID:Structure and distribution of N-glycans on the S7-allele stylar self-incompatibility ribonuclease of Nicotiana alata. 956 34

Oligosaccharides derivatized with 4-aminobenzoic acid 2-(diethylamino) ethyl ester (ABDEAE) can be analyzed by ESI (Yoshino, K.; et al. Anal. Chem. 1995, 67, 4028-4031) and MALDI (Takao, T.; et al. Rapid Commun. Mass Spectrom. 1996, 10, 637-640) mass spectrometry. In this study, oligosaccharides derived from the enzymatic cleavage of the sugar chains of glycoproteins ribonuclease B, erythropoietin, and transferrin were subjected to ABDEAE derivatization, prior to analysis on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) for high-resolution mass measurement and a postsource decay (PSD) experiment. In the mass measurement of ABDEAE derivatives, quasi-molecular ion species have been observed in monoisotopic resolution using 2,5-dihydroxybenzoic acid as the matrix from spots that contain 50-200 fmol of sample; in the PSD analyses from the spots contained 500 fmol-1 pmol of sample, the predominant backbone ion series which covers the entire mass range for all the derivatives, the internal ion series which reflect the branched trimannosyl core structure of N-glycans, and the low m/z fingerprint ion of ABDEAE were consecutively observed, permitting structure elucidation of the oligosaccharides. Given the effectiveness of this derivatization in terms of its high sensitivity and resolution with respect to MALDI-TOF MS, current methodology is clearly applicable to the sensitive detection and accurate structural analysis of N-glycans.
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PMID:Structural analysis of oligosaccharides derivatized with 4-aminobenzoic acid 2-(diethylamino)ethyl ester by matrix-assisted laser desorption/ionization mass spectrometry. 982 11

High-performance liquid chromatography with on-line electrospray ionization mass spectrometry (ESI-LC/MS) was investigated for the analysis of carbohydrate heterogeneity using RNase B as a model glycoprotein. Oligosaccharides released from RNase B with endoglycosidase H were reduced and separated on a graphitized carbon column (GCC). GCC-HPLC/MS in the positive-ion mode was successful in the identification of one Man5GlcNAc, three Man6GlcNAc, three Man7GlcNAc, three Man8GlcNAc, one Man9GlcNAc, and an oligosaccharide having six hexose units (Hex) and two N-acetylhexosamine units (HexNAc). The branch structures of the three Man7GlcNAc isomers were determined by liquid chromatography with tandem mass spectrometry (LC/MS/MS). LC/MS/MS analysis was shown to be useful for the detection and identification of a trace amount of Hex6HexNAc2 alditol as a hybrid-type oligosaccharide. Its structure was confirmed by the combination of LC/MS with enzymatic digestion using beta-galactosidase and N-acetyl-beta-glucosaminidase. The relative quantities of high-mannose-type oligosaccharides in RNase B detected by ESI-LC/MS are in reasonable agreement with those by UV, high-pH anion-exchange chromatography with pulsed amperometric detection, fluorophore-assisted carbohydrate electrophoresis. Our results indicate that LC/MS and LC/MS/MS can be utilized to elucidate the distribution of oligosaccharides and their structures, which differ in molecular weight, sugar sequence, and branch structure.
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PMID:Analysis of carbohydrate heterogeneity in a glycoprotein using liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry. 1022 1

Mutation of the transthyretin (TTR) plasma protein and gene in a Japanese patient with amyloid polyneuropathy was investigated by electrospray ionization mass spectrometry (ESI-MS) and nonisotopic RNase cleavage assay (NIRCA), respectively. ESI-MS analysis showed normal TTR peaks and additionally a variant TTR with 12-dalton-higher molecular weight than normal TTR. NIRCA suggested that the mutation existed near either the 5' or 3' end of exon 3. Direct DNA sequencing revealed both a normal ACC (threonine) and a variant ATC (isoleucine) at codon 49, which was located near the 5' end of exon 3. The molecular weight shift of this mutation was 12 D, consistent with the result of ESI-MS.
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PMID:Identification of a new transthyretin variant (Ile49) in familial amyloidotic polyneuropathy using electrospray ionization mass spectrometry and nonisotopic RNase cleavage assay. 1043 78

The modification of ribonuclease A with the heterobifunctional cross-linker, 4-succinimdyloxycarbonyl-methyl-alpha-[2-pyridyldithio]-toluene (SMPT) is described. RNase A has 11 potential sites of modification by the SMPT reagent. Tracking the two-dimensional separation and proteolytic digestion of SMPT-modified RNase A with ESI/FTICR-MS and HPLC/ESI/QIT-MS demonstrates the detailed information about number of SMPT modifications and sites of modification that can be obtained by application of these techniques. Analysis of native and modified RNase A tryptic digests by ESI/FTICR-MS resulted in the identification of the sites of modification. Semiquantitative results of the reactivity of certain lysine residues toward the coupling reagent SMPT are presented. Two sites (lysines 1 and 37) are highly reactive, while three sites (lysines 41, 61, and 104) appear to be unreactive toward SMPT under the conditions used. Experimental results demonstrate that quantitative comparison of relative intensities of peptide sequences of different charge states is not possible. No correlation was found between number of basic residues and sensitivity to detection. Digestion of the modified and unmodified RNase A by subtilisin followed by examination by HPLC/ESI/QIT-MS and MS(n) enabled further investigation of modification on lysines 1 and 7, including modification at the epsilon- and alpha-amino positions on lysine 1.
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PMID:Bioconjugation of ribonuclease A: a detailed chromatographic and mass spectrometric analysis of chemical modification by a cross-linking reagent. 1072 95

The characterization of high-mannose-type N-glycosylation by capillary electrophoresis-electrospray mass spectrometry (CE-ESI MS) was described. In addition to the use of a cationic noncovalent capillary coating, strong acidic buffer, and charge reversal to increase the glycoform resolving power, N-glycosidase F (PNGase F) combined with a basic protease and alpha-mannosidase combined with an acidic protease were used to analyze the high-mannose-type N-glycosylation in ribonuclease B (RNase B) and in a novel C-type lectin from the venom of Trimeresurus stejnegeri (TSL). The structures of oligosaccharide, glycosylation sites, and glycoform distributions were determined simultaneously, and the differential oxidation of Met residues in glycopeptides obtained from TSL protease digestion was also characterized successfully by CE-MS/MS. The results showed that the oligosaccharide attached to RNase B has a structure of GlcNAc2Man5 approximately 9, and that attached to TSL has a structure of GlcNAc2Min5 approximately 8. The glycoform distributions in these glycoproteins are quite different, with the GlcNAc2Man5 type predominant in RNase B, and the GlcNAc2Man8 type, in TSL This method may be useful not only for the characterization of glycosylation sites and glycan structures, but also for the determination of the relative abundance of individual glycoforms.
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PMID:Capillary electrophoresis-electrospray mass spectrometry for the characterization of high-mannose-type N-glycosylation and differential oxidation in glycoproteins by charge reversal and protease/glycosidase digestion. 1179 56

Electrospray ionization mass spectrometry (ESI-MS) has proven to be an extremely powerful tool for studying biomolecular structures and noncovalent interactions. Here we report a method using a fully automated, chip-based nanoESI-MS system to determine the dissociation constants (Kd) for the complexes of two different proteins with their ligands. The automated nanoelectrospray system, consisting of the NanoMate and ESI chip, serves functionally as a combination of autosampler and nanoelectrospray ionization source. This system provides all the advantages of conventional nanoelectrospray plus automated, high-throughput analyses without carryover. The automated nanoESI system was used to investigate quantitative noncovalent interactions between ribonuclease A (RNase A) and cytidylic acid ligands (2'-CMP, CTP), a well-characterized model protein-ligand complex, and between an inactive endocellulase mutant (Thermobifida fusca Cel6A D117Acd) and four oligosaccharide ligands (cellotriose, cellotetraose, cellopentaose, cellohexaose). Both titration and competitive binding approaches were performed prior to automated nanoESI-MS analysis with a Q-TOF mass spectrometer. Dissociation constants for each complex were calculated from the sum of ion peak areas of free and complexed proteins during the titration and competition experiments. The measured Kd values for the RNase A-CMP and Cel6A D117Acd-G3 complexes were found to be in excellent agreement with the available published values obtained by standard spectroscopic titration techniques. To our knowledge, this is the first report of using an ESI-MS approach to study the interactions between a cellulase and oligosaccharides. The results provide new insights for understanding the nature of cellulase-cellulose interactions.
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PMID:Quantitative determination of noncovalent binding interactions using automated nanoelectrospray mass spectrometry. 1296 45


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