Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.3 (RNase T1)
 1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. When ribonuclease T1 [EC 3.1.4.8] was treated with trypsin [EC 3.4.21.4] at pH 7.5 and 37 degrees, activity was lost fairly slowly. At higher temperatures, however, the rate of inactivation was markedly accelerated. The half life of the activity was about 2.5 h at 50 degrees and 1 h at 60 degrees. 3'-GMP and guanosine protected the enzyme significantly from tryptic inactivation. 2. Upon tryptic digestion at 50 degrees, the Lys-Tyr (41-42) and Arg-Val (77-78) bonds were cleaved fairly specifically, yielding two peptide fragments. One was a 36 residue peptide comprizing residues 42 to 77. The other was a 68 residue peptide composed of two peptide chains cross-linked by a disulfide bond between half-cystines -6 and -103, comprizing residues 1 to 41 and 78 to 104. 3. When the trinitrophenylated enzyme, in which the alpha-amino group of alanine-1 and the episolone-amino group of lysine 41 were selectively modified, was treated with trypsin at 37 degrees, the activity was lost fairly rapidly with a half life of about 4 h. In this case, tryptic hydrolysis occurred fairly selectively at the single Arg-Val bond. Thus the enzyme could be inactivated by cleavage of a single peptide bond in the molecule, an indication of the importance of the peptide region involving the single arginine residue at position 77 in the activity of ribonuclease T1.
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PMID:The structure and function of ribonuclease T1. XXII. Tryptic cleavages of the single lysyl and arginyl bonds in ribonuclease T1. 19 42

Ribonuclease T1 [EC 3.1.4.8] was selectively hydrolyzed by digestion with trypsin at the peptide bond of the single arginine residue at position 77. Selective hydrolysis was achieved by blocking the xi-amino group of Lys-41 with 2-methoxy-5-nitrotropone and subsequent digestion with trypsin in the presence of 2M urea. The trypsin-digested ribonuclease T1 was composed of two polypeptide chains containing 77 and 27 residues, though the two chains were covalently linked by a disulfide bond between Cys-6 and Cys-103. The modified enzyme lost enzymatic activity toward RNA and the ability to bind to 3'-GMP. The circular dichroic spectrum of the modified protein suggested that its conformation was extensively destroyed. It is concluded from the present results that the continuity of the peptide chain at the arginine residue is extremely important for maintaining the active conformation of the enzyme protein and for the enzymatic function of ribonuclease T1.
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PMID:Preparation and properties of trypsin-digested ribonuclease T1 split at the single arginyl peptide bond. 82 Jun 95

The nature of the interaction between the RNA and the protein component in the yeast 5 S rRNA-L1a complex was assessed using fluorescence and controlled proteolytic and RNase digestion. (a) Influence of L1a on the RNA conformation was monitored by ethidium fluorescence and controlled RNase T1 digestion. The complex was digested with alpha-chymotrypsin, Staphylococcus aureus protease V8, subtilisin, or trypsin. Both termini of L1a in the complex were readily accessible to proteases. Proteolytic digestion of the complex resulted in a reduction in fluorescence intensity if ethidium was added after proteolysis. No change was observed when ethidium was allowed to react with the complex prior to proteolysis. Neither the rate of proteolysis nor the resultant peptide pattern was affected by the presence of ethidium. T1 digestion of intact RNP and trypsin-treated RNP produced different oligonucleotide patterns. Both the fluorescence and the T1 digestion data suggest that the conformation of the RNA moiety was influenced by the protein. (b) Influence of the RNA molecule on L1a conformation in the complex was monitored by limited proteolysis. Whereas the protein in the complex was relatively sensitive to proteases, free protein was completely resistant to digestion under identical conditions. The trypsin sensitivity of L1a in complexes containing different truncated 5 S RNA molecules was studied also. Upon removal of residues 31-49 of the 5 S RNA molecule, L1a in the complex became resistant to proteolysis. These results are interpreted in a model in which specific regions of both the RNA and the protein are involved in the interaction.
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PMID:Probing the yeast 5 S RNA-protein complex by fluorescence and controlled proteolytic digestion. 240 92

High-performance liquid chromatography on an Asahipak GS-320 column using isocratic elution with 0.1 M acetic acid has proven effective for fractionation of peptides of molecular weights lower than 3000. This technique enabled the separation of the peptides derived from digestion of native ribonuclease F1 by trypsin and chymotrypsin in combination with conventional gel filtration through Sephadex G-25 and reversed-phase HPLC. Amino acid analysis of the cystine-containing peptides thus obtained revealed the disulfide linkages Cys-6-Cys-102 and Cys-24-Cys-84 in this protein. The behavior of a number of peptides in the HPLC on an Asahipak GS-320 column is described and the separation mechanism is discussed.
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PMID:Peptide fractionation by high-performance liquid chromatography on an Asahipak GS-320 column: application to determination of the disulfide pairings in ribonuclease F1. 310 81

Extracellular RNase Fl1 has been purified from the culture filtrate of Fusarium lateritium. The enzyme has been obtained in the electrophoretically homogeneous state with the yield about 90% and 300 fdd degree of purification. RNase Fl1 is a guanyl specific enzyme (EC 3.1.27.3) with the specific activity on RNA 1420 units/mg of protein. The total primary structure of the RNase has been determined by the automated Edman degradation of two non-fractionated peptide hydrolysates produced by trypsin and Staphylococcus aureus protease and of the hydroxylamine cleavage products of the protein. It was shown that hydroxylamine converts the RNase Fl1 N-terminal residue, pyroglutamic acid, into the hydroxyamic acid derivative sensitive to Edman degradation. RNase Fl1 consists of 105 amino acid residues (Mr 10,852) and is a structural homologue of the Fus. moniliforme RNase F1, differing from the latter by 15 amino acid substitutions outside the enzyme active site.
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PMID:[Ribonuclease Fl1 from Fusarium lateriticum. Isolation, substrate specificity and amino acid sequence]. 314 86

The complete amino acid sequence of ribonuclease U1 (RNase U1), a guanine-specific ribonuclease from a fungus, Ustilago sphaerogena, was determined by conventional protein sequencing, using peptide fragments obtained by several enzymatic cleavages of the performic acid-oxidized protein. The oxidized protein was first cleaved by trypsin and the resulting peptides were purified and their amino acid sequences were determined. These tryptic peptides were aligned with the aid of overlapping peptides isolated from a chymotryptic digest of the oxidized protein. The amino acid sequence thus deduced was further confirmed by isolation and analysis of peptides obtained by digestion of the oxidized protein with lysyl endopeptidase. The location of the disulfide bonds was deduced by isolation and analysis of cystine-containing peptides from a chymotryptic digest of heat-denatured RNase U1. These results showed that the protein is composed of a single polypeptide chain of 105 amino acid residues cross-linked by two disulfide bonds, having a molecular weight of 11,235, and that the NH2-terminus is blocked by a pyroglutamate residue. It has an overall homology with other guanine-specific or related ribonucleases, and shows 48% identity with RNase T1 and 38% identity with RNase U2.
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PMID:The amino acid sequence of ribonuclease U1, a guanine-specific ribonuclease from the fungus Ustilago sphaerogena. 316 89

The biochemical basis for variation in foot-and-mouth disease virus (FMDV) has been explored by analysis of the virus RNA and the virus-induced and structural proteins of three isolates of the virus. Two of the isolates were from serotype A and the third was from serotype O. Hybridization studies of the RNAs showed greater than 80% homology between the two type A viruses and about 65% homology between the two type A viruses and the virus of type O. The ribonuclease T1 maps of the three viruses gave distinct patterns typical of FMDV, but did not show that any two of the three viruses were more closely related. The virus-induced primary translation products, P88, P52 and P100 isolated from infected cells, were compared by tryptic peptide analysis. Combinations of 3H- and 14C-leucine-labelled polypeptides were hydrolysed with trypsin and resolved on an ion-exchange column. Much greater differences were found in P88 than in P52 or P100, indicating that the major variation occurs in the region of the genome coding for the structural proteins. Similar analysis of combinations of the structural proteins of the three viruses showed that there were differences in VP1, VP2 and VP3 and these results were supported by those obtained by PAGE analysis of the Staphylococcus aureus V8 protease cleavage products.
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PMID:Biochemical aspects of variation in foot-and-mouth disease virus. 624 41

The proteins specified by four Theiler's murine encephalomyelitis virus isolates in infected BHK-21 cells were studied. Their processing, sensitivity to trypsin, and the changeover after viral infection from synthesis of cellular proteins to synthesis of viral proteins were determined by one- and two-dimensional gel electrophoreses. The molecular weights and isoelectric points of the structural and nonstructural proteins of DA and WW isolates, which represent the less virulent subgroup of Theiler's murine encephalomyelitis virus, and of GDVII and FA isolates, which represent the virulent subgroup, were found to be the same. The sensitivity of DA and GDVII isolates to trypsin, as purified virions, and in infected cell extracts was similar. The shut-off of cellular protein synthesis in cells infected with the same two isolates and the changeover to the synthesis of viral proteins appeared to have the same pattern. These findings are interesting since the two subgroups of Theiler's murine encephalomyelitis virus differ in their pathogenicity, intracellular development in infected BHK-21 cells, and RNA composition, as determined by RNase T1 fingerprinting analysis.
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PMID:Proteins induced in tissue culture by four isolates of Theiler's murine encephalomyelitis virus. 630 Apr 15

We have used ribonuclease T1 and its chemically modified derivatives as substrates, and trypsin as proteinase, to investigate the kinetics of proteolysis of a specific peptide bond in the folded and unfolded conformations of a protein. Steady-state kinetic studies showed that Km = 0.27 mM and Kcat. = 2.45 s-1 for the tryptic hydrolysis of the Arg(77)-Val(78) peptide bond in unfolded ribonuclease T1. This Km is somewhat lower than, and the kcat. value similar to, values found for the tryptic hydrolysis of comparable bonds in small peptides. Our data for the initial velocity of hydrolysis of the Arg(77)-Val(78) bond in a solution of the folded protein indicate that the bond is at least 1700 times less rapidly hydrolysed in the folded than in the unfolded conformation of ribonuclease T1, and do not exclude the possibility that the bond is completely resistant to hydrolysis in the folded protein.
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PMID:Kinetics of tryptic hydrolysis of the arginine-valine bond in folded and unfolded ribonuclease T1. 643 Feb 67

Flocculent densities appear in the mitochondrial matrix during ischemic damage. These densities were examined in kidneys after 2 hours of autolysis. The samples were embedded in water-miscible glycol methacrylate (GMA). Ultrathin sections on copper grids were digested with pronase, trypsin, ribonuclease T1, various phospholipases (A2, C, D), lipase, and desoxyribonuclease I. For statistical analysis the mean number (+/- SD) of densities per mitochondrion was determined in tubular epithelial cells. A statistically significant (p less than 0.001) decrease in the number of densities was apparent after treatment with pronase (75.5%) of the densities digested completely) and with trypsin (43.3% of the densities digested completely). Digestion with other enzymes did not significantly decrease the number of densities. This study provides quantitative support for the hypothesis that flocculent densities are composed of proteins, most probably mitochondrial membrane proteins or matrix proteins. The results do not support the hypothesis that RNA, neutral lipids, phospholipids or DNA are major constituents of these structures.
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PMID:Mitochondrial flocculent densities in ischemia. Digestion experiments. 666 82


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