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)

We have determined the nucleotide sequence changes caused by three missense mutations leading to the production of inactive colicin E3 proteins. The ceaC1 mutation, affecting the translocation of colicin E3 through bacterial membranes, is caused by a serine to phenylalanine change at position 37 within the glycine-rich region at the N-terminal part of colicin E3. This confirms previous results suggesting a role for this domain in colicin uptake by sensitive cells. The ceaC2 and ceaC3 mutations, abolishing colicin E3 RNase activity, affect the C-terminal enzymatic domain of the molecule. In the ceaC2 mutant, serine at position 529 was converted to leucine. The ceaC3 mutation replaced a glycine residue at position 524 with an aspartic acid residue. The two mutations ceaC2 and ceaC3 yield information on the amino acid residues involved in the RNase activity of colicin E3.
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PMID:DNA sequence analysis of three missense mutations affecting colicin E3 bactericidal activity. 283 30

The amino acid sequence and disulfide bond pairing of human tumor derived angiogenin, the first tumor angiogenesis factor to be isolated in pure form from human sources, have been determined by conventional sequencing techniques adapted and applied to nanomole and subnanomole levels of material. Angiogenin, obtained from conditioned media of a human colonic adenocarcinoma cell line, is a single-chain protein consisting of 123 amino acids with the following sequences: less than Glu1-Asp-Asn-Ser-Arg-Tyr-Thr-His- Phe-Leu-Thr-Gln-His-Tyr-Asp15-Ala-Lys-Pro-Gln-Gly-Arg-Asp-Asp- Arg-Tyr-Cys-Glu-Ser-Ile-Met30- Arg-Arg-Arg-Gly-Leu-Thr-Ser-Pro-Cys-Lys-Asp-Ile-Asn-Thr- Phe45-Ile-His-Gly-Asn-Lys-Arg-Ser -Ile-Lys-Ala-Ile-Cys-Glu-Asn-Lys60-Asn-Gly-Asn-Pro-His-Arg-Glu-Asn -Leu-Arg-Ile -Ser-Lys-Ser-Ser75 -Phe-Gln-Val-Thr-Thr-Cys-Lys-Leu-His-Gly-Gly-Ser-Pro-Trp-Pro90-Pro -Cys-Gln-Tyr -Arg-Ala-Thr-Ala -Gly-Phe-Arg-Asn-Val-Val-Val105-Ala-Cys-Glu-Asn-Gly-Leu-Pro-Val- His-Leu-Asp-Gln-Ser-Ile-Phe120-Arg-Arg-Pro123-OH. Three disulfide bonds link the half-cystinyl residues 26-81, 39-92, and 57-107. The sequence is homologous to that of the pancreatic ribonucleases with 35% identity and many of the remaining residues conservatively replaced. Similarities are especially apparent around the major active-site residues His-12, Lys-41, and His-119 of ribonuclease which are conserved as are three of the four disulfide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Amino acid sequence of human tumor derived angiogenin. 286 94

The genetic basis of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency has been identified by nucleotide sequence analysis of HPRT cDNAs cloned from a patient with gout. A single nucleotide change was identified in two independent clones: an A to G transition at nucleotide 602. Confirmation of a mutation at this site was provided by RNase mapping analysis. The predicted consequence of this transition is an aspartic acid to glycine substitution at amino acid 201. We have designated this variant HPRTAshville. Prior to this report, enzyme activity in HPRTAshville had not been detected by routine assay. Using more sensitive techniques, including an in situ gel assay for HPRT activity, we were able to demonstrate electrophoretic, kinetic, and structural differences between HPRTAshville and normal HPRT. Electrophoretic migration of HPRTAshville has elevated Michaelis constants for 5-phosphoribosyl-1-pyrophosphate and hypoxanthine. Predicted secondary structural alterations may result from the aspartic acid to glycine substitution.
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PMID:Human hypoxanthine-guanine phosphoribosyltransferase deficiency. The molecular defect in a patient with gout (HPRTAshville). 290 37

A new method is described for locating the specific sites of attachment of Asn-linked carbohydrates in glycoproteins. The molecular weights of peptides released from the glycoprotein with proteases of known specificity are determined by fast atom bombardment mass spectrometry and fitted to the known or DNA-derived sequence. Oligosaccharides attached to Asn are released either before or after proteolysis with a glycosidase, usually peptide: N-glycosidase F, an enzyme that cleaves the beta-aspartylglycosylamine linkage of all known types of Asn-linked sugars and converts the attachment-site Asn to Asp. New peaks appearing in the mass spectra after treatment with glycosidase correspond to formerly glycosylated sites. Conversely, signals which disappear after glycosidase treatment correspond to glycopeptides. The differences in mass between these sets of signals define the composition of the carbohydrate at the given site in terms of deoxyhexose, hexose, N-acetylhexosamine, and sialic acid content. The extent of glycosylation at a given site can be estimated from the ratio of the peak heights corresponding to the Asn- vs Asp-containing peptides which differ by 1 Da in mass. This rapid and sensitive (low nmol) technique is illustrated here for ribonuclease B and for tissue plasminogen activator, a multiply glycosylated glycoprotein.
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PMID:Carbohydrate mapping by mass spectrometry: a novel method for identifying attachment sites of Asn-linked sugars in glycoproteins. 309 66

A procedure of large-scale isolation of homogeneous ribonuclease Th1 from cultural filtrates of Trichoderma harzianum with a yield over 50% has been developed. Three ion-exchange chromatographies on CM- and DEAE-cellulose gave 7500 fold purification of the protein with a specific activity of ca. 4500 U/mg. The RNase Th1 is shown to be a basic protein (pI 9.5) with Mr 10,747; it contains 106 amino acid residues (2 Asp, 6 Asn, 9 Thr, 12 Ser, 2 Glu, 1 Gln, 4 Pro, 16 Gly, 14 Ala, 4 Cys, 7 Val, 5 Ile, 2 Leu, 7 Tyr, 6 Phe, 2 His, 4 Lys, 3 Arg). The total amino acid sequence of RNase Th1 was determined and, on comparison with other guanyl-specific fungal RNases, showed a significant degree of homology, thus indicating probability of a common origin. By means of the equilibrium dialysis, crystals of RNase Th1 were obtained with the space group P3(2)21, a = b = 55.7, c = 80.1 A. A preliminary X-ray study of RNase Th1 was undertaken.
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PMID:[Isolation, analysis of amino acid sequence and crystallization of the extracellular ribonuclease Th1 from Trichoderma harzianum-01]. 313 1

Recognition by ribonuclease T1 of guanine bases via multidentate hydrogen bonding and stacking interactions appears to be mediated mainly by a short peptide segment formed by one stretch of a heptapeptide, Tyr42-Asn43-Asn44-Tyr45-Glu46-Gly47- Phe48. The segment displays a unique folding of the polypeptide chain--consisting of a reverse turn, Asn44-Tyr45-Glu46-Gly47, stabilized by a hydrogen-bond network involving the side chain of Asn44, the main-chain atoms of Asn44, Gly47 and Phe48 and one water molecule. The segment is connected to the C terminus of a beta-strand and expands into a loop region between Asn43 and Ser54. Low values for the crystallographic thermal parameters of the segment indicate that the structure has a rigidity comparable to that of a beta-pleated sheet. Replacement of Asn44 with alanine leads to a far lower enzymatic activity and demonstrates that the side chain of Asn44 plays a key role in polypeptide folding in addition to a role in maintaining the segment structure. Substitution of Asn43 by alanine to remove a weak hydrogen bond to the guanine base destabilized the transition state of the complex by 6.3 kJ/mol at 37 degrees C. In contrast, mutation of Glu46 to alanine to remove a strong hydrogen bond to the guanine base caused a destabilization of the complex by 14.0 kJ/mol. A double-mutant enzyme with substitutions of Asn43 by a histidine and Asn44 by an aspartic acid, to reproduce the natural substitutions found in ribonuclease Ms, showed an activity and base specificity similar to that of the wild-type ribonuclease Ms. The segment therefore appears to be well conserved in several fungal ribonucleases.
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PMID:Conformational properties of the guanine-binding site of ribonuclease T1 inferred from the X-ray structure and protein engineering. 315 Oct 17

The amino acid sequence of angiogenin isolated from bovine milk was deduced by gas-phase sequencing of the protein and its fragments. The protein contains 125 residues and has a calculated molecular mass of 14,577 Da. The sequence is highly homologous (65% identity) to the sequence of human angiogenin, most of the differences being the result of conservative replacements. Like human angiogenin, the bovine protein is also homologous to bovine pancreatic RNase A (34% identity) and the three major active site residues known to be involved in the catalytic process, His-14, Lys-41 and His-115, are conserved. When tested against conventional substrates for RNase A activity, bovine angiogenin displays the same selective ribonucleolytic activity as human angiogenin. The sequence of bovine angiogenin contains the cell recognition tripeptide Arg-Gly-Asp which is not present in the human protein.
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PMID:The complete amino acid sequence of bovine milk angiogenin. 319 38

An inactivated gene for Bacillus amyloliquefaciens extracellular ribonuclease (barnase) has previously been cloned and sequenced following transposon mutagenesis. The intact gene could not be assembled in Escherichia coli and is presumed to be lethal. Therefore, we introduced specific mutations into the barnase gene to prevent its lethal effect. A Gln-73 mutant gene was stable in E. coli but only produced low amounts of barnase antigen. Mutants containing Asp, Gln or Arg, instead of His-102, at the active site were identified by immunological screening for barnase antigen. None of the mutant proteins with alterations at aa residue 102 possessed RNase activity. The level of barnase (Asp-102) was higher in E. coli than in B. subtilis but the protein was not processed to the correct size in E. coli. To obtain correct processing, the barnase (Asp-102) structural gene was fused to the E. coli alkaline phosphatase promoter and signal sequence (phoA). Cells containing this construct secreted correctly processed barnase (Asp-102) into the periplasmic space and culture supernatant at a level of 20 mg/l. Barnase (Asp-102) was purified and found to have an identical N-terminus and a thermal unfolding curve that was nearly identical to that of active barnase (His-102). The cloning and expression of barnase in E. coli will allow detailed analysis of barnase protein folding by molecular genetic approaches.
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PMID:Expression of Bacillus amyloliquefaciens extracellular ribonuclease (barnase) in Escherichia coli following an inactivating mutation. 329 26

The highly refined 1.26 A structure (R = 0.15) of phosphate-free bovine pancreatic ribonuclease A was modeled with 13 residues having discrete multiple conformations of side chains. These residues are widely distributed over the protein surface, but only one of them, Lys 61, is involved in crystal packing interactions. The discrete conformers have no unusual torsion angles, and their interactions with the solvent and with other atoms of the protein are similar to those residues modeled with a single conformation. For three of the residues--Val 43, Asp 83, and Arg 85--two correlated conformations are found. The observed multiple conformations on the protein surfaces will be of significance in analyzing structure-function relationships and in performing protein engineering.
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PMID:Multiple conformations of amino acid residues in ribonuclease A. 344 61

Selective condensation of the unprotected fragments of alpha-globin--namely, alpha 1-30 and alpha 31-141--is catalyzed by Staphylococcus aureus V8 protease in the presence of 25% 1-propanol. The propensity of 1-propanol to induce the alpha-helical conformation and to generate a "native-like" topology for the polypeptide chain has been now investigated in an attempt to understand the molecular basis of this enzyme-catalyzed stereospecific condensation. Removal of heme from the alpha-chain decreases the overall alpha-helical conformation of the protein considerably. A significant amount of the alpha-helical conformation is restored in the presence of 25% 1-propanol and the digestion of alpha-globin by V8 protease becomes more selective concomitant with the increase in helicity. V8 protease digestion of alpha-globin at pH 6.0 and 4 degrees C occurs at Glu-30, Asp-47, Glu-27, and Glu-23 in the absence of 1-propanol. In the presence of 25% 1-propanol, the digestion is selective to the peptide bond of Glu-30. This selectivity appears to be a characteristic feature of the native conformation of alpha-chain (polypeptide chain with bound heme). 1-Propanol induces the alpha-helical conformation into RNase S peptide also. However, this increased helical conformation did not protect the RNase S peptide from V8 protease digestion at the Glu-9-Arg-10 peptide bond. RNase S peptide is an alpha-helical conformation in RNase S, an interacting fragment-complementing system of S protein and S peptide. S peptide is resistant to V8 protease hydrolysis in this conformation. Thus, the resistance of a peptide bond in a segment of a protein to protease digestion appears to be a consequence of the secondary structure as well as the tertiary interactions of this segment with the rest of the molecule. The results suggest that the 1-propanol induces alpha-helical conformation into segments of alpha-globin as well as packing of these helices in a native-like topology.
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PMID:Conformational studies of alpha-globin in 1-propanol: propensity of the alcohol to limit the sites of proteolytic cleavage. 347 77


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