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)

Lysozyme, cytochrome c, poly(L-lysine), myelin basic protein and ribonuclease were used to form multilayer dispersions containing about 50% protein (by weight) with bovine brain diacyl phosphatidylserine (PS). 31P nuclear magnetic resonance shift anisotropies, spin-spin (T2) and spin-lattice (T1) relaxation times for the lipid headgroup phosphorus were measured at 36.44 MHz. At pH 7.5, lysozyme, cytochrome c, poly(L-lysine) and ribonuclease were shown to increase the chemical shift anisotropy of PS by between 12-20%. Myelin basic protein altered the shape of the phosphate resonance, suggesting the presence of two lipid components, one of which had a modified headgroup conformation. The presence of cytochrome c led to the formation of a narrow spike at the isotropic shift position of the spectrum. Of the various proteins or peptides we have studied, only poly(L-lysine) and cytochrome c had any effect on the T1 of PS (1050 ms). Both caused a 20-30% decrease in T1 of the lamellar-phase phosphate peak. The narrow peak in the presence of cytochrome c had a very short T1 of 156 ms. The possibility is considered that the cytochrome Fe3+ contributes to the phosphate relaxation in this case. The effect of all proteins on the T2 of the phosphorus resonance was to cause an increase from the value for pure PS (1.6 ms) to between 2 and 5 ms. The results obtained with proteins are compared with the effects of small ions and intrinsic membrane proteins on the order and motion of the headgroups of lipids in bilayers.
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PMID:31P nuclear magnetic resonance studies of the association of basic proteins with multilayers of diacyl phosphatidylserine. 619 74

Earlier studies have indicated the marked resistance of two pronase endopeptidases to denaturation in high concentrations of urea or guanidine hydrochloride (Siegel, S., and Awad, W. M., Jr. (1973) J. Biol. Chem. 248, 3233--3240). One component has only a single residue of lysine and the other has none. The consideration arose that lysine-containing peptide segments may be less stable than those containing arginine because of the fluctuations of the side groups of the former residue. The small epsilon amino groups may not be able to sustain solvation of the hydrophobic arm in an aqueous medium. Arginine residues have shorter hydrophobic arms, larger hydrophilic groups, and higher pKa values and, thus may be less motile than lysine. The hypothesis was tested by guanidination of seven globular proteins (bovine carbonic anhydrase, chymotrypsinogen, alpha-lactalbumin, serum albumin, ribonuclease, hen egg lysozyme, and horse heart cytochrome c). Conversion of lysine residues to homoarginine was between 90 and 99%. Tritium-hydrogen isotope exchange revealed that all proteins except lysozyme demonstrated reduced out-exchange after guanidination. The results with lysozyme were not unexpected since only this protein has a high arginine to lysine ratio. These findings suggest that high arginine to lysine ratios contribute to protein stability.
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PMID:Stabilization of proteins by guanidination. 625 87

A unique resonance in the 13C NMR spectrum of [13C]methylated ribonuclease A has been assigned to a N epsilon, N-dimethylated active site residue, lysine 41. The chemical shift of this resonance was studied over the pH range 3 to 11, and the titration curve showed two inflection points, at pH 5.7 and 9.0. The higher pKa, designated pKa1, was assigned to the ionization of the lysyl residue itself while the pKa of 5.7, designated pKa2, was assigned on the basis of its pKa to the ionization of a histidyl residue which is somehow coupled to lysine 41. Both pKa values are measurably perturbed by the binding of active site ligands including nucleotides, nucleosides, phosphate, and sulfate. In most cases, the alterations in pKa values induced by the ligands were larger for pKa2. The ligand-induced perturbations in pKa2 generally paralleled those reported for histidine 12, another active site residue (Griffin, J. H., Schechter, A. N., and Cohen, J. S. (1973) Ann. N. Y. Acad. Sci. 222, 693-708). The sensitivity of the N epsilon, N-dimethylated lysine 41 resonance to the histidyl ionization may result from a conformational change in the active site region of ribonuclease which is coupled to the histidyl ionization. This coupling between lysine 41 and another ribonuclease residue, which has not been documented previously, offers new insight into the interrelationship between residues in the active site of this well characterized enzyme.
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PMID:[13C]Methylated ribonuclease A. 13C NMR studies of the interaction of lysine 41 with active site ligands. 625 47

We have designed and synthesized a model pentadecapeptide predicted to have the essential sequence information needed to form a stable and enzymatically active noncovalent complex with bovine pancreatic ribonuclease S-protein. The model peptide sequence, based on the conformational approach of simplifying the native sequence in a manner consistent with retention of essential noncovalent contacts and of secondary structure features, contained alanine at all positions except for Glu 2, Lys 7, Phe 8, Arg 10, His 12, and Met 13. The peptide was synthesized by the Merrifield solid phase method. The circular dichroism spectra of the purified model peptide in water and trifluoroethanol indicated a tendency to form an alpha-helical structure similar to that found for native S-peptide. The model peptide formed a stable complex with ribonuclease S-protein. With 12-fold excess of the peptide, the complex exhibited 36% of the specific activity of fully native ribonuclease S against the substrate cyclic cytidine 2':3'-monophosphate at pH 7.15. The dissociation constant of the model peptide for S-protein was found to be 1.1 x 10(-6) M, compared with 0.1 x 10(-6) M for native S-peptide. Crystals grown of the model peptide-S-protein complex were found to be isomorphous with those of native complex. The activity, stability, and structural integrity of the model complex verify the deductions made about essential sequence information in the NH2-terminal region of ribonuclease. Further, the results emphasize the general usefulness of the conformational approach in designing simplified sequences for other peptides and proteins.
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PMID:Sequence modeling using semisynthetic ribonuclease S. 627 8

The amino groups of beta-lactoglobulins A and B, cytochrome c and ribonuclease were progressively converted to acidic groups by reaction with succinic anhydride. The mixtures of modified proteins generated in this way were analyzed by urea-gradient electrophoresis, which separates the molecules on the basis of their net charge and demonstrates visually their urea-induced unfolding transitions. Molecules succinylated to varying extents were resolved by the electrophoresis, so purification of the many modified species was not required. It is demonstrated that accurate estimates of the stability of the folded state of an individual species may be estimated very easily from its urea-gradient electrophoretic pattern. Changes in ionization of the protein upon unfolding may also be detected. The general electrostatic effect of varying the net charge on these proteins was small. Converting the normally basic ribonuclease and cytochrome c to neutral and then to acidic proteins caused the net stabilities of their folded states to vary by no more than a few kJ/mol. However, specific interactions between a few ionized groups appear to be more important in some instances. Succinylation of the 19th, and final, lysine residue of cytochrome c produced unfolding even in the absence of urea, whereas reaction of the first 18 had very little effect. Reaction of the initial amino groups of beta-lactoglobulins A and B produced a small increase in stability in a few instances, a decrease in others; modification of more than about ten groups abruptly caused unfolding in the absence of urea.
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PMID:Effect on protein stability of reversing the charge on amino groups. 627 60

A radiochemical method for the determination of the amino terminus on very small amounts (0.5-5 nmol) of protein is described. The high sensitivity of the method is achieved by using undiluted 1-fluoro-2,4-dinitro-[3,5-3H]benzene [( 3H]Dnp-F) as the labelling reagent under conditions in which a maximum amount of radioactive label is incorporated. Chemical homogeneity is achieved by reacting with excess unlabelled Dnp-F. High recovery is obtained by adding Dnp-albumin as carrier protein. A mixture of Dnp 14C-labelled amino acids is added prior to hydrolysis and identification of the amino terminus is made on the basis of the 3H/14C ratios of the separated Dnp-amino acids. The method was tested on insulin, pancreatic ribonuclease, and lysozyme which gave high 3H/14C ratios only in the expected amino-terminal amino acids. Application to multiple forms of poly(C)-avid ribonuclease gave only amino-terminal lysine. Two of four putative isozymes of 17 beta-hydroxysteroid dehydrogenase had serine as the amino terminus while the other two had aspartic acid or asparagine.
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PMID:A highly sensitive method for identification of amino termini of proteins: application to multiple forms of poly(C)-avid ribonuclease and 17 beta-hydroxysteroid dehydrogenase. 630 40

The relative affinities of all Escherichia coli amino-acyl-tRNAs for E. coli elongation factor (EF) Tu-GTP have been measured by two independent applications of the competition form of the ribonuclease resistance assay. The set of aminoacyl-tRNAs includes at least one tRNA for each of the 20 amino acids as well as purified isoacceptor tRNA species for arginine, glycine, leucine, lysine, and tyrosine. In the first competition study, [3H]Phe-tRNA was used as the competing aminoacyl-tRNA against [14C]aminoacyl-tRNA in the set of all tRNAs; in the second study, [3H]Leu-tRNALeu4 was used as the competing aminoacyl-tRNA. The relative order of aminoacyl-tRNA affinities for EF-Tu-GTP was the same in each study. The results indicate that the affinity of EF-Tu-GTP at 4 degrees C, pH 7.4, is strongest for Gln-tRNA and weakest for Val-tRNA. Both Gly-tRNA and Pro-tRNA bind very strongly to EF-Tu-GTP relative to other aminoacyl-tRNAs. Various models of ternary complex interactions are discussed in light of the new data. Although the properties of the amino acid substituent are primarily responsible for the differences in relative affinities among the noninitiator aminoacyl-tRNAs, the results for the four isoacceptor species of Leu-tRNALeu indicate that the secondary structural features of the tRNA are also influential.
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PMID:Relative affinities of all Escherichia coli aminoacyl-tRNAs for elongation factor Tu-GTP. 637 Sep 98

S-Peptide combines with S-protein during the refolding of ribonuclease S. The kinetics of combination have now been measured by a specific probe, the absorbance (492 nm) of a fluoresceinthiocarbamyl (FTC) group on lysine-7 of S-peptide. pK changes of the FTC group detect both initial combination and later, first-order, stages in folding. Combination with the slow-folding species of S-protein occurs with a half-time of 0.4 s at 50 microM, whereas complete folding takes 50 s (pH 6.8, 31 degrees C). Thus combination takes place at an early stage in folding. The second-order rate constant of the refolding combination reaction (5 X 10(4) M-1 s-1) is 100-fold smaller than that for combination with folded S-protein, which probably reflects the lower affinity of S-protein for S-peptide in the initial complex. Inhibition by S-peptide of combination between FTC-S-peptide and S-protein shows that the refolding combination reaction is specific and reversible. Both the fast-folding and slow-folding species of unfolded S-protein participate in the refolding combination reaction.
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PMID:Measurement of the refolding combination reaction between S-peptide and S-protein. 640 7

The results of X-ray analyses of three ribonuclease-A-nucleotide complexes, at 2.3 A, are reported. A modified purine mononucleotide, 8-oxo-guanosine 2'-phosphate in a syn conformation, binds at the pyrimidine-binding site of the catalytic cleft. Solvent molecules are expelled from the active site due to inhibitor binding. The positions of the side-chains of the active-site residues Gln-11, His-12 and Thr-45 are well defined and do not alter on inhibitor binding. The mobility of Lys-41 is greatly reduced in the protein-nucleotide complexes and the terminal amino group interacts directly with the 2'-phosphate group of the nucleotides. In the complex of the enzyme with a modified pyrimidine, cytidine-N(3)-oxide 2'-phosphate, His-119 is stabilised in the minor site of the native protein [Borkakoti, N., Moss, D.S. and Palmer, R.A. (1982) Acta Crystallogr. B38, 2210-2217], while in the protein-purine derivative the imidazole group is located in the major site. Inhibitor binding induces movements in the side-chains of Lys-7 and Lys-66 which also modify the conformation of the active-site cleft of ribonuclease A.
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PMID:The active site of ribonuclease A from the crystallographic studies of ribonuclease-A-inhibitor complexes. 640 32

Lysozyme, ribonuclease and insulin were exposed to dry heating for 1 to 24 h at temperatures between 80 and 180 degrees C. Amino acid analyses of the heated samples showed that most of the amino acids are stable up to 120 degrees C. Initially, at higher temperatures, an almost rectilinear decrease took place which reached a critical stage at 160 degrees C. Nonpolar aliphatic, acidic and aromatic amino acids were all relatively stable (maximum loss less than 20% after 24 h at 180 degrees C). The lability of the other amino acids increased in the order proline, arginine, histidine, cysteine, threonine, lysine, tryptophan, serine, and methionine. Methionine was 86% decomposed after 24 h at 180 degrees C. Loss of trinitrobenzene sulfonic acid-reactive lysine ("available lysine") reached 20% at 100 degrees C and essentially 100% after 24 h at 180 degrees C. Maximum loss in weight during heating was 11%, although maximum protein loss was between 20 and 35%. Reaction orders and activation energies were estimated for some of the amino acid losses. Of the atypical amino acids ("hot spots") lysinoalanine, allo-isoleucine and ornithine that were detected, only lysinoalanine is useful as an indicator to detect amino acid damage after dry heating.
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PMID:Model studies on the heating of food proteins. Amino acid composition of lysozyme, ribonuclease and insulin after dry heating. 641 75


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