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 identified a pentapeptide region of microinjected ribonuclease A that is required for enhanced degradation of this protein during serum withdrawal. We introduced reductively methylated [3H]ribonuclease A, [3H]ribonuclease S-protein (residues 21-124), and [3H]ribonuclease S-peptide (residues 1-20) into the cytosol of human fibroblasts by red cell-mediated microinjection and osmotic lysis of pinosomes. The degradative rates of ribonuclease A and ribonuclease S-peptide are increased 2-fold upon withdrawal of serum, while catabolism of ribonuclease S-protein is not regulated in this manner. Certain fragments of ribonuclease S-peptide are also degraded in a serum-dependent fashion (residues 1-14 and 4-13), while other fragments are not (residues 1-10 and 2-8). [3H]Ribonuclease S-peptide is cleaved into two smaller radioactive peptides during loading into red cell ghosts. We tentatively identified the larger fragment as residues 7-11 based on its molecular weight determined by Sephadex chromatography in the presence of 8 M urea combined with sequential Edman degradation to identify the position of radioactive lysines. The smaller peptide fragment appears to be the amino-terminal dipeptide, Lys-Glu, and/or residues 7-8, Lys-Phe. After microinjection into fibroblasts, the pentapeptide is degraded at an enhanced rate in the absence of serum, while degradation of the dipeptide is not affected. We confirmed that residues 7-11 constitute the larger hydrolysis product of S-peptide by synthesizing this pentapeptide and radiolabeling it by reductive methylation. It migrated at the expected position after Sephadex chromatography in 8 M urea and was further hydrolyzed only slightly during loading into red cells. Finally, degradation of this pentapeptide after injection into fibroblasts was enhanced 2-fold upon serum withdrawal. These results, combined with our other recent studies (McElligott, M. A., Miao, P., and Dice, J. F. (1985) J. Biol. Chem. 260, 11986-11993), suggest that the pentapeptide, Lys-Phe-Glu-Arg-Gln, targets microinjected ribonuclease A to lysosomes for enhanced degradation during serum deprivation.
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PMID:Regulation of catabolism of microinjected ribonuclease A. Identification of residues 7-11 as the essential pentapeptide. 370 Apr 19

The S-peptide and S-protein fragments of ribonuclease S (RNase S, no EC no. assigned) have been immobilized onto separate Sepharose gels via a "leash" of polycytidylic acid substrate. Each of these gels releases its RNase fragment when treated with the complementary enzyme fragment or with RNase A (EC 3.1.27.5), and the released fragments recombine to give RNase S activity. Thus this system provides substrate-leash amplification (SLA), such that more enzymatic activity is eluted from the system than is applied. For example, 100 pg of RNase applied to the S-peptide gel is amplified by 1.9 X 10(4) to the equivalent of 1.9 micrograms of activity in 20 h, when followed by combination of the released S-peptide with excess S-protein. We also tested a three-stage amplification system, with a pair of S-peptide and S-protein gels at each stage. In this system the cumulative amplification of the initial 1-ng dose of RNase A is 4.9, 52, and 25-fold after each stage, respectively. Only 2 mg of each SLA gel is used per stage in these experiments, reflecting the magnitude of their production of RNase S activity.
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PMID:Substrate-leash amplification with ribonuclease S-peptide and S-protein. 374 90

X-ray diffraction methods were used to test a synthetic-modeling approach to the sequence engineering of bovine pancreatic ribonuclease. A model of RNase S-peptide (residues 1-20), having a simplified amino acid sequence but retaining elements deduced to be essential for conformation and function, was previously synthesized and found to form a catalytically active and stable complex with native S-protein (residues 21-24). We have now obtained a 3-A-resolution electron density map of this semisynthetic complex which reveals that the conformation of model peptide closely mimics that of native S-peptide, as intended by sequence design. Some small differences from the native structure are observed: Glu-2 and Arg-10 of the model complex are not close enough to form a salt bridge, the position of the His-12 imidazole ring is slightly shifted in the active site, and the peptide's amino terminus is reoriented. Nonetheless, the major structural features predicted to be essential by computer-aided peptide-design analysis are preserved in the model peptide portion of the complex. These include (i) the alpha-helical framework involving residues 3-13, (ii) the catalytically competent orientation of His-12, and (iii) complex-stabilizing non-bonding interactions involving Phe-8 and Met-13 of S-peptide and hydrophobic residues in the cleft region of S-protein. Further, sequence simplification has not introduced any non-native, potentially stabilizing contacts between the model peptide and S-protein. The results emphasize the usefulness, in redesigning native proteins, of categorizing sequence into residues providing conformational framework and those determining intra-and intermolecular surface recognition.
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PMID:Crystallographic structure of an active, sequence-engineered ribonuclease. 386 3

There are 33 invariant amino acid positions out of 132 positions in 42 investigated sequences of ribonucleases from a number of mammalian species and a reptile (snapping turtle, Chelydra serpentina). These invariant residues are unequally distributed over 3 different parts of the molecule. The lobe of the S-protein part of the molecule, which lacks one disulfide bridge and has two shortened loops in turtle ribonuclease, has the lowest percentage of invariant residues, although the active-site residue His 119 is located in this part.
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PMID:Comparison of the structure of turtle pancreatic ribonuclease with those of mammalian ribonucleases. 394 Sep 1

In order to examine the effect of a defined enantiomeric sequence on protein structure, the all-D model ribonuclease S-peptide, H-Ala-Glu-Ala4-Lys-Phe-Ala-Arg-Ala-His-Met-Ala2-OH, has been synthesized by the solid phase method. The all-L peptide has been synthesized previously and shown to possess 36% of ribonuclease S activity when added to ribonuclease S-protein (Komoriya, A. & Chaiken, I.M. (1982) J. Biol. Chem 257, 2599-2604). The synthetic D-peptide was purified by gel filtration and semipreparative reverse phase HPLC. Amino acid composition of the synthetic peptide was in agreement with theory and gas chromatographic analysis showed that no significant racemization had occurred during synthesis. Circular dichroism (CD) studies of the D-peptide showed a peak of positive ellipticity in the 220-230 nm region, whereas a negative ellipticity peak for the L-peptide was observed. The effects of temperature and trifluoroethanol on the far-ultraviolet CD spectra of D- and L-peptides were similar but of opposite sign, confirming the expectation that the D-peptide has the propensity to form an alpha-helical structure which is enantiomeric with respect to that formed by the L-peptide. In the presence of S-protein, the L-peptide showed hydrolytic activity against the substrate cytidine-2':3'-monophosphate, whereas the D-peptide was inactive. Addition of the D-peptide to mixtures of L-peptide and S-protein did not lead to inhibition of enzymatic activity. These results indicate lack of binding of D-peptide to S-protein to produce either an active or inactive species.
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PMID:Synthesis and properties of an all-D model ribonuclease S-peptide. 399 53

We have analyzed the subcellular localization of 125I-labeled ribonuclease A and ribonuclease S-protein (residues 21-124) after erythrocyte-mediated microinjection into confluent cultures of IMR-90 human lung fibroblasts. Microinjected cells were fractionated by two consecutive Percoll gradients, and the distribution of radioactive ribonuclease A and S-protein was compared to patterns for known enzyme markers. Ribonuclease A is localized in the cytosol immediately after microinjection, but thereafter a portion of the microinjected enzyme is associated with lysosomes. We obtained similar results for ribonuclease S-protein except extensive association with a nonlysosomal intracellular structure is also evident. The effects of ammonium chloride on proteolysis indicate that ribonuclease A and ribonuclease S-protein are degraded at least in part by lysosomal pathways. Degradation of long-lived cellular proteins is inhibited by 17% in the presence of serum and by 35% in the absence of serum. The effects of ammonium chloride on catabolism of microinjected proteins are more variable. Inhibition in the presence and absence of serum ranged between 43 and 64% for both ribonuclease A and ribonuclease S-protein. To quantitatively assess the role of lysosomal and cytosolic pathways in the degradation of microinjected proteins, we have tagged proteins with the inert trisaccharide, [3H] raffinose. The radioactive degradation products of such proteins are completely retained within lysosomes since the lysosomal membrane is impermeable to [3H] raffinose coupled to lysine or small peptides. These studies show that ribonuclease A and S-protein are degraded almost entirely by lysosomes while bovine serum albumin is degraded principally in the cytosol. A mixture of rat liver cytosolic proteins is degraded approximately 60% in the cytosol and 40% by lysosomes confirming that both lysosomal and nonlysosomal pathways of proteolysis are important in confluent human fibroblasts.
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PMID:Lysosomal degradation of ribonuclease A and ribonuclease S-protein microinjected into the cytosol of human fibroblasts. 404 85

1. A method is described for measuring the concentration of periodate over the range 0.2-20mum by adding 1,2-di-(p-dimethylaminophenyl)ethane-1,2-diol to a sample solution. Periodate cleaves this compound to from two molecules of p-dimethylaminobenzaldehyde, the extinction of which is then read at 352mmu. 2. The method has been used to follow the course of periodate oxidations of serine methyl ester, ribonuclease A and ribonuclease S-protein. Addition of the reagent stops further periodate reaction by reducing the remaining periodate to iodate. 3. The presence of protein does not interfere with the assay.
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PMID:A spectrophotometric method for the microdetermination of periodate. 429 21

We make use of the known exchange rates of individual amide proton in the S-peptide moiety of ribonuclease S (RNAase S) to determine when during folding the alpha-helix formed by residues 3 to 13 becomes stable. The method is based on pulse-labeling with [3H]H2O during the folding followed by an exchange-out step after folding that removes 3H from all amide protons of the S-peptide except from residues 7 to 14, after which S-peptide is separated rapidly from S-protein by high performance liquid chromatography. The slow-folding species of unfolded RNAase S are studied. Folding takes place in strongly native conditions (pH 6.0, 10 degrees C). The seven H-bonded amide protons of the 3-13 helix become stable to exchange at a late stage in folding at the same time as the tertiary structure of RNAase S is formed, as monitored by tyrosine absorbance. At this stage in folding, the isomerization reaction that creates the major slow-folding species has not yet been reversed. Our result for the 3-13 helix is consistent with the finding of Labhardt (1984), who has studied the kinetics of folding of RNAase S at 32 degrees C by fast circular dichroism. He finds the dichroic change expected for formation of the 3-13 helix occurring when the tertiary structure is formed. Protected amide protons are found in the S-protein moiety earlier in folding. Formation or stabilization of this folding intermediate depends upon S-peptide: the intermediate is not observed when S-protein folds alone, and folding of S-protein is twice as slow in the absence of S-peptide. Although S-peptide combines with S-protein early in folding and is needed to stabilize an S-protein folding intermediate, the S-peptide helix does not itself become stable until the tertiary structure of RNAase S is formed.
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PMID:Amide proton exchange used to monitor the formation of a stable alpha-helix by residues 3 to 13 during folding of ribonuclease S. 609 89

Intracellular serine protease, termed ISP-103, was isolated from Bacillus subtilis, strain 103. The substrate specificity of the enzyme was compared to that of secretory subtilisins. Similar to subtilisins, ISP-103 cleaves a single peptide bond Ala20-Ser21 within the native pancreatic ribonuclease A, which results in the accumulation of trypsin-sensitive ribonuclease S, consisting of a non-covalently bound S-peptide (20 amino acid residues) and S-protein (104 amino acid residues). The enzyme hydrolyzes a single peptide bond Leu15-Tyr16 of the B-chain of oxidized bovine insulin, in contrast to the subtilisins cleaving four additional bonds. ISP prefers Leu rather than Phe in the P1 binding site of the rho-nitroanilide peptide substrates and shows a more strict dependence of the activity on the presence of the hydrophobic residues in the P2 and P3 sites. The data obtained indicate that the substrate specificity of ISP, being within the borders of subtilisin specificity, is nevertheless much more restricted.
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PMID:[Substrate specificity of Bacillus subtilis intracellular serine protease. Hydrolysis of insulin beta-chain, native ribonuclease A and p-nitroanilide peptide substrates]. 626 Feb 44

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


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