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:3.1.27.4 (ribonuclease)
6,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

For Petunia inflata and Nicotiana alata, which display gametophytic self-incompatibility, S proteins (the products of the multiallelic S gene in the pistil) have been shown to control the pistil's ability to recognize and reject self-pollen. The biochemical mechanism for rejection of self-pollen by S proteins has been shown to involve their ribonuclease activity; however, the molecular basis for self/non-self recognition by S proteins is not yet understood. Here, we addressed whether the glycan chain of the S3 protein of P. inflata is involved in self/non-self recognition by producing a nonglycosylated S3 protein in transgenic plants and examining the effect of deglycosylation on the ability of the S3 protein to reject S3 pollen. The S3 gene was mutagenized by replacing the codon for Asn-29, which is the only potential N-glycosylation site of the S3 protein, with a codon for Asp, and the mutant S3 gene was introduced into P. inflata plants of the S1S2 genotype. Six transgenic plants that produced a normal level of the nonglycosylated S3 protein acquired the ability to reject S3 pollen completely. These results suggest that the carbohydrate moiety of the S3 protein does not play a role in recognition or rejection of self-pollen and that the S allele specificity determinant of the S3 protein and those S proteins that contain a single glycan chain at the same site as the S3 protein must reside in the amino acid sequence itself.
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PMID:Carbohydrate moiety of the Petunia inflata S3 protein is not required for self-incompatibility interactions between pollen and pistil. 786 34

All of the individual carboxyl groups (the side-chain carboxyl groups of Asp and Glu, and the C-terminal alpha-carboxyl group) in Escherichia coli ribonuclease HI, which is an enzyme that cleaves the RNA strand of a RNA/DNA hybrid, were pH-titrated, and their ionization constants (pKa) were determined from an analysis of the pH-dependent chemical shifts of the carboxyl carbon resonances obtained from 1H-13C heteronuclear two-dimensional NMR. The pKa values in the enzyme varied widely among individual residues, for example, in the unusual pKa values for two important catalytic residues, Asp10 (pKa 6.1) and Asp70 (pKa 2.6). Moreover, remarkable two-step titrations were observed for these carboxylates. The binding of Mg2+ ion to the enzyme, which is the cofactor necessary for catalytic activity, caused no significant change in the pKa values of the carboxyl groups, except for that of Asp10. The variations of the pKas that were dependent on the microenvironment in the protein were theoretically reproduced to compare with the experimental results by a numerical calculation, using a continuum electrostatic model. Most of the significant pKa decreases were brought about through strong electrostatic interactions with the neighboring basic amino acids, Arg or Lys. The pKa shifts and the two-step titrations of Asp10 and -70, which are close to each other, were interpreted to be due to the neighboring effect of two functional groups, as observed in the interacting titratable groups of a dicarboxyl compound or in the active site carboxylates of lysozyme and aspartic protease. The role of Asp10 in the catalytic action is either to be the proton donor to the RNA moiety or the binding partner of the Mg2+ ion cofactor. Asp70, on the other hand, is considered to be the proton acceptor from a water molecule.
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PMID:Individual ionization constants of all the carboxyl groups in ribonuclease HI from Escherichia coli determined by NMR. 790 91

Ribonuclease inhibitor (RI) was purified about 1300-fold from human cerebrum (including a small portion of midbrain) by a combination of ammonium sulfate precipitation, ribonuclease A-Sepharose chromatography, and high-performance anion-exchange chromatography. The purified RI appeared to be homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using the same method, a homogeneous RI was also obtained from human hindbrain (brainstem and cerebellum). The cerebral RI appeared to be virtually identical with the hindbrain RI on the basis of the following properties: (a) Molecular mass was estimated to be 50 kDa on SDS-PAGE under reducing conditions. (b) Composition analysis revealed that the RI was rich in leucine and cysteine residues and included no amino sugars. (c) The N-terminus was blocked and probably modified by N-acetylation. After treatment with trifluoroacetic acid, it became susceptible to Edman degradation and was sequenced as Ser-Leu-Asp-Ile-Gln-Ser-Leu-Asp-Ile-Gln-(Cys)-Glu-Glu-. (d) The RI, which showed sulfhydryl-dependent inhibitory activity on both secretory-type and nonsecretory-type ribonucleases, bound tightly to ribonuclease to form a 1:1 complex on a molar basis. (e) The RI cross-reacted strongly with anti-human placental RI antibody. These findings also indicate that human brain RI is quite similar to human placental RI. In contrast to the abundance of RI in human brain tissue (about 0.08% (w/w) of total protein), RI was undetectable in human cerebrospinal fluid, suggesting that brain RI may not be a secreted protein.
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PMID:Purification and characterization of human brain ribonuclease inhibitor. 803 55

Complete primary structure of an extracellular low molecular mass ribonuclease of Bacillus thuringiensis was determined using Edman degradation and mass-spectrometry analysis of individual peptides obtained after hydrolysis of the protein by cyanogen bromide and staphylococcal protease. The peptides were isolated and purified by HPLC and denaturing PAGE. The enzyme consists of 109 amino acid residues (Asp 8, Asn 6, Thr 6, Ser 10, Glu 3, Gln 1, Pro 3, Gly 9, Ala 12, Val 7, Ile 7, Leu 7, Tyr 7, Phe 4, His 1, Arg 10, Trp 3 and Lys 5) and has a molecular weight of 12182 Da. A single difference was detected between primary structures of the enzyme and an extracellular ribonuclease of B. intermedius.
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PMID:[Complete primary structure of Bacillus thuringiensis extracellular ribonuclease]. 825 Sep 78

S-peptide (residues 1-20) and S-protein (residues 21-124) are the enzymatically inactive products of the limited digestion of ribonuclease A by subtilisin. S-peptide binds S-protein with high affinity to form ribonuclease S, which has full enzymatic activity. Recombinant DNA technology was used to produce a fusion protein having three parts: carrier, spacer, and target. The two carriers used were the first 15 residues of S-peptide (S15) and a mutant S15 in which Asp 14 had been changed to Asn (D14N S15). The spacer consisted of three proline residues and a four-residue sequence recognized by factor Xa protease. The target was beta-galactosidase. The interaction between the S-peptide portion of the fusion protein and immobilized S-protein allowed for affinity purification of the fusion protein under denaturing (S15 as carrier) or nondenaturing (D14N S15 as carrier) conditions. A sensitive method was developed to detect the fusion protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis by its ribonuclease activity following activation with S-protein. S-peptide has distinct advantages over existing carriers in fusion proteins in that it combines a small size (> or = 15 residues), a tunable affinity for ligand (Kd > or = 10(-9) M), and a high sensitivity of detection (> or = 10(-16) mol in a gel).
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PMID:Ribonuclease S-peptide as a carrier in fusion proteins. 845 73

Barnase, the guanine specific ribonuclease of Bacillus amyloliquefaciens, was subjected to mutations in order to alter the electrostatic properties of the enzyme. Ser-85 was mutated into Glu with the goal to introduce an extra charge in the neighborhood of His-102. A double mutation (Ser-85-Glu and Asp-86-Asn) was introduced with the same purpose but without altering the global charge of the enzyme. A similar set of mutations was made using Asp at position 85. For all mutants the pI was determined using the technique of isoelectric focusing and calculated on the basis of the Tanford-Kirkwood theory. When Glu was used to replace Ser-85, the correlation between the experimental and the calculated values was perfect. However, in the Ser-85-Asp mutant, the experimental pI drop is bigger than the calculated one, and in the double mutant (Ser-85-Asp and Asp-86-Asn) the compensation is not achieved. The effect of the mutations on the pKa of His-102 can be determined from the pH dependence of the kcat/KM for the hydrolysis of dinucleotides, e.g., GpC. The effect can also be calculated using the the method of Honig. In this case the agreement is very good for the Glu-mutants and the single Asp-mutant, but less for the double Asp-mutant. The global stability of the Asp-mutants is, however, the same as the wild type, as shown by stability studies using urea denaturation. Molecular dynamics calculations, however, show that in the double Asp-mutant His-102 (H+) swings out of its pocket to make a hydrogen bridge with Gin-104 which should cause an additional pKa rise. The effect of the Glu-mutations was also tested on all the kinetic parameters for GpC and the cyclic intermediate G > p at pH 6.5, for RNA at pH 8.0, and for poly(A) at pH 6.2. The effect of the mutations is rather limited for the dinucleotide and the cyclic intermediate, but a strong increase of the KM is observed in the case of the single mutant (extra negative charge) with polymeric substrates. These results indicate that the extra negative charge has a strong destabilizing effect on the binding of the polymeric substrates in the ground state and the transition state complex. A comparison with the structure of bound tetranucleotides (Buckle, A.M. and Fersht, A.R., Biochemistry 33:1644-1653, 1994) shows that the extra negative charge points towards the P2 site.
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PMID:Experimental and theoretical study of electrostatic effects on the isoelectric pH and the pKa of the catalytic residue His-102 of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase). 877 84

The LH/CG receptor (LH/CG-R) belongs to the family of glycoprotein hormone G protein-coupled receptors. The extracellular domain of LH/CG-R is associated with high ligand-binding affinity and contains leucine-rich repeats (LRRs). With the goal of identifying essential amino acid residues involved in ligand binding, we replaced several conserved ionizable residues in the rat LH/CG-R with ones of opposite charge. The expression of these mutants was assessed by binding studies and Western blots after COS-7 cells were transiently transfected with wild type and mutant receptor cDNAs. The charge inversion of each of Lys40, Lys104, Asp118, Glu132, and Asp135 with Asp or Lys resulted in no detectable human CG binding in intact or solubilized cells; as control, a Lys40-->Arg replacement yielded a mutant with characteristics of the wild type receptor. Western analysis showed that the Lys40-->Arg mutant expressed at a level comparable to that of wild type receptor and, like wild type, exhibited a predominant immunoreactive mature form of LH/CG-R. Each of the five charge inversion mutants expressed at a lower level than wild type as assessed by immunoreactivity, and the levels of the Lys40-->Asp and Glu132-->Lys mutants were particularly low. The ratio of the mature to immature form of the receptor was high, i.e. like that of wild type, for the Glu132-->Lys and Asp135-->Lys replacements; the three other charge inversion mutants exhibited less mature than immature forms of the receptor. To aid in interpreting these results, we developed a model incorporating residues 27-235 of the extracellular domain of the rat LH/CG-R based on the crystal structure of the porcine ribonuclease inhibitor. Sequence homology and alignment revealed nine LRRs, with flanking cysteine clusters as found in a number of LRR proteins. Our model suggested that the Lys replacements of Glu132 and Asp135, i.e. those mutants that formed mature receptors, would disrupt the regional negative charge of the receptor. We propose that these residues are either directly involved in hormone binding or indirectly by disruption of the charge of an important binding surface.
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PMID:Determination of residues important in hormone binding to the extracellular domain of the luteinizing hormone/chorionic gonadotropin receptor by site-directed mutagenesis and modeling. 888 49

Onconase is a cytotoxic ribonuclease with antitumor properties. A semisynthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and last six of the 104 amino acid residues to a genomic clone that encoded the remaining amino acid residues. Additionally, the 15 N-terminal amino acid residues of onconase were replaced with the first 21 amino acid residues of the homologous human RNase, eosinophil-derived neurotoxin, EDN. Two versions of the hybrid EDN-onconase protein were cloned, expressed and purified. The chimera that contained a glycine in lieu of the aspartic acid present in native onconase (position 26 in the chimera) exhibited enzymatic activity more characteristic of EDN than native onconase and was considerably more active with respect to both RNase activity and cellular cytotoxicity than recombinant onconase. In contrast to native or recombinant onconase, the EDN chimera was recognized by anti-EDN polyclonal antibodies, demonstrating that the chimera also shared structural antigenic determinants to the human enzyme. These results demonstrate that a chimeric ribonuclease has cytotoxicity comparable to onconase in two out of four cell lines tested. The implications with regard to cancer therapy are presented.
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PMID:Expression and characterization of a cytotoxic human-frog chimeric ribonuclease: potential for cancer therapy. 919 72

A major allergen/antigen, Asp fl, secreted by Aspergillus fumigatus exhibits cytotoxicity towards eukaryotic cell lines. Asp fl inhibited protein synthesis in RAW cells with an IC50 of 4.5 nM and also degraded ribosomal RNA of RAW cells at a similar concentration. Ribosomal inactivation by Asp fl may be the probable mechanism for protein synthesis inhibition. Specific ribonuclease activity of Asp fl was observed to be 100,000 U/mg. Presence of strong RNase activity in Asp fl was further confirmed by agar gels containing yeast RNA. Electrophoretic run on agarose gels showed that Asp fl degrades all species of naked RNA. Modification of histidine residues of Asp fl with diethyl pyrocarbonate and alkylation of cysteines with iodoacetamide resulted in loss of ribonuclease activity and cytotoxicity of Asp fl. The current study establishes the ribonuclease activity of a purified major allergen of A. fumigatus that inhibits protein synthesis and kills the eukaryotic cells.
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PMID:Ribonuclease activity dependent cytotoxicity of Asp fl, a major allergen of A. fumigatus. 935 29

Crystal structures of adenine-specific Ustilago sphaerogena ribonuclease U2 complexed with the substrate analogues, d(ApG), d(ApGpG), and d(ApGpC), with the intermediate analogue, 2',3'-O-isopropylidene-adenosine, and with the product, 3'-AMP, have been determined. In each structure, the adenine base is recognized by the enzyme with four hydrogen-bonds. In the substrate analogue structures, the second base of guanine is sandwiched between His 101 and Tyr 107 side-chains, and forms two hydrogen-bonds with Tyr 107 O and Asp 108 O delta 1 atoms. The third base of the trinucleotides is in van der Waals interaction with the Tyr 78 side-chain. The phosphate group between the second and third nucleosides forms two hydrogen-bonds with the side chains of Asp 37 and Tyr 78. Oxygen atoms of the scissile phosphate group are involved in interactions with catalytic residues of Tyr 39, His 41, Glu 62, Arg 85, and His 101. These interactions indicate that either His 41 or Glu 62 acts as a general base and His 101 acts as a general acid in the first step of RNA hydrolysis.
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PMID:Crystal structures of nucleic acid complexes of ribonuclease U2. 958 11


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