Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human immunodeficiency virus-1 Tat protein can efficiently enter cells when added exogenously in tissue culture. Using the transactivation activity of Tat as a measure of intracellular delivery, we found that the addition of hydrophobic groups to Tat potentiated its uptake. Biotin was the most promising of the reagents tested and we characterized this effect in more detail. When coupled through a cysteine thiol, the addition of a single biotin to Tat increased activity by about six-fold. Increased activity was only seen with reducible biotin analogs, as modification with noncleavable analogs is known to block Tat transactivation activity. Biotin had no effect on Tat uptake when mixed with Tat without cross-linking. Recently, Tat was used as a carrier to direct the uptake of heterologous proteins into cells. We have used RNase as a model system for studying Tat-mediated uptake and found that biotin also increased the delivery of a Tat37-58-RNase conjugate. The increased uptake of Tat and Tat conjugates by addition of hydrophobic groups may significantly enhance the usefulness of Tat as a delivery vehicle, and the approach may be applicable to other systems.
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PMID:Increased cellular uptake of the human immunodeficiency virus-1 Tat protein after modification with biotin. 766 78

Although the hepatitis delta virus genome contains multiple open reading frames, only one of these reading frames is known to be expressed during replication of the virus. This open reading frame encodes two distinct molecular species of hepatitis delta antigen (HDAg), p24 delta and p27 delta, depending on the location of the stop codon which terminates translation. We found antibody specific for p27 delta to be capable of precipitating p24 delta in extracts of infected liver, indicating that p27 delta and p24 delta form heterologous complexes in vivo. After cross-linking with 0.05% glutaraldehyde, specific HDAg dimers were detected in antigen prepared from both the liver and serum of an HDV-infected woodchuck carrier of woodchuck hepatitis virus. Guanidine HCl-denatured HDAg extracted from liver and dialyzed against phosphate-buffered saline sedimented in rate-zonal sucrose density gradients as 15S multimeric complexes. These 15S multimers were stable in the presence of 1.2% Nonidet P-40. After RNase digestion, the 15S complex was reduced to a 12S complex without associated RNA, while boiling for 3 min in 1% sodium dodecyl sulfate-0.5% 2-mercaptoethanol further reduced the 15S complex to 3S HDAg monomers. In the absence of glutaraldehyde cross-linking, HDAg extracted from liver migrated as monomer species in reducing and nonreducing gels, suggesting that the conserved cysteine residue present in p27 delta does not play a role in the formation of either dimers or multimers. On the other hand, an amino-terminal chymotrypsin-digested HDAg fragment, with a predicted length of 81 or less amino acids, retained the ability to form dimers, consistent with the hypothesis that a coiled-coil motif present between residues 27 and 58 may play a role in HDAg protein interactions in vivo.
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PMID:Hepatitis delta virus antigen forms dimers and multimeric complexes in vivo. 767 57

Glutaredoxin (Grx) contains a redox-active disulfide and catalyzes thiol-disulfide interchange reactions with specificity for GSH. The dithiol form of Grx reduces mixed disulfides involving GSH or protein disulfides. During oxidative refolding of 8 microM reduced and denatured ribonuclease RNase-(SH)8 in a redox buffer of 1 mM GSH and 0.2 mM GSSG to yield native RNase-(S2)4, a large number of GSH-mixed disulfide species are formed. A lag phase that precedes formation of folded active RNase at a steady-state rate was shortened or eliminated by the presence of a catalytic concentration (0.5 microM) of Escherichia coli Grx together with protein disulfide-isomerase (PDI), its procaryotic equivalent E. coli DsbA, or the PDI analogue the E. coli thioredoxin mutant protein P34H. A mutant Grx in which one of the active site cysteine residues (Cys-11 and Cys-14) had been replaced by serine, C14S Grx, had similar effect compared with its wild-type counterpart. This demonstrated that Grx acted by a monothiol mechanism involving only Cys-11 and that RNase-S-SG-mixed disulfides were the substrates. Grx displayed synergistic activity together with PDI only in GSH/GSSG redox buffers with sufficiently low redox potential (E'0 of -208 or -181 mV) to allow reduction of the active site of Grx. In refolding systems that do not depend on glutathione, like cystamine/cysteamine or in the presence of selenite (SeO3(2-)), no synergistic activity of Grx was observed with PDI. We conclude that Grx acts by reducing mixed disulfides between GSH and RNase that are rate-limiting in enzyme-catalyzed refolding.
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PMID:Glutaredoxin accelerates glutathione-dependent folding of reduced ribonuclease A together with protein disulfide-isomerase. 771 72

The production of several single-chain Fv (sFv) antibody proteins was examined by three modes of mammalian cell expression. Our primary model was the 741F8 anti-c-erbB-2 sFv, assembled as either the VH-VL or VL-VH, and expressed alone, with C-terminal cysteine for dimerization, or as fusion proteins with carboxyl-terminal effector domains, including interleukin-2, the B domain of staphylococcal protein A, the S-peptide of ribonuclease S, or hexa-histidine metal chelate peptide. Constructs were expressed and secreted transiently in 293 cells and stably in CHO or Sp2/0 cell lines, the latter yielding up to 10 mg per liter. Single-chain constructs of MOPC 315 myeloma and 26-10 monoclonal antibodies were also expressed, as were hybrids comprising unrelated VH and VL regions. Our results suggest that mammalian expression is a practical and valuable complement to the bacterial expression of single-chain antibodies.
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PMID:Mammalian cell expression of single-chain Fv (sFv) antibody proteins and their C-terminal fusions with interleukin-2 and other effector domains. 776 52

A novel S-alkylating reagent, N-(3-bromopropyl)-N,N,N',N',N'-pentamethyl-1,3-propanedi(ammonium bromide) (TAP2-Br) which carries two positive charges in the molecule, was prepared to increase the solubility or to decrease the hydrophobicity of cysteine-containing denatured proteins (or peptides). S-Alkylation with TAP2-Br introduces two positive charges per cysteine residue, which will effectively shift the net charge of a protein in the positive direction. Disulfide-containing proteins, such as hen egg-white lysozyme, RNase A, BSA, and soybean trypsin inhibitor (Kunitz type), were reduced and S-alkylated with TAP2-Br to evaluate the potential of this reagent compared with other S-alkylating reagents such as monoiodoacetic acid, bromosuccinic acid and (3-bromopropyl)trimethylammonium bromide. The solubilities of these denatured proteins in the pH range of 2-10 indicated that S-alkylation with TAP2-Br effectively solubilized not only basic proteins (lysozyme and RNase) but also an acidic protein containing a fairly large number of cysteine residues (BSA). Moreover, the retentions of cysteine-containing tryptic peptides derived from lysozyme on reversed-phase HPLC were greatly reduced by S-alkylation with TAP2-Br. These results indicate that TAP2-Br is very useful to increase the solubility of some cysteine-containing denatured proteins and to decrease the hydrophobicity of peptides containing cysteine residue(s).
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PMID:An S-alkylating reagent with positive charges as an efficient solubilizer of denatured disulfide-containing proteins. 788 61

Complementary DNA clones representing two alleles of the self-incompatibility (S) locus of apple (Malus x domestica Borkh.) have been isolated and characterised. One of the alleles corresponds to a 29 kDa ribonuclease (S-RNase) that was purified from pistil tissue. On northern blots, both cDNAs hybridized to a transcript that was only present in pistils and not in the other plant tissues analysed. Corresponding genomic sequences, amplified by PCR, were found to contain a single intron of 138 bp and 1100 bp respectively. Comparison of both sequences shows that the cDNAs encode mature proteins containing 65% of identical residues. Eight invariable cysteine residues, conserved regions around two histidines thought to play a role in RNA catalysis, and a number of other distinct residues are conserved between the apple S-RNases and similar proteins in the family Solanaceae. As this is the first report of sequences of S-alleles from a species belonging to a family that is not related with the Solanaceae, the structural features of S-RNases deduced from a comparison of their sequences are discussed.
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PMID:cDNA cloning and molecular analysis of two self-incompatibility alleles from apple. 789 15

Derivatives of ribonuclease A (RNase A) with modifications in positions 1 and/or 7 were prepared by subtilisin-catalyzed semisynthesis starting from synthetic RNase 1-20 peptides and S-protein (RNase 21-124). The lysyl residue at position 1 was replaced by alanine, whereas Lys-7 was replaced by cysteine that was specifically modified prior to semisynthesis. The enzymes obtained were characterized by protein chemical methods and were active toward uridylyl-3',5'-adenosine and yeast RNA. When Lys-7 was replaced by S-methyl-cysteine or S-carboxamido-contrast, the catalytic properties were only slightly altered. The dissociation constant for the RNase A-RI complex increased from 74 fM (RNase A) to 4.5 pM (Lys-1, Cys-7-methyl RNase), corresponding to a decrease in binding energy of 10 kJ mol-1. Modifications that introduced a positive charge in position 7 (S-aminoethyl- or S-ethylpyridyl-cysteine) led to much smaller losses. The replacement of Lys-1 resulted in a 4-kJ mol-1 loss in binding energy. S-protein bound to RI with Ki = 63.4 pM, 800-fold weaker than RNase A. This corresponded to a 16-kJ mol-1 difference in binding energy. The results show that the N-terminal portion of RNase A contributes significantly to binding of ribonuclease inhibitor and that ionic interactions of Lys-7 and to a smaller extent of Lys-1 provide most of the binding energy.
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PMID:Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor. 800 61

A cDNA coding for bovine pancreatic RNase A was mutagenized to insert a proline, a leucine, and 2 cysteine residues, i.e. the residues present at corresponding positions in the subunit of seminal RNase, the only dimeric RNase of the pancreatic-type superfamily. The mutant, expressed in Escherichia coli, eventually aggregated into catalytically active dimers. Like naturally dimeric seminal RNase, at equilibrium the mutant dimeric RNase A adopted two quaternary structures (one with an exchange of the N-terminal segments between partner subunits, the other with no exchange) and displayed a selective toxicity for malignant cells, absent in the monomeric, parent protein.
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PMID:Ribonuclease A can be transformed into a dimeric ribonuclease with antitumor activity. 802 Dec 40

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

Although ribonucleases fold into correct tertiary conformation in vitro guided solely by information contained in the primary amino acid sequence (Sela, M., White, F. H., and Anfinsen, C. B. (1957) Science 124, 691-693), it is not clear whether folding of these proteins proceeds unassisted in a complex intracellular environment. We describe here the specific and high affinity binding of groEL, the prokaryotic homolog of the heat shock protein 60 family of molecular chaperones, to recombinant eosinophil cationic protein and eosinophil-derived neurotoxin, two members of the human ribonuclease gene family. We have determined that groEL binds to a unique peptide sequence near the amino terminus of nascent eosinophil cationic protein that includes the first of eight cysteine residues. This binding site functions independently and can confer groEL binding activity on an unrelated carrier protein. GroEL dissociates from the binding site upon addition of ATP and Mg2+; no other cations or cofactors are necessary. These findings suggest the possibility that interaction with a groEL-like molecular chaperone may be a requirement for correct folding and/or translocation of eukaryotic ribonucleases in vivo.
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PMID:Characterization of a distinct binding site for the prokaryotic chaperone, GroEL, on a human granulocyte ribonuclease. 809 49


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