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)

The primary structures of the two isoforms of ribonuclease U2, RNAases U2-A and U2-B, were analysed and compared with each other. Among the chymotryptic peptides obtained from the reduced and S-carboxymethylated enzymes, only peptides C-3 were different from each other in terms of chromatographic behaviour on reverse-phase h.p.l.c. On the basis of chemical analyses of these peptides, it was shown that RNAase U2-B had an isopeptide bond in which Asp-32 was linked to Gly-33 through the beta-carboxy group in its side chain instead of the alpha-carboxy group. Deamidation of Asn-32 in RNAase U2-A led to the formation of this unusual linkage. The previously reported sequence of RNAase U2 [Sato & Uchida (1975) Biochem. J. 145, 353-360] was corrected by changing amino acid residues at eight different positions and by inserting an asparagine residue at position 32. The numbering of the positions of amino acid residues located downstream of Asn-32 was therefore shifted by 1. Accordingly, RNAase U2-A was shown to be composed of 114 amino acid residues.
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PMID:Comparison of the primary structures of ribonuclease U2 isoforms. 382 36

n-Hexane is metabolized to the gamma-diketone 2,5-hexanedione (2,5-HD), a derivative that covalently binds to lysine residues in neurofilament (NF) protein to yield 2,5-dimethylpyrrole adducts. Studies comparing the pyrrole-forming potential and neurotoxic potency of gamma-diketones have demonstrated that pyrrolylation is an absolute requirement in the neuropathogenesis. Autoxidative cross-linking of pyrrolylated NF proteins occurs and is proposed as a second required event. In the present study, the role of nucleophilic thiols and amines in the pyrrole-mediated cross-linking reaction was investigated. When pyrrolylated ribonuclease was incubated with N-acetyllysine, N-acetylcysteine, or glutathione in physiologic buffer (pH 7.4) under air, pyrrole-to-pyrrole cross-linking was inhibited only by the thiol-containing compounds. Stable thiol--pyrrole conjugates containing a bridge from the pyrrole ring at C-3 to the sulfur atom of the thiol were characterized by thermospray LC/MS and 1H-NMR spectroscopy. In contrast to low-molecular-mass thiols, SDS--PAGE studies indicated that, under the same incubation conditions, free thiols present in proteins did not undergo reaction with pyrrole adducts to form cross-links. Further experiments using a low-molecular-mass pyrrole derivative indicated that glutathione may also able to suppress pyrrole dimerization without conjugate formation, possibly via inhibition of a free radical-dependent mechanism. The results suggest the following: (1) 2,5-HD-induced protein cross-linking is mediated primarily by pyrrole-to-pyrrole bridging under physiologic conditions, and (2) glutathione and other low-molecular-mass thiols may inhibit the pyrrole dimerization reaction by two distinct pathways. These findings have significant implications for the mechanism of gamma-diketone neuropathy.
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PMID:Inhibition of 2,5-hexanedione-induced protein cross-linking by biological thiols: chemical mechanisms and toxicological implications. 754 60

2,5-Hexanedione (2,5-HD) is the neurotoxic gamma-diketone metabolite of the industrial solvent n-hexane. Substantial evidence indicates that 2,5-HD reacts with neurofilament protein lysine epsilon-amines to yield 2,5-dimethylpyrrole adducts and that this reaction is critical to the mechanism of toxicity. Alkylpyrroles are susceptible to autoxidative dimerization, a process that has also been suggested as an obligatory step in 2,5-HD neuropathy. In the present study, we characterized pyrrole autoxidation products of a 2,5-HD-treated lysine analogue and of a model, lysine-containing dipeptide and examined mechanistic aspects of pyrrole-mediated protein cross-linking. Incubation of 2,5-HD with N alpha-acetyllysine or the dipeptide N alpha-acetylglycyllysine methyl ester in physiological buffer (pH 7.4) under oxidative conditions resulted in time-dependent formation of the N epsilon-pyrrole derivative and two major pyrrole autoxidation products, as demonstrated by HPLC, on-line thermospray MS, and UV photodiode array detection. An autoxidative pyrrole dimer containing a methylene bridge between C-2 of one pyrrole ring and C-3 of a second ring was characterized by thermospray MS and 1H-NMR spectroscopy. 13C-NMR spectroscopy provided evidence for an identical pyrrole-to-pyrrole bridge in autoxidized, pyrrolylated ribonuclease (RNase). MS analysis also revealed a second major product--a stable, oxygen-containing monomeric pyrrole derivative. This product exhibited a UV absorbance maximum (lambda max = 355 nm) consistent with extended conjugation. Polymerization of pyrrolylated acetyllysine was accelerated by persulfate, a free-radical initiator, and inhibited by ascorbate, an antioxidant.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Formation and structure of cross-linking and monomeric pyrrole autoxidation products in 2,5-hexanedione-treated amino acids, peptides, and protein. 798 20