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Query: C30B5 .7
 413,019 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

5-Iodouracil was a substrate for bovine liver dihydropyrimidine dehydrogenase (DHPDHase) and was a potent inactivator of the enzyme. NADPH increased the rate of inactivation and thymine protected against inactivation. These findings suggest that 5-iodouracil was a mechanism-based inactivator. However, dithiothreitol and excess 5-iodouracil protected the enzyme against inactivation. Thus, a reactive product, presumably 5-iodo-5,6-dihydrouracil generated through the enzymatic reduction of 5-iodouracil, was released from DHPDHase during processing of 5-iodouracil. Since only 18% of [6-3H]5-iodouracil reduced by DHPDHase was covalently bound to the enzyme and radiolabel was not lost to the solvent as tritium, the partition coefficient for inactivation was 4.5. However, the enzymatic activity was completely titrated with 1.7 mol of 5-iodouracil per mol of enzyme-bound flavin. These results indicate that there was 0.31 mol of enzyme-bound inactivator per mol of enzyme flavin. This suggests there were 3.2 flavins per active site, which is consistent with the report of multiple flavins per enzymic subunit (Podschun, B., Wahler, G., and Schnackerz, K. D. (1989) Eur. J. Biochem. 185, 219-224). DHPDHase was inactivated by 2.1 mol of racemic 5-iodo-5,6-dihydrouracil per mol of active sites. The stoichiometry for inactivation of the enzyme by the nonenzymatically generated enantiomer of 5-iodo-5,6-dihydrouracil was calculated to be 1. Two radiolabeled fragments were isolated from a tryptic digest of DHPDHase inactivated with radiolabeled 5-iodouracil. The amino acid sequences of these peptides were Asn-Leu-Ser-X-Pro-His and Asn-Leu-Ser-X-Pro-His-Gly-Met-Gly-Glu-Arg where X was the modified amino acid containing radiolabel from [6-3H]5-iodouracil. Fast atom bombardment mass spectral analysis of the smaller peptide yielded a protonated parent ion mass of 782 daltons that was consistent with X being a S-(hexahydro-2,4-dioxo-5-pyrimidinyl)cysteinyl residue.
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PMID:Inactivation of dihydropyrimidine dehydrogenase by 5-iodouracil. 193 61

The complete primary structures of the major Asn-linked oligosaccharides from the type II variant surface glycoproteins (VSGs), MITat 1.2 and MITat 1.7, and the type III VSG, MITat 1.5, were determined using a combination of exo- and endoglycosidase digestions, methylation analysis, acetolysis, and 500 MHz 1H NMR spectroscopy. Each variant contained classical branched oligomannose-type and biantennary complex oligosaccharides, a proportion of the latter substituted with terminal alpha(1-3)-linked galactose residues, the first report of the presence of this epitope in Trypanosoma brucei. In addition both the type II variants contained relatively large amounts of the unusual small oligomannose-type oligosaccharides, Man4GlcNAc2 and Man3GlcNAc2, and a diverse array of novel branched poly-N-acetyllactosamine oligosaccharides, similar but not identical to those from mammalian glycoproteins. These latter structures were also partially substituted with terminal alpha(1-3)-linked galactose residues. Glycosylation in the type II variants showed site specificity in that the poly-N-acetyllactosamine and Man(9-5)GlcNAc2 oligosaccharides were located exclusively at Asn-glycosylation site 1 very close to the C terminus, whereas the Man(4-3)GlcNAc2 and biantennary complex oligosaccharides were located exclusively at site 2. This is the first report of the presence of poly-N-acetyllactosamine oligosaccharides in protozoa.
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PMID:Structural characterization of the asparagine-linked oligosaccharides from Trypanosoma brucei type II and type III variant surface glycoproteins. 193 85

Asp176, Glu179 and Glu180 of Aspergillus awamori glucoamylase appeared by differential labeling to be in the active site. To test their functions, they were replaced by mutagenesis with Asn, Gln and Gln respectively, and kinetic parameters and pH dependencies of all enzyme forms were determined. Glu179----Gln glucoamylase was not active on maltose or isomaltose, while the kcat for maltoheptaose hydrolysis decreased almost 2000-fold and the KM was essentially unchanged from wild-type glucoamylase. The The Glu180----Gln mutation drastically increased the KM and moderately decreased the kcat with maltose and maltoheptaose, but affected isomaltose hydrolysis less. Difference in substrate activation energies between Glu180----Gln and wild-type glucoamylases indicate that Glu180 binds D-glucosyl residues in subsite 2. The Asp176----Asn substitution gave moderate increases and decreases in KM and kcat respectively, and therefore similar increases in activation energies for the three substrates. This and the differences in subsite binding energies between Asp176----Asn and wild-type glucoamylases suggest that Asp176 is near subsite 1, where it stabilizes the transition state and interacts with Trp120 at subsite 4. Glu179 and Asp176 are thus proposed as the general catalytic acid and base of pKa 5.9 and 2.7 respectively. The charged Glu180 contributes to the high pKa value of Glu179.
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PMID:Catalytic mechanism of fungal glucoamylase as defined by mutagenesis of Asp176, Glu179 and Glu180 in the enzyme from Aspergillus awamori. 197 Apr 34

Two distinct forms of botrocetin, the von Willebrand factor (vWF)-dependent platelet coagglutinin isolated from the venom of the snake Bothrops jararaca, were purified and characterized structurally and functionally. The apparent molecular mass of the one-chain botrocetin was 28 kDa before and 32 kDa after reduction of disulfide bonds, while that of the two-chain botrocetin was 27 kDa before and 15/14.5 kDa after reduction. Amino acid composition of the two species revealed a similar high content of potentially acidic residues (greater than 60 Asx and Glx residues/molecule) but significant differences in the content of Cys and Phe residues. The NH2-terminal sequence of the one-chain botrocetin was Ile-Ile/Val-Ser-Pro-Pro-Val-Cys-Gly-Asn-Glu-. Two constituent polypeptides of the two-chain botrocetin showed similar but different NH2-terminal sequences, distinct from that of the one-chain species: (alpha) Asp-Cys-Pro-Ser-Gly-Trp-Ser-Ser-Tyr-Glu- and (beta) Asp-Cys-Pro-Pro-Asp-Trp-Ser-Ser-Tyr-Glu-. The carbohydrate content of both species was less than 2% of the total mass, and the pI was 4.0-4.1 for the one-chain species, and 4.6, 5.3-5.4, and 7.7-7.8 for the two-chain species. No free sulfhydryl group was detected in each species. Both types of botrocetin were resistant to proteolysis at neutral pH. Incubation of 125I-labeled one-chain botrocetin with the crude venom solution resulted in no detectable structural change. On a weight basis, the two-chain botrocetin was 34 times more active than the one-chain form in promoting vWF binding to platelets.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation and chemical characterization of two structurally and functionally distinct forms of botrocetin, the platelet coagglutinin isolated from the venom of Bothrops jararaca. 199 6

By means of Fractogel TSK-50, CM-Sephadex C-50 column chromatography, gel filtrations on Sephadex G-75 and Sephacryl S-200 columns and reverse-phase HPLC, an antiplatelet peptide, arietin, was purified from venom of Bitis arietans. Arietin was shown to be an Arg-Gly-Asp-containing peptide with a NH2-terminus, Ser-Pro-Pro-Val-Cys-Gly-Asn-Lys- (Mr 8500). Arietin dose-dependently inhibited aggregation of human platelet suspension stimulated by ADP, thrombin, collagen and U46619 with IC50, 1.3-2.7.10(-7) M, while it had no effect on the initial shape changes and only slightly affected ATP release of platelets caused by thrombin and collagen. Arietin also blocked platelet aggregation in platelet-rich plasma and whole blood, and inhibited thrombin-induced clot retraction of platelet-rich plasma. Furthermore, arietin (6.5.10(-8) M) completely blocked the fibrinogen-induced aggregation of elastase-treated platelets, indicating that arietin interferes with the fibrinogen binding to fibrinogen receptors on platelet membranes. In conclusion, arietin, an Arg-Gly-Asp-containing peptide, inhibits platelet aggregation probably through the blockade of fibrinogen binding to the activated platelets.
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PMID:Purification and characterization of an antiplatelet peptide, arietin, from Bitis arietans venom. 204 63

Three geometric isomers of a single triantennary glycopeptide, each containing two fluorophores attached to terminal positions in the molecule, were used to probe distance and flexibility of the oligosaccharide in solution. A dansyl group (energy acceptor) was attached to the C6 of Gal at either position 6', 6, or 8, and a naphthyl-2-acetyl group (energy donor) was coupled to the N terminus of the Ala-Asn peptide. (formula; see text) Resonance energy-transfer measurements revealed an average distance of approximately 22, 18, and 17 A between the donor and the acceptor attached to either the 6, 8, or 6' Gal residue, respectively. The lifetime of the donor's emission was nearly a single-exponential decay of 27 ns (96%), whereas the decay of the donor with proximally attached acceptor was fit by nonlinear least-squares analysis to a multiexponential for each glycopeptide probe. Fitting with a Lorentzian function revealed spatially distinct donor/acceptor distances presumably arising from glycopeptide branch flexibility. The results suggest that the acceptor located at Gal 8 is the most rigid relative to the donor with a single population of distances centered at 18.4 A. In contrast, the acceptor attached to either Gal 6' or 6 displayed two populations of different distances from the donor. The Gal 6 isomer contained a major population with average donor/acceptor separation distance of 21.7 A and a minor population with average separation distance of 9.7 A. Similarly, the Gal 6' isomer showed a major population with donor/acceptor separation distance of 18.3 A and a minor population with separation distance of 11.7 A. These data support the earlier conclusions that the Man alpha(1----6)Man linkage found in the core pentasaccharide of all branched N-linked oligosaccharides is flexible. In addition, the data suggest that the branch containing Gal 6 is also flexible in the triantennary glycopeptide.
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PMID:Interterminal distance and flexibility of a triantennary glycopeptide as measured by resonance energy transfer. 206 52

A peptide of acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7) from the venom of the cobra Naja naja oxiana labeled by the affinity reagent N,N-dimethyl-2-phenylaziridinium (DPA) has been identified. The sequence is Gly-Ala-Glu-Met-Trp-Asn-Pro-Asn. In AcChoEase from Torpedo californica, a homologous peptide was labeled and isolated. Its sequence is Ser-Gly-Ser-Glu-Met-Trp-Asn-Pro-Asn, representing positions 79 through 87. In both cases labeling can be prevented by 0.1 mM edrophonium, indicating that the respective peptides form part of the anionic subsite of the catalytic center. The modified residue was tryptophan (Trp-84 in Torpedo AcChoEase) in both enzymes. In contrast to AcChoEase from Torpedo, the enzyme from cobra venom does not contain a peripheral anionic binding site.
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PMID:Anionic subsites of the catalytic center of acetylcholinesterase from Torpedo and from cobra venom. 206 91

We have isolated a 2.7-kilobase rat liver cDNA clone that contains the entire 544-amino acid coding sequence for matrin F/G. This protein has previously been localized to the internal, fibrogranular areas of the nuclear matrix and shown to bind to DNA on nitrocellulose blots. The predicted amino acid sequence from the coding region of this cDNA shows that this protein contains approximately 50% hydrophobic amino acids with secondary structure predictions suggesting a large percentage of beta-sheet regions. No significant homologies were found with any other known proteins, including the nuclear lamins. The predicted amino acid sequence was also searched for DNA binding motifs. Two putative zinc finger motifs were found. In addition, a 7-mer palindromic sequence (Ser-Ser-Thr-Asn-Thr-Ser-Ser) was discovered within one of these zinc finger DNA binding regions. A possible regulatory role for this element is discussed.
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PMID:Molecular cloning of matrin F/G: A DNA binding protein of the nuclear matrix that contains putative zinc finger motifs. 206

Isolated rat brain synaptosomes accumulated L-asparagine with a Km value of 348 microM and a Vmax value of 3.7 nmol/mg of protein/min at 28 degrees C. Uptake of L-asparagine was inhibited by the presence of L-glutamine, whereas transport of L-glutamine was blocked by L-asparagine. Alanine, serine, cysteine, threonine, and, in particular, leucine were also inhibitory whereas alpha-(methylamino)isobutyrate, ornithine, lysine, arginine, and glutamate were much less effective blockers. Transport of L-asparagine had a substantial sodium-dependent component, whereas that of the D-stereoisomer was almost unaffected by the presence or absence of the cation. L-Asparagine was accumulated to a maximal gradient, [L-Asn]i/[L-Asn]o, of 20-30, and this value was reduced to 5-6 by withdrawal of sodium or addition of high [KCI]. A plot of log [Na+]o/[Na+]i against the log [L-Asn]i/[L-Asn]o had a slope close to I, which indicates that a single sodium ion is transported inward with each asparagine molecule. It is postulated that uptake of L-asparagine occurs, to a large extent, in cotransport with Na+ and that it utilizes the sodium chemical gradient and the membrane electrical potential as the source of energy. The similarity between the L-asparagine and L-glutamine transport systems and the reciprocal inhibition of influx of the two amino acids suggest that the same mechanism is responsible for glutamine accumulation. This could explain the high [Gln]i maintained by the brain in vivo.
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PMID:Transport of asparagine by rat brain synaptosomes: an approach to evaluate glutamine accumulation. 207 99

Boc-L-Asn-L-Pro-OBzl: C21H29O6N3.CH3OH, Mr = 419.48 + CH3 OH, monoclinic, P2(1), a = 10.049(1), b = 10.399(2), c = 11.702(1) A, beta = 92.50(1)degrees, V = 1221.7(3) A3, dx = 1.14 g.cm-3, Z = 2, CuK alpha (lambda = 1.54178 A), F(000) = 484 (with solvent), 23 degrees, unique reflections (I greater than 3 sigma(I)) = 1745, R = 0.043, Rw = 0.062, S = 1.66. Boc-beta-cyano-L-alanine-L-Pro-OBzl: C21H27O5N3, Mr = 401.46, orthorhombic, P2(1)2(1)2(1), a = 15.741(3), b = 21.060(3), c = 6.496(3) A, V = 2153(1) A3, dx = 1.24 g.cm-3, Z = 4, CuK alpha (lambda = 1.54178 A), F(000) = 856, 23 degrees, unique reflections (I greater than 3 sigma(I)) = 1573, R = 0.055, Rw = 0.078, S = 1.86. The tert.-butyloxycarbonyl (Boc) protected dipeptide benzyl ester (OBzl), Boc-L-Asn-L-Pro-OBzl, prepared from a mixed anhydride reaction using isobutylchloroformate, Boc-L-asparagine, and HCl.L-proline-OBzl, crystallized with one methanol per asymmetric unit in an extended conformation with the Asn-Pro peptide bond trans. Intermolecular hydrogen bonding occurs between the methanol and the Asn side chain and between the peptide backbone and the Asn side chain. A minor impurity due to the dehydration of the Asn side chain to a beta-CNala crystallized with a similar extended conformation and a single intermolecular hydrogen bond.
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PMID:Synthesis and crystal structures of Boc-L-Asn-L-Pro-OBzl.CH3OH and dehydration side product, Boc-beta-cyano-L-alanine-L-Pro-OBzl. 209 Jun 45


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