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101.
S M Janes M M Palcic C H Scaman A J Smith D E Brown D M Dooley M Mure J P Klinman 《Biochemistry》1992,31(48):12147-12154
The nature of the active site cofactor and the amino acid sequence flanking this structure have been determined in a range of copper amine oxidases. For enzymes from porcine plasma, porcine kidney, and pea seedlings, proteolytic digestion was performed on phenylhydrazone or p-nitrophenylhydrazone derivatives. Thermolysin treatment leads to relatively small active site peptides, which have been characterized by Edman degradation and by resonance Raman spectroscopy. Resonance Raman spectra of peptides show identical peak positions and intensities relative to each other and to a model p-nitrophenylhydrazone derivative of topaquinone hydantoin, establishing topaquinone as the cofactor in each instance. Edman degradation of peptides provides active site sequences for comparison to previous determinations with bovine serum and yeast amine oxidases. The available data establish a consensus sequence of Asn, Topa, Asp/Glu. Trypsin leads to significantly longer peptides, which reveal a high degree of sequence identity between plasma proteins from bovine and porcine sources (89%), with significantly decreased identity between the porcine serum and intracellular amine oxidases (56%). A lower degree of identity (45%) is observed between the pea seedling and mammalian enzymes. As an alternative to the isolation of active site peptides for topaquinone identification, visible spectra of intact proteins have been investigated. It is shown that p-nitrophenylhydrazone derivatives of native enzymes, active site-derived peptides, and a topaquinone model exhibit identical behavior, absorbing at 457-463 nm at neutral pH (pH 7.2) and at 575-587 nm in basic solution (1-2 M KOH). These spectral properties, which appear unique to topaquinone, provide a rapid and simple test for the presence of this cofactor in intact enzymes.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
102.
Transition state binding theory utilizes non-enzymic and enzymic rate ratios to predict the ratio of transition state analog dissociation constants to substrate dissociation constants. In this paper we show that enzyme rate accelerations due solely to lessened entropy requirements, arising from the juxtaposition of a catalytic group and a substrate binding site at an enzyme active site, will result in a ratio of transition state and substrate dissociation constants which is different, in general, from the ratio of non-enzymic and enzymic rate constants. The arguments presented in this paper provide a possible explanation for the frequently observed large discrepancy between the measured and predicted values for transition state analog dissociation constants. 相似文献
103.
Copper amine oxidase (CAO) is a dual-functioning enzyme that catalyzes the biosynthesis of a self-derived coenzyme and subsequent oxidative deamination of primary amines. The organic cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), is generated from the post-translational modification of an active site tyrosine (Y405) in a reaction shown to be dependent on both molecular oxygen and a mononuclear copper center. Previous investigations of Cu(II)-dependent cofactor formation in the Hansenula polymorpha amine oxidase (HPAO) provided evidence for the coordination of the precursor tyrosine in forming a ligand-to-metal charge transfer complex as a means of activating the tyrosyl ring for direct attack by triplet-state dioxygen. To further delineate the role of the metal in facilitating this complex series of reactions, apo-HPAO was reconstituted with alternate metals of varying reduction potentials and Lewis acidities [Ni(II), Co(II), Mn(II), Fe(II), and Fe(III)] and the consequence of each substitution on TPQ biogenesis examined. Ni(II) was found to support the transformation of the precursor tyrosine to the quinone cofactor to yield a mature enzyme competent for methylamine oxidation. Detailed kinetic analysis of the mechanism of TPQ biogenesis for the Ni(II)-substituted enzyme has led to the proposal of a direct electron transfer from the metal-coordinated tyrosinate to dioxygen as the dominant rate-limiting step. 相似文献
104.
Sen G Flora M Chattopadhyay G Klinman DM Lees A Mond JJ Snapper CM 《Cellular immunology》2004,232(1-2):64-74
Double- and single-stranded oligodeoxynucleotides containing unmethylated cytosine-guanosine (CpG) dinucleotides (CpG-ODN) activate immune cells via TLR9. In this report we synthesized hybrid DNA-RNA molecules (HDR) in order to further explore the structure-immune function relationship of CpG-ODN in TLR9 signaling and the potential immunomodulatory properties of RNA. We demonstrate that replacement of the deoxyadenosine flanking sequences, critical for the immune activating properties of CpG-ODN, with a similar number of adenosines, although not guanosines, cytosines, or uracils, maintains complete immunostimulatory activity of the hybrid oligonucleotide in vitro, whereas a similar RNA replacement of even 1 base of the required unmethylated 6 base DNA motif (purine-purine-CpG-pyrimidine-pyrimidine) results in a complete loss of activity. Regardless of whether the critical flanking sequence was RNA or DNA there was no significant change in the quantitative or qualitative immune-stimulating activity, or TLR-specificity of the resulting sequences, thus underscoring the relatively permissive functional role of the flanking sequence, and the more specific role of the motif in mediating TLR9 signaling. These data further support a potential role for RNA in immunomodulation. 相似文献
105.
106.
Clara Correia‐Melo Francisco DM Marques Rhys Anderson Graeme Hewitt Rachael Hewitt John Cole Bernadette M Carroll Satomi Miwa Jodie Birch Alina Merz Michael D Rushton Michelle Charles Diana Jurk Stephen WG Tait Rafal Czapiewski Laura Greaves Glyn Nelson Mohammad Bohlooly‐Y Sergio Rodriguez‐Cuenca Antonio Vidal‐Puig Derek Mann Gabriele Saretzki Giovanni Quarato Douglas R Green Peter D Adams Thomas von Zglinicki Viktor I Korolchuk João F Passos 《The EMBO journal》2016,35(7):724-742
107.
D M Klinman 《Journal of immunology (Baltimore, Md. : 1950)》1990,144(7):2529-2534
Autoimmune MRL-lpr/lpr and NZB/W mice spontaneously secrete large quantities of pathogenic IgG1 and IgG2a autoantibodies. NZB mice also produce autoantibodies but these tend to be of the IgM H chain class. This work examines whether differences in the isotype of autoantibody produced by lupus-prone mice reflects differences in the sensitivity of autoreactive B cells to lymphokine-mediated IgG secretion. Twenty-five percent of normal BALB/c B cells produced IgG1 when stimulated in vitro with IL-4 plus LPS. This was comparable with the effect of IL-4 on small resting B cells from MRL-lpr/lpr and NZB/W mice. In contrast, less than 8% of the resting B cells from NZB mice produced IgG1 under these conditions. LPS plus IFN-gamma induced 5% of BALB/c and NZB/W but only 1% of NZB B cells to secrete IgG2a. Because lymphocytes from both young and old NZB mice showed diminished IgG1 and IgG2a secretion after lymphokine treatment, B cells from this strain appeared to be intrinsically resistant to the effects of IL-4 and IFN-gamma. In contrast, a disproportionately large proportion (22%) of B cells from adult MRL-lpr/lpr mice produced IgG2a when treated with IFN-gamma in vitro. Only B cells from MRL-lpr/lpr mice with active disease responded with such high levels of IgG2a production: cells from animals that had not yet developed clinical disease produced normal levels of IgG2a. Within each strain, B cells producing antibodies against autoantigens such as DNA, bromelain-treated mouse RBC and Sm responded to treatment with IL-4 and IFN-gamma in a manner indistinguishable from B cells producing antibodies against conventional Ag such as TNP and ARS. 相似文献
108.
Plasma amine oxidases catalyze the oxidative deamination of amines to aldehydes, followed by a 2e- reduction of O2 to H2O2. Pyrroloquinoline quinone (PQQ), previously believed to be restricted to prokaryotes, has recently been proposed to be the cofactor undergoing reduction in the first half-reaction of bovine plasma amine oxidase (Ameyama, M., Hayashi, U., Matsushita, K., Shinagawa, E., and Adachi, O. (1984) Agric. Biol. Chem. 48, 561-565; Lobenstein-Verbeek, C. L., Jongejan, J. A., Frank, J., and Duine, J. A. (1984) FEBS Lett. 170, 305-309). This result is unexpected, since model studies with PQQ implicate Schiff's base formation between a reactive carbonyl and substrates, whereas experiments with bovine plasma amine oxidase have failed to provide evidence for a carbonyl cofactor. We have, therefore, re-examined putative adducts between substrate and enzyme-bound cofactor, employing a combination of [14C]benzylamine and [3H]NaCNBH3. The use of the relatively weak reductant, NaCNBH3, affords Schiff's base specificity and permits the study of enzyme below pH 7.0. As we show, enzyme can only be inactivated by NaCNBH3 in the presence of substrate, leading to the incorporation of 1 mol of [14C]benzylamine/mol of enzyme subunit at complete inactivation. By contrast, we are unable to detect any labeling with [3H]NaCNBH3, analogous to an earlier study with [3H]NaCNBH4 (Suva, R. H., and Abeles, R. H. (1978) Biochemistry 17, 3538-3545). We conclude, first, that our inability to obtain adducts containing both carbon 14 and tritium rules out the reductive trapping either of amine substrate with pyridoxal phosphate or of aldehyde product with a lysyl side chain and, second, that the observed pattern of labeling is fully consistent with the presence of PQQ at the active site of bovine plasma amine oxidase. 相似文献
109.
The mechanism of the first electron transfer from reduced cofactor to O2 in the catalytic cycle of copper amine oxidases (CAOs) remains controversial. Two possibilities have been proposed. In the first mechanism, the reduced aminoquinol form of the TPQ cofactor transfers an electron to the copper, giving radical semiquinone and Cu(I), the latter of which reduces O2 (pathway 1). The second mechanism invokes direct transfer of the first electron from the reduced aminoquinol form of the TPQ cofactor to O2 (pathway 2). The debate over these mechanisms has arisen, in part, due to variable experimental observations with copper amine oxidases from plant versus other eukaryotic sources. One important difference is the position of the aminoquinol/Cu(II) to semiquinone/Cu(I) equilibrium on anaerobic reduction with amine substrate, which varies from almost 0% to 40% semiquinone/Cu(I). In this study we have shown how protein structure controls this equilibrium by making a single-point mutation at a second-sphere ligand to the copper, D630N in Hansenula polymorpha amine oxidase, which greatly increases the concentration of the cofactor semiquinone/Cu(I) following anaerobic reduction by substrate. The catalytic properties of this mutant, including 18O kinetic isotope effects, point to a conservation of pathway 2, despite the elevated production of the cofactor semiqunone/Cu(I). Changes in kcat/Km[O2] are attributed to an impact of D630N on an increased affinity of O2 for its hydrophobic pocket. The data in this study indicate that changes in cofactor semiquinone/Cu(I) levels are not sufficient to alter the mechanism of O2 reduction and illuminate how subtle features are able to control the reduction potential of active site metals in proteins. 相似文献
110.
Characterization of new mouse V kappa groups 总被引:3,自引:0,他引:3
A lambda gt10 BXSB spleen cDNA library was screened with a DNA probe for the C kappa region. Forty individual C kappa+ phages were tested for hybridization with DNA probes representing 11 V kappa region groups. Of the phage inserts large enough to contain V kappa region sequences, 3 were negative for hybridization with all 11 V kappa region probes. The inserts from those three were subcloned, sequenced, and compared with V kappa region sequences in the gene bank. One was identical to 87.92.6 for the region sequenced (a member of V kappa RF). The second showed 93.8% sequence similarity with AN04 and called V kappa 32. The third called V kappa 33 showed 76% sequence similarity with the human sequence V52 and 73.2% sequence similarity with the mouse sequence L6. An insert from V kappa 32 containing the 5' untranslated regions through the codon for Cys 88 of the V kappa region was used as a probe in Southern blot analysis of genomic DNA from inbred and congenic strains of mice. V kappa 32 is a four to eight member group and some of the members are retained in the B6.PL-Ly2a congenic and missing from the B6.PL (85NS) congenic consistent with a map location near V kappa 28. The same filters were hybridized with the insert from V kappa 33 containing 5' untranslated region through the codon for Ser 93 of the V kappa region. V kappa 33 is a one to three member group and using the B6.PL congenics maps with the polymorphic fragments of V kappa 32 and V kappa 28. 相似文献