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Growth ofPseudomonas testosteroni in a medium containing 1mm Cu(II) causes a color change from blue to green. The spectrum of the supernatant solution from the blue culture shows an absorption at 660 nm, identical to that of 1mm [Cu(II)] in the medium. The green supernatant solution shows a UV absorption, which tails into the visible and so is responsible for the green color, and ad-d absorption at 720 nm. The absorption at 660 nm for the blue supernatant solution is probably due to [Cu(NH3)3(H2O)3]2+. Growth of the organism causes loss of ammonia and a speciation change to [Cu(NH3)2(H2O)4]2+, with a shift in absorption maximum from 660 to 720 nm. These conclusions are based upon the spectra of known aquaammine complexes of Cu(II) and calculations of the speciation of Cu(II) before and after growth. Change in metal speciation owing to nutrient uptake by an organism does not appear to have been recognized previously. 相似文献
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Human plasma alpha1-antitrypsin inhibits human pancreatic trypsin, chymotrypsin and elastase, which are massively released into the blood stream during acute pancreatitis. To examine whether the plasma proteins of individuals with genetic deficiency of alpha1-antitrypsin are protected against the deleterious action of these enzymes by other inhibitors, we have tested their inhibition by alpha2-antiplasmin and antithrombin. We have determined the inhibition rate constants kass and calculated d(t), the in vivo inhibition time. Surprisingly, trypsin is inhibited faster by alpha2-antiplasmin [kass=2.5 x 10(6) M(-1)S(-1), d(t)=2.3 s] and antithrombin [kass=1.7 x 10(5) M(-1)s(-1), d(t)=5.8 s] than by alpha1-antitrypsin [d(t)=17 s or 116 s in alpha1-antitrypsin-sufficient or alpha1-antitrypsin-deficient individuals, respectively]. Low molecular weight heparin accelerates the inhibition of trypsin by antithrombin by a factor of 16 [d(t)=0.36 s]. Antithrombin and alpha2-antiplasmin are not physiological inhibitors of chymotrypsin and elastase. These enzymes are, however, physiologically inhibited by alpha1-antitrypsin and alpha1-antichymotrypsin even in alpha1-antitrypsin-deficient individuals. We conclude that (i) low molecular weight heparin may be helpful in the management of acute pancreatitis, and (ii) genetically determined alpha1-antitrypsin deficiency probably does not lead to a significantly increased risk of plasma protein degradation during this disease. 相似文献
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Mohammad Saeid Hejazi Kamal Kazemi Tabar Reza Azarbaijani Laleh Zereshki Nobar 《Annals of microbiology》2007,57(2):259-263
Cloning, sequencing and analysis of partial segment of cholesterol oxidase(cho) encoding gene fromStreptomyces luridus using PCR technique is described. The primers used for PCR were designed on the basis of inter-species homology, and the pTZ57R/T vector was used for molecular cloning. The sequencing results lead to identification of the oxidoreductase domain ofcho enzyme with 818 nucleotide length encoding 272 amino acids, which was submitted in NCBI with DQ480365 accession number. The submitted sequence in cludes complete FAD-linked reductase C terminal domain with 170 amino acids size and N-terminal of GMC-oxidoreductase domain ofcho enzyme. 相似文献
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Marie-Louise Zani Shila M Nobar Sandrine A Lacour Soazig Lemoine Christian Boudier Joseph G Bieth Thierry Moreau 《European journal of biochemistry》2004,271(12):2370-2378
Elafin and its precursor, trappin-2 or pre-elafin, are specific endogenous inhibitors of human neutrophil elastase and proteinase 3 but not of cathepsin G. Both inhibitors belong, together with secretory leukocyte protease inhibitor, to the chelonianin family of canonical protease inhibitors of serine proteases. A cDNA coding either elafin or its precursor, trappin-2, was fused in frame with yeast alpha-factor cDNA and expressed in the Pichia pastoris yeast expression system. Full-length elafin or full-length trappin-2 were secreted into the culture medium with high yield, indicating correct processing of the fusion proteins by the yeast KEX2 signal peptidase. Both recombinant inhibitors were purified to homogeneity from concentrated culture medium by one-step cationic exchange chromatography and characterized by N-terminal amino acid sequencing, Western blot and kinetic studies. Both recombinant elafin and trappin-2 were found to be fast-acting inhibitors of pancreatic elastase, neutrophil elastase and proteinase 3 with k(ass) values of 2-4 x 10(6) m(-1).s(-1), while dissociation rate constants k(diss) were found to be in the 10(-4) s(-1) range, indicating low reversibility of the complexes. The equilibrium dissociation constant K(i) for the interaction of both recombinant inhibitors with their target enzymes was either directly measured for pancreatic elastase or calculated from k(ass) and k(diss) values for neutrophil elastase and proteinase 3. K(i) values were found to be in the 10(-10) molar range and virtually identical for both inhibitors. Based on the kinetic parameters determined here, it may be concluded that both recombinant elafin and trappin-2 may act as potent anti-inflammatory molecules and may be of therapeutic potential in the treatment of various inflammatory lung diseases. 相似文献
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Neutrophil proteinase-mediated lung tissue destruction is prevented by inhibitors, including elafin and its precursor, trappin. We wanted to establish whether neutrophil-derived oxidants might impair the inhibitory function of these molecules. Myeloperoxidase/H(2)O(2) and N-chlorosuccinimide oxidation of the inhibitors was checked by mass spectrometry and enzymatic methods. Oxidation significantly lowers the affinities of the two inhibitors for neutrophil elastase (NE) and proteinase 3 (Pr3). This decrease in affinity is essentially caused by an increase in the rate of inhibitory complex dissociation. Oxidized elafin and trappin have, however, reasonable affinities for NE (K(i) = 4.0-9.2 x 10(-9) M) and for Pr3 (K(i) = 2.5-5.0 x 10(-8) M). These affinities are theoretically sufficient to allow the oxidized inhibitors to form tight binding complexes with NE and Pr3 in lung secretions where their physiological concentrations are in the micromolar range. Yet, they are unable to efficiently inhibit the elastolytic activity of the two enzymes. At their physiological concentration, fully oxidized elafin and trappin do not inhibit more than 30% of an equimolar concentration of NE or Pr3. We conclude that in vivo oxidation of elafin and trappin strongly impairs their activity. Inhibitor-based therapy of inflammatory lung diseases must be carried out using oxidation-resistant variants of these molecules. 相似文献
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An intracellular serpin regulates necrosis by inhibiting the induction and sequelae of lysosomal injury 总被引:5,自引:0,他引:5
Luke CJ Pak SC Askew YS Naviglia TL Askew DJ Nobar SM Vetica AC Long OS Watkins SC Stolz DB Barstead RJ Moulder GL Brömme D Silverman GA 《Cell》2007,130(6):1108-1119
Extracellular serpins such as antithrombin and alpha1-antitrypsin are the quintessential regulators of proteolytic pathways. In contrast, the biological functions of the intracellular serpins remain obscure. We now report that the C. elegans intracellular serpin, SRP-6, exhibits a prosurvival function by blocking necrosis. Minutes after hypotonic shock, srp-6 null animals underwent a catastrophic series of events culminating in lysosomal disruption, cytoplasmic proteolysis, and death. This newly defined hypo-osmotic stress lethal (Osl) phenotype was dependent upon calpains and lysosomal cysteine peptidases, two in vitro targets of SRP-6. By protecting against both the induction of and the lethal effects from lysosomal injury, SRP-6 also blocked death induced by heat shock, oxidative stress, hypoxia, and cation channel hyperactivity. These findings suggest that multiple noxious stimuli converge upon a peptidase-driven, core stress response pathway that, in the absence of serpin regulation, triggers a lysosomal-dependent necrotic cell death routine. 相似文献
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