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A new enzyme which catalyzes the oxidation of the side chain of tryptophan and other indole derivatives, has been purified to apparent homogeneity from Pseudomonas and crystallized. The overall purification was about 25-fold with a yield of 4.5%. The purified enzyme was apparently homogeneous as judged by polyacrylamide gel electrophoresis. The molecular weight estimated by gel filtration was approximately 280,000 and sedimentation coefficient (S20,w) was 11 by sucrose density gradient ultracentrifugation. The absorption spectra indicated that the enzyme was a hemoprotein. The purified enzyme was shown to catalyze the reaction in which 1 mol each of NH3 and CO2 was formed at the expense of 1 mol each of L-tryptophan and molecular oxygen. Neither peroxidase nor catalase activity was detected in the purified enzyme and no formation of H2O2 was observed during the enzyme reaction. The product(s) of the reaction was unstable but was converted to and was identified as its stable quinoxaline derivative, 2-(3-indolyl)quinoxaline, in the presence of o-phenylenediamine. These results indicate that the product of the reaction was 3-indolylglycoaldehyde or 3-indolylglyoxal. A variety of other indole derivatives such as D-tryptophan, 5-hydroxyl-L-tryptophan, tryptamine, serotonin, melatonin, N-acetyl-L-tryptophan, N-acetyl-L-tryptophanamide, 3-indoleacetamide, 3-indolelactic acid, 3-indolepropionic acid, 3-indoleethanol, and skatole were also substrates.  相似文献   
64.
Summary Specificity of reception on 11 electrolytes in the slime moldPhysarum polycephalum was investigated in the presence of polyvalent cations in media. Membrane potential and motive force of tactic movement were examined with the aid of the double chamber method, and the zeta potential at the membrane surface of the slime mold was measured by electrophoretic mobility. The results obtained are summarized as follows: (1) The presence of polyvalent cations (e.g., Ca2+, Mg2+, Sr2+, Ba2+, La3+, Th4+) in medium led to an increase in threshold concentration,C th , determined from the potential measurements for Na- or Li-salts, and to a decrease inC th for K-, Rb-, or NH4-salts,C th for 11 electrolytes changed discontinuously when the concentration of polyvalent cations in medium exceeded their respective thresholds. (2) TheC th determined from chemotaxis agreed with that from the potential response both in the presence and absence of polyvalent cations. (3) Sequence of selectivity of univalent cations varied extensively in the presence of polyvalent cations. (4) Changes in the zeta potential induced by NaCl reception agreed with those in the membrane potential even in the presence of Ca2+ in medium. (5) TheC th for reception of NaCl changed sharply at about 12 °C in the presence of polyvalent cations, while that for KCl was independent of the temperature.Conformational changes in surface membrane of the slime mold in response to reception of polyvalent cations were then discussed in relation to the discrimination of univalent cations.  相似文献   
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We have established a procedure for converting porcine insulin into human insulin using a serine protease from Achromobacterlyticus M497-1 which shows unique specificity against lysine residues on the carboxyl side of the splitting point. Desalanine-(B30)-insulin (DAI) was prepared by digestion of porcine insulin with Achromobacter protease. The coupling between DAI and Thr-OBut was performed by the same enzyme at pH 6.5 with a large excess of the amine component (Thr-OBut) in the presence of high concentrations of organic co-solvents. The highest yield was 85% by 20 h reaction at 37°C. The synthesized [Thr-OBut-B30]-insulin was isolated, then deprotected with trifluoroacetic acid in the presence of anisole to obtain semisynthetic human insulin.  相似文献   
67.
The isolation and reconstitution of two electron transfer components of tryptophan side chain oxidase from Pseudomonas (ATCC 29574) are described. The dehydrogenase component abstracts electrons from the substrate and transfers them to oxidation-reduction dyes such as potassium ferricyanide and 2,6-dichlorophenolindophenol but not to molecular oxygen. It is composed of a single polypeptide chain with a molecular weight of 72,000 and exhibits the absorption spectrum of a reduced b-type cytochrome with maxima at 563, 532, 433, 323, and 278 nm. The oxidase component transfers electrons, derived from the former component, to oxygen, and has a molecular weight of 48,000. The absorption spectrum exhibits broad peaks at 680, 438, and 358 nm, and a peak at 280 nm. On sucrose gradient centrifugation and polyacrylamide gel electrophoresis, these two components are shown to form a molecular complex, which has the reconstituted oxidase activity. The turnover number of the reconstituted enzyme is comparable to that of the native enzyme.  相似文献   
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Partially purified β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans showed high activity towards both pure lactose and lactose in skim milk, and a better thermal stability than the enzyme from yeast or Escherichia coli. During the course of hydrolysis of lactose catalysed by the enzyme, considerable amounts of oligosaccharides were produced. β-d-Galactosidase from B. circulans was immobilized onto Duolite ES-762, Dowex MWA-1 and sintered alumina by adsorption with glutaraldehyde treatment. The highest activity for hydrolysis of lactose was obtained with immobilization onto Duolite ES-762. During a continuous hydrolysis of lactose, the immobilized enzyme was reversibly inactivated, probably due to oligosaccharides accumulating in the gel. The inactivation was reduced when a continuous reaction was operated at a high percent conversion of lactose in a continuous stirred tank reactor (CSTR). The half-life of the immobilized enzyme was estimated to be 50 and 15 days at 50 and 55°C, respectively, when the reaction was carried out in a CSTR with a percent conversion of lactose >70%.  相似文献   
70.
IL-4-supported induction of cytolytic T lymphocytes requires IL-2 and IL-6   总被引:1,自引:0,他引:1  
Previous work indicated that a CTL response can be generated by the combination of IL-2 plus IL-6 or IL-4 alone. Because of the ubiquitous production of IL-6 and its apparent ability to induce IL-2, we explored the interdependence of these lymphokines in supporting a CTL response from murine thymocytes. For thymocytes cultured in IL-4, further addition of IL-6 enhanced thymocyte proliferation. In addition, a role for IL-6 in thymocyte activation was indicated by the ability of anti-IL-6 mAb to block both IL-4-directed proliferation and the cytotoxic response found in the presence of IL-4. The addition of IL-2 to limiting doses of IL-4 augmented the CTL response; however, the response to high levels of IL-4 was not augmented by addition of IL-2. Consistent with this apparent involvement of IL-2 in the IL-4-mediated response we found: (a) that mAb to IL-2 significantly reduced the CTL response generated in the presence of IL-4; (b) that IL-2 activity was present in culture supernatant following incubation of thymocytes with high levels of IL-4; and (c) that enhanced IL-2 receptor expression found in the presence of IL-4 was blocked with the addition of anti-IL-2 antibody to the thymocyte culture. In contrast to the data for proliferation, anti-IL-4 mAb had no effect on the generation of CTL in the presence of IL-2 + IL-6 but readily blocked the CTL response to IL-4. These results indicate that, for thymocyte responders, the CD8+ CTL generated in the presence of IL-4 require both IL-2 and IL-6.  相似文献   
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