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81.
Okubo A Li M Ashida M Oyama H Gustchina A Oda K Dunn BM Wlodawer A Nakayama T 《The FEBS journal》2006,273(11):2563-2576
Kumamolisin-As is an acid collagenase with a subtilisin-like fold. Its active site contains a unique catalytic triad, Ser278-Glu78-Asp82, and a putative transition-state stabilizing residue, Asp164. In this study, the mutants D164N and E78H/D164N were engineered in order to replace parts of the catalytic machinery of kumamolisin-As with the residues found in the equivalent positions in subtilisin. Unlike the wild-type and D164N proenzymes, which undergo instantaneous processing to produce their 37-kDa mature forms, the expressed E78H/D164N proenzyme exists as an equilibrated mixture of the nicked and intact forms of the precursor. X-ray crystallographic structures of the mature forms of the two mutants showed that, in each of them, the catalytic Ser278 makes direct hydrogen bonds with the side chain of Asn164. In addition, His78 of the double mutant is distant from Ser278 and Asp82, and the catalytic triad no longer exists. Consistent with these structural alterations around the active site, these mutants showed only low catalytic activity (relative k(cat) at pH 4.0 1.3% for D164N and 0.0001% for E78H/D164N). pH-dependent kinetic studies showed that the single D164N substitution did not significantly alter the logk(cat) vs. pH and log(k(cat)/Km) vs. pH profiles of the enzyme. In contrast, the double mutation resulted in a dramatic switch of the logk(cat) vs. pH profile to one that was consistent with catalysis by means of the Ser278-His78 dyad and Asn164, which may also account for the observed ligation/cleavage equilibrium of the precursor of E78H/D164N. These results corroborate the mechanistic importance of the glutamate-mediated catalytic triad and oxyanion-stabilizing aspartic acid residue for low-pH peptidase activity of the enzyme. 相似文献
82.
Hydrogen Peroxide-dependent Synthesis of Flavonols in Mesophyll Cells of Vicia faba L. 总被引:2,自引:0,他引:2
Mesophyll cells of Vicia faba contain kaempferol and quercetinglycosides. When isolated mesophyll cells were treated with0.1 mM H2O2 for 2 h, the levels of these flavonols increasedby 1070% of the control values (mean values, 19.6% and34.4% for kaempferol and quercetin glycosides, respectively).Such increases in levels of flavonols were also observed inisolated vacuoles of mesophyll cells. However, when mesophyllcells and vacuoles were treated with 10 mM H2O2)degradationof flavonols was observed. These data suggest that H2O2 hastwo effects on the metabolism of flavonols: induction of theirsynthesis and stimulation of their oxidation. (Received March 6, 1989; Accepted July 10, 1989) 相似文献
83.
84.
Akira Watanabe Kazumi Hiraga Masako Suda Hideaki Yukawa Masayuki Inui 《Applied and environmental microbiology》2015,81(12):4173-4183
The Corynebacterium alkanolyticum
xylEFGD gene cluster comprises the xylD gene that encodes an intracellular β-xylosidase next to the xylEFG operon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover for p-nitrophenyl-β-d-xylopyranoside of 111 ± 4 s−1), weak α-l-arabinofuranosidase activity (turnover for p-nitrophenyl-α-l-arabinofuranoside of 5 ± 1 s−1), and high tolerance to product inhibition (Ki for xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutive tac promoter in the Corynebacterium glutamicum xylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended on cgR_2369, a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguous xylD gene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability of C. glutamicum to take up xylooligosaccharides, an ability that is enhanced by in the presence of a functional xylEFG-encoded xyloside ABC transporter. The finding that xylEFG imparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates. 相似文献
85.
Masako Osumi Fusako Fukuzumi Yutaka Teranishi Atsuo Tanaka Saburo Fukui 《Archives of microbiology》1975,103(1):I-II
Development of microbodies in Candida tropicalis pK 233 was studied mainly by electron microscopical observation. The yeast cells, precultured on malt extract, scarcely contained microbodies and showed very low catalase activity. When the precultured cells were transferred to a n-alkane medium and incubated with shaking, the number of microbodies increased and concomitantly the activity of catalase was enhanced. That is, both the area ratio of microbodies in the cell and the ratio of microbodies to cytoplasm in area increased significantly during the utilization of n-alkanes for 8 hrs. Localization of catalase in the microbodies was demonstrated cytochemically by use of 3,3'-diaminobenzidine, but other organella in the cell, except for vacuoles appearing in the early growth phase and mitochondria, were not stained with this reagent. Microbodies seemed to grow by division. Biogenesis of microbodies in the yeast cells is also discussed. 相似文献
86.
T Kitagawa T Kanamaru H Wakamatsu H Kato S Yano Y Asanuma 《Journal of biochemistry》1978,84(2):491-494
A new method for the preparation of ampicillin-BSA conjugate by a three step procedure was developed. The first step is the introduction of a maleimide residue to ampicillin by a cross-linking reagent, MBS. The second step is reductive cleavage of disulfide bonds in BSA. The third step is thioether formation between the introduced maleimide residues and the reduced thiol groups. The obtained ampicillin-BSA conjugated raised an anti-ampicillin serum in rabbits. A new reagent, MPGS, was used for enzyme labelling of ampicillin to avoid immunological cross reaction. Using the enzyme labelled ampicillin and anti-ampicillin serum, enzyme immunoassay of ampicillin was successful in detecting 4 ng to 1 mug. Cross reactivities of anti-ampicillin to ampicillin analogs were studied by the enzyme immunoassay to find that the antiserum is specific to penicillin especially to ampicillin but hardly reacts with cephalosporins or the penicilloic acid derivative of ampicillin. 相似文献
87.
Wakamatsu Kunimitsu; Ikehara Norikatsu; Nishimura Mitsuo 《Plant & cell physiology》1974,15(4):601-610
The light-induced absorbance change at 515 nm, light-inducedhydrogen ion uptake and ATP formation were compared in chloroplastsand different types of sonicated subchloroplast particles. Noparallel relationship among the activities for ATP formation,hydrogen ion uptake and the 515-nm change was observed in differenttypes of preparations. NH4Cl inhibited ATP formation in chloroplastsbut had little effect on subchloroplast particles. In contrast,the light-induced hydrogen ion uptake was inhibited by NH4Clin a similar manner. Tetraphenylboron (TPB), at 1 µM, inhibited ATP formationby about 30% in both chloroplasts and subchloroplast particles.In the presence of TPB, ATP formation in chloroplasts was stronglyinhibited by NHC4Cl, but in subchloroplast particles the additionalinhibitory effect of NH4Cl was small. A synergistic inhibitionof photophosphorylation by valinomycin plus NH4Cl was much clearer.Although acceleration of the recovery of the 515-nm change byNH4Cl or valinomycin was moderate, the 515-nm change virtuallydisappeared when NH4Cl and valinomycin were added simultaneously. Although the membrane potential has a major role as the principaldriving force for ATP formation in subchloroplast particles,the simultaneous abolishment of the pH gradient and membranepotential may be required to uncouple ATP formation.
1Present address: Fukuoka Women's University, Kasumigaoka, Fukuoka813, Japan.
2Present address: Ryukyu University, Naha, Okinawa 903, Japan. (Received February 5, 1974; ) 相似文献
88.
Heme Orientation of Cavity Mutant Hemoglobins (His F8 → Gly) in Either α or β Subunits: Circular Dichroism, 1H NMR,and Resonance Raman Studies 下载免费PDF全文
Masako Nagai Yukifumi Nagai Yayoi Aki Hiroshi Sakurai Naoki Mizusawa Takashi Ogura Teizo Kitagawa Yasuhiko Yamamoto Shigenori Nagatomo 《Chirality》2016,28(8):585-592
Native human adult hemoglobin (Hb A) has mostly normal orientation of heme, whereas recombinant Hb A (rHb A) expressed in E. coli contains both normal and reversed orientations of heme. Hb A with the normal heme exhibits positive circular dichroism (CD) bands at both the Soret and 260‐nm regions, while rHb A with the reversed heme shows a negative Soret and decreased 260‐nm CD bands. In order to examine involvement of the proximal histidine (His F8) of either α or β subunits in determining the heme orientation, we prepared two cavity mutant Hbs, rHb(αH87G) and rHb(βH92G), with substitution of glycine for His F8 in the presence of imidazole. CD spectra of both cavity mutant Hbs did not show a negative Soret band, but instead exhibited positive bands with strong intensity at the both Soret and 260‐nm regions, suggesting that the reversed heme scarcely exists in the cavity mutant Hbs. We confirmed by 1H NMR and resonance Raman (RR) spectroscopies that the cavity mutant Hbs have mainly the normal heme orientation in both the mutated and native subunits. These results indicate that the heme Fe‐His F8 linkage in both α and β subunits influences the heme orientation, and that the heme orientation of one type of subunit is related to the heme orientation of the complementary subunits to be the same. The present study showed that CD and RR spectroscopies also provided powerful tools for the examination of the heme rotational disorder of Hb A, in addition to the usual 1H NMR technique. Chirality 28:585–592, 2016. © 2016 Wiley Periodicals, Inc. 相似文献
89.
90.
Yutaka Teranishi Susumu Kawamoto Atsuo Tanaka Masako Osumi Saburo Fukui 《Bioscience, biotechnology, and biochemistry》2013,77(6):1221-1225
The catalase activity of Candida tropicalis pK 233 was induced by hydrocarbons but not by glucose, galactose, ethanol, acetate or lauryl alcohol.
The induction of the catalase activity depending upon hydrocarbons was sensitive to cycloheximide but not to chloramphenicol.
Glucose repressed strongly the induction of the catalase activity by hydrocarbons but galactose did not affect seriously.
When C. tropicalis was incubated with hydrocarbons, the appearance of microbodies was observed electronmicroscopicaliy.