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T. J. Lyons A. Nersissian H. Huang H. Yeom C. R. Nishida J. A. Graden E. B. Gralla J. S. Valentine 《Journal of biological inorganic chemistry》2000,5(2):189-203
We have investigated factors that influence the properties of the zinc binding site in yeast copper-zinc superoxide dismutase
(CuZnSOD). The properties of yeast CuZnSOD are essentially invariant from pH 5 to pH 9. However, below this pH range there
is a change in the nature of the zinc binding site which can be interpreted as either (1) a change in metal binding affinity
from strong to weak, (2) the expulsion of the metal bound at this site, or (3) a transition from a normal distorted tetrahedral
ligand orientation to a more symmetric arrangement of ligands. This change is strongly reminiscent of a similar pH-induced
transition seen for the bovine protein and, based on the data presented herein, is proposed to be a property that is conserved
among CuZnSODs. The transition demonstrated for the yeast protein is not only sensitive to the pH of the buffering solution
but also to the occupancy and redox status of the adjacent copper binding site. Furthermore, we have investigated the effect
of single site mutations on the pH- and redox-sensitivity of Co2+ binding at the zinc site. Each of the mutants H46R, H48Q, H63A, H63E, H80C, G85R, and D83H is capable of binding Co2+ to a zinc site with a distorted tetrahedral geometry similar to that of wild-type. However, they do so only if Cu+ is bound at the copper site or if the pH in raised to near physiological levels, indicating that the change at the zinc binding
site seen in the wild-type is conserved in the mutants, albeit with an altered pK
a. The mutants H71C and D83A did not bind Co2+ in a wild-type-like fashion under any of the conditions tested. This study reveals that the zinc binding site is exquisitely
sensitive to changes in the protein environment. Since three of the mutant yeast proteins investigated here contain mutations
analogous to those that cause ALS (amyotrophic lateral sclerosis) in humans, this finding implicates improper metal binding
as a mechanism by which CuZnSOD mutants exert their toxic gain of function.
Received: 17 September 1999 / Accepted: 8 December 1999 相似文献
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Summary The molecular weight of a polymer is an important parameter characterizing the physical properties of the polymer. The effect of pH on the molecular weight of poly-3-hydroxybutyric acid (PHB) produced by Alcaligenes sp. K-912 were investigated. PHB having higher molecular weight was obtained when cultivated under high pH conditions compared to the results cultivated under low pH conditions. The polydispersity index was almost the same as 1.6. But the cell mass was decreased as the pH became higher. The molecular weight was mainly determined in the early cultivation period and molecular weight difference could be explained by calculating the number of polymer chains. 相似文献
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The map position of Oct-4 on mouse chromosome 17 is between Q and T regions in the Major Histocompatibility Complex (MHC), and it is physically located within 35 kb of a class I gene. Several Oct-4-related genes are present in the murine genome; one of them maps to chromosome 9. The genomic structure and sequence of Oct-4 determined in t-haplotypes reveals five exons, and shows no significant changes in the t12 mutant haplotype making it unlikely that Oct-4 and the t12 early embryonic lethal are the same gene. By in situ hybridization, detectable onset of zygotic Oct-4 expression does not occur until compaction begins at 8-cells, suggesting that there might be other regulatory factors responsible for initiating Oct-4 expression. 相似文献
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An investigation into the influence of low temperature thermo-chemical pretreatment on sludge reduction in a semi-continuous anaerobic reactor was performed. Firstly, effect of sludge pretreatment was evaluated by COD solubilization, suspended solids reduction and biogas production. At optimized condition (60 °C with pH 12), COD solubilization, suspended solids, reduction and biogas production was 23%, 22% and 51% higher than the control, respectively. Secondly, semi-continuous process performance was studied in a lab-scale semi-continuous anaerobic reactor (5 L), with 4 L working volume. With three operated SRTs, the SRT of 15 days was found to be most appropriate for economic operation of the reactor. Combining pretreatment with anaerobic digestion led to 80.5%, 117% and 90.4% of TS, SS and VS reduction respectively, with an improvement of 103% in biogas production. Thus, low temperature thermo-chemical can play an important role in reducing sludge production. 相似文献
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A putative fatty acid hydratase gene from Macrococcus caseolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was a 68 kDa dimer with a molecular mass of 136 kDa. The enzymatic products formed from fatty acid substrates by the putative enzyme were isolated with high purity (>99%) by solvent fractional crystallization at low temperature. After the identification by GC–MS, the purified hydroxy fatty acids were used as standards to quantitatively determine specific activities and kinetic parameters for fatty acids as substrates. Among the fatty acids evaluated, specific activity and catalytic efficiency (kcat/Km) were highest for oleic acid, indicating that the putative fatty acid hydratase was an oleate hydratase. Hydration occurred only for cis-9-double and cis-12-double bonds of unsaturated fatty acids without any trans-configurations. The maximum activity for oleate hydration was observed at pH 6.5 and 25 °C with 2% (v/v) ethanol and 0.2 mM FAD. Without FAD, all catalytic activity was abolished. Thus, the oleate hydratase is an FAD-dependent enzyme. The residues G29, G31, S34, E50, and E56, which are conserved in the FAD-binding motif of fatty acid hydratases (GXGXXG(A/S)X(15–21)E(D)), were selected by alignment, and the spectral properties and kinetic parameters of their alanine-substituted variants were analyzed. Among the five variants, G29A, G31A, and E56A showed no interaction with FAD and exhibited no activity. These results indicate that G29, G31, and E56 are essential for FAD-binding. 相似文献