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81.
Cytoplasmic alpha-glycerol-3-phosphate dehydrogenase from fruit-bat-breast muscle was purified by ion-exchange and affinity chromatography. The specific activity of the purified enzyme was approximately 120 units/mg of protein. The apparent molecular weight of the native enzyme, as determined by gel filtration on Sephadex G-100 was 59,500 +/- 650 daltons; its subunit size was estimated to be 35,700 +/- 140 by SDS-polyacrylamide gel electrophoresis. The true Michaelis-Menten constants for all substrates at pH 7.5 were 3.9 +/- 0.7 mM, 0.65 +/- 0.05 mM, 0.26 +/- 0.06 mM, and 0.005 +/- 0.0004 mM for L-glycerol-3-phosphate, NAD(+), DHAP, and NADH, respectively. The true Michaelis-Menten constants at pH 10.0 were 2.30 +/- 0.21 mM and 0.20 +/- 0.01 mM for L-glycerol-3-phosphate and NAD(+), respectively. The turnover number, k(cat), of the forward reaction was 1.9 +/- 0.2 x 10(4)s(-1). The treatment of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) under denaturing conditions indicated that there were a total of eight cysteine residues, while only two of these residues were reactive towards DTNB in the native enzyme. The overall results of the in vitro experiments suggest that alpha-glycerol-3-phosphate dehydrogenase of the fruit bat preferentially catalyses the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate. 相似文献
82.
The influence of chaotropic and kosmotropic salts on Mucuna pruriens protein isolates was investigated. Protein solubility profile indicated that solubility was minimal at the isoelectric point of the protein isolate (4.0) while the solubility was maximal at pH 10.0 in all salt solutions. Chaotropes (I(-), ClO(4)(-) and SCN(-)) exhibit better protein solubility than the kosmotropes (SO(4)(2-), Cl(-) and Br(-)). Increase in protein solubility follows the Hofmeister series: NaSO(4)相似文献
83.
The coupling of succinate oxidation to the reduction of ubiquinone by succinate dehydrogenase (SDH) constitutes a pivotal reaction in the aerobic generation of energy. In Saccharomyces cerevisiae, SDH is a tetramer composed of a catalytic dimer comprising a flavoprotein subunit, Sdh1p and an iron-sulfur protein, Sdh2p and a heme b-containing membrane-anchoring dimer comprising the Sdh3p and Sdh4p subunits. In order to investigate the role of heme in SDH catalysis, we constructed an S. cerevisiae strain expressing a mutant enzyme lacking the two heme axial ligands, Sdh3p His-106 and Sdh4p Cys-78. The mutant enzyme was characterized for growth on a non-fermentable carbon source, for enzyme assembly, for succinate-dependent quinone reduction and for its heme b content. Replacement of both Sdh3p His-106 and Sdh4p Cys-78 with alanine residues leads to an undetectable level of cytochrome b(562). Although enzyme assembly is slightly impaired, the apocytochrome SDH retains a significant ability to reduce quinone. The enzyme has a reduced affinity for quinone and its catalytic efficiency is reduced by an order of magnitude. To better understand the effects of the mutations, we employed atomistic molecular dynamic simulations to investigate the enzyme's structure and stability in the absence of heme. Our results strongly suggest that heme is not required for electron transport from succinate to quinone nor is it necessary for assembly of the S. cerevisiae SDH. 相似文献
84.
Declining food production in African agroecosystems is attributable to changes in weather patterns, soil infertility and limited farming inputs. The exploitation of plant growth-promoting soil microbes could remedy these problems. Such microbes include Azotobacter; free-living, nitrogen-fixing bacteria, which confer stress tolerance, avail phytohormones and aid in soil bioremediation. Here, we aimed to isolate, characterize and determine the biodiversity of native Azotobacter isolates from soils in semi-arid Eastern Kenya. Isolation was conducted on nitrogen-free Ashby''s agar and the morphological, biochemical and molecular attributes evaluated. The isolates were sequenced using DNA amplicons of 27F and 1492R primers of the 16S rRNA gene loci. The Basic Local Alignment Search Tool (BLASTn) analysis of their sequences revealed the presence of three main Azotobacter species viz., Azotobacter vinelandii, Azotobacter salinestris and Azotobacter tropicalis. Kitui County recorded the highest number of recovered Azotobacter isolates (45.4%) and lowest diversity index (0.8761). Tharaka Nithi County showed the lowest occurrence (26.36%) with a diversity index of (1.057). The diversity was influenced by the soil pH, texture and total organic content. This study reports for the first time a wide diversity of Azotobacter species from a semi-arid agroecosystem in Kenya with potential for utilization as low-cost, free-living nitrogen-fixing bioinoculant. 相似文献
85.
The Saccharomyces cerevisiae succinate dehydrogenase (SDH) provides an excellent model system for studying the assembly, structure, and function of a mitochondrial succinate:quinone oxidoreductase. The powerful combination of genetic and biochemical approaches is better developed in yeast than in other eukaryotes. The yeast protein is strikingly similar to other family members in the structural and catalytic properties of its subunits. However, the membrane domain and particularly the role of the single heme in combination with two ubiquinone-binding sites need further investigation. The assembly of subunits and cofactors that occurs to produce new holoenzyme molecules is a complex process that relies on molecular chaperones. The yeast SDH provides the best opportunity for understanding the biogenesis of this family of iron-sulfur flavoproteins. 相似文献
86.
Ezekiel Ze Ken Cheong Jun Ping Quek Liu Xin Chaoqiang Li Jing Yi Chan Chong Wai Liew Yuguang Mu Jie Zheng Dahai Luo 《The Journal of biological chemistry》2022,298(8)
Rubella, a viral disease characterized by a red skin rash, is well controlled because of an effective vaccine, but outbreaks are still occurring in the absence of available antiviral treatments. The Rubella virus (RUBV) papain-like protease (RubPro) is crucial for RUBV replication, cleaving the nonstructural polyprotein p200 into two multifunctional proteins, p150 and p90. This protease could represent a potential drug target, but structural and mechanistic details important for the inhibition of this enzyme are unclear. Here, we report a novel crystal structure of RubPro at a resolution of 1.64 Å. The RubPro adopts a unique papain-like protease fold, with a similar catalytic core to that of proteases from Severe acute respiratory syndrome coronavirus 2 and foot-and-mouth disease virus while having a distinctive N-terminal fingers domain. RubPro has well-conserved sequence motifs that are also found in its newly discovered Rubivirus relatives. In addition, we show that the RubPro construct has protease activity in trans against a construct of RUBV protease–helicase and fluorogenic peptides. A protease–helicase construct, exogenously expressed in Escherichia coli, was also cleaved at the p150–p90 cleavage junction, demonstrating protease activity of the protease–helicase protein. We also demonstrate that RubPro possesses deubiquitylation activity, suggesting a potential role of RubPro in modulating the host''s innate immune responses. We anticipate that these structural and functional insights of RubPro will advance our current understanding of its function and help facilitate more structure-based research into the RUBV replication machinery, in hopes of developing antiviral therapeutics against RUBV. 相似文献
87.
88.
Bamidele I. Olu-Owolabi Foluso O. Agunbiade Ekemena O. Oseghe Kayode O. Adebowale 《人类与生态风险评估》2012,18(4):902-918
There are numerous statistical models for evaluating the degree of pollution in an environment. This study presents a fuzzy logic–based model—simple fuzzy classification (SFC)—for evaluating contamination of Ni and V species in the sediments of Nigeria's Ondo coastal area. Concentrations of five species of these metals were obtained from 10 sampling sites following sequential extractions from sediments. The results were formulated into a fuzzy membership function matrix based on three classifications relative to regulatory standards and sediments’ degree of contamination. The results of the SFC show that the estuary is moderately enriched by Ni species in a range of 61–84% and further introduction of Ni may shift its contamination level into the highly polluted category. The SFC results also show that the estuary is clean of V species contamination in a range of 77–99%. The Ni and V were associated with the organic specie notably at the crude oil exploration site and at the coastal discharge point. Crude oil exploration and domestic wastes discharges are notable sources of metal contaminations into the estuary. However, the salinity incursion from the coastal ocean and prevailing biogeochemistry affect the species in which the metals exist. 相似文献
89.
Z Zhang KK Ojo SM Johnson ET Larson P He JA Geiger A Castellanos-Gonzalez AC White M Parsons EA Merritt DJ Maly CL Verlinde WC Van Voorhis E Fan 《Bioorganic & medicinal chemistry letters》2012,22(16):5264-5267
Calcium-dependent protein kinase-1 (CDPK1) from Cryptosporidium parvum (CpCDPK1) and Toxoplasma gondii (TgCDPK1) have become attractive targets for discovering selective inhibitors to combat infections caused by these protozoa. We used structure-based design to improve a series of benzoylbenzimidazole-based compounds in terms of solubility, selectivity, and potency against CpCDPK1 and TgCDPK1. The best inhibitors show inhibitory potencies below 50nM and selectivity well above 200-fold over two human kinases with small gatekeeper residues. 相似文献
90.