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1.
Kuntal Dey Tapati Chakraborti Soumitra Roy Biswarup Ghosh Pulak Kar Sajal Chakraborti 《Life sciences》2010,86(13-14):473-481
AimsWe sought to identify, purify and partially characterize a protein inhibitor of Na+/K+-ATPase in cytosol of pulmonary artery smooth muscle.Main methods(i) By spectrophotometric assay, we identified an inhibitor of Na+/K+-ATPase in cytosolic fraction of pulmonary artery smooth muscle; (ii) the inhibitor was purified by a combination of ammonium sulfate precipitation, diethylaminoethyl (DEAE) cellulose chromatography, hydroxyapatite chromatography and gel filtration chromatography; (iii) additionally, we have also purified Na+/K+-ATPase α2β1 and α1β1 isozymes for determining some characteristics of the inhibitor.Key findingsWe identified a novel endogenous protein inhibitor of Na+/K+-ATPase having an apparent mol mass of ~ 70 kDa in the cytosolic fraction of the smooth muscle. The IC50 value of the inhibitor towards the enzyme was determined to be in the nanomolar range. Important characteristics of the inhibitor are as follows: (i) it showed different affinities toward the α2β1 and α1β1 isozymes of the Na+/K+-ATPase; (ii) it interacted reversibly to the E1 site of the enzyme; (iii) the inhibitor blocked the phosphorylated intermediate formation; and (iv) it competitively inhibited the enzyme with respect to ATP. CD studies indicated that the inhibitor causes an alteration of the conformation of the enzyme. The inhibition study also suggested that the DHPC solubilized Na+/K+-ATPase exists as (αβ)2 diprotomer.SignificanceThe inhibitor binds to the Na+/K+-ATPase at a site different from the ouabain binding site. The novelty of the inhibitor is that it acts in an isoform specific manner on the enzyme, where α2 is more sensitive than α1. 相似文献
2.
【目的】通过改造谷氨酸棒杆菌JNR中双功能尿苷酰转移/去除酶GlnD,减弱尿苷酰去除酶的活性,增强NH_4~+的转运和利用,提高L-精氨酸的合成。【方法】本文对来源于谷氨酸棒杆菌的突变菌株JNR中的双功能尿苷酰转移/去除酶GlnD进行整合突变,采用同源重组的方法将H_(414)和D_(415)位点突变为两个丙氨酸AA,在此菌株的基础上过量表达PII蛋白GlnK,并对其进行尿苷酰化研究,离子色谱检测摇瓶发酵过程中NH4+的浓度,并对最终的改造菌株进行连续流加发酵分析。【结果】该双功能尿苷酰转移/去除酶在谷氨酸棒杆菌中成功进行整合突变,有效减弱了尿苷酰去除酶的活性;同时过表达PII蛋白GlnK,其酰基化程度明显增强。摇瓶发酵结果表明菌株L4消耗NH_4~+增加,L-精氨酸产量为36.2±1.2 g/L,比对照菌株L3高出22.7%。5-L发酵罐实验结果显示改造菌株L4的L-精氨酸的产量为52.2 g/L,较野生型菌株L0提高了25.3%。【结论】谷氨酸棒杆菌合成L-精氨酸的过程中氮源是必不可少的。减弱GlnD尿苷酰去除酶的活性后,胞内尿苷酰化的GlnK-UMP增加,GlnK-UMP与氮转录调控因子AmtR结合,转运至胞内的NH_4~+浓度提高,促使L-精氨酸产量显著提高。 相似文献
3.
《Biochimica et Biophysica Acta (BBA)/General Subjects》2016,1860(2):345-353
BackgroundThe mitochondrial F1FO-ATP synthase has not only the known life function in building most cellular ATP, but also, as recently hinted, an amazing involvement in cell death. Accordingly, the two-faced enzyme complex, which catalyzes both ATP synthesis and ATP hydrolysis, has been involved in the mitochondrial permeability transition, the master player in apoptosis and necrosis. Nitrite, a cellular nitric oxide reservoir, has a recognized role in cardiovascular protection, through still unclear mechanisms.MethodsIn swine heart mitochondria the effect of nitrite on the F1FO-ATPase activity activated by Ca2 +, henceforth defined as Ca-ATPase(s), or by the natural cofactor Mg2 +, was investigated by evaluating ATP hydrolysis under different assay conditions.ResultsCa2 + is far less efficient than the natural cofactor Mg2+ in the ATPase activation. However, when activated by Ca2+ the ATPase activity is especially responsive to nitrite, which acts as uncompetitive inhibitor and up to 2 mM inhibits the Ca2+-activated-ATPase(s), probably by promoting dytirosine formation on the enzyme proteins, leaving the Mg-ATPase(s) unaffected. Most likely these ATPases refer to the same F1FO complex, even if coexistent ATPases may overlap.ConclusionsThe preferential inhibition by nitrite of the Ca-ATPase(s), due to post-translational tyrosine modifications, may prevent the calcium-dependent functionality of the mitochondrial F1FO complex and related events.General significanceIn mitochondria the preferential inhibition of the Ca-ATPase activity/ies by nitrite concentrations which do not affect the coexistent Mg-ATPase(s) may quench the negative events linked to the calcium-dependent functioning mode of the F1FO complex under pathological conditions. 相似文献
4.
Renato Bianchetti 《Plant biosystems》2013,147(1-2):203-210
Abstract PHOSPHOGLUCOISOMERASE FROM PEA COTYLEDONS. — 6-P-glucose iso-merase has been purified from pea cotyledons. A 70-fold purification has been obtained by means of acetone fractionation and two absorption-elution steps on calcium phosphate gel. The partially purified enzyme is free of interfering activities. KM values of 2.5×10?4 and 10?4 been measured for glucose-6-P and fructose-6-P respectively. reaction, measured at pH 7.8 and 30° C., is 3.7 (Gl-6-PIFr-6-P). The enzyme is not inhibited by p-chloro-mercurybenzoate up to 10?3 M. Besides the substances already known to inhibit competitively the isomerase from animal tissues, the pea enzyme has been found to be competitively inhibited by ribose-5-P and by triosespho-sphates, the K1, being respectively 7×10?4 and 2.5×10?4. The properties of the pea enzyme are compared to those of animal tissues isomerase. The possible physiological significance of these properties is discussed. 相似文献
5.
Abstract Guanylyl (3′→5′)-5-methylcytidine (Gpm5C) has been synthesized enzymatically through the use of T1RNAse at high enzyme dilution. In contrast with GpC, the methylated dinucleoside monophosphate is shown to be inactive as a primer for RNA synthesis by the RNA-dependent RNA polymerase of Influenza A virus. 相似文献
6.
[目的] 为探究重金属对淡水绿藻生长的影响。[方法] 选取对水质检测具有明显指示作用的普通小球藻(Chlorella vulgaris)为实验材料,CdCl2·2H2O和CrCl3·7H2O提供重金属离子,探究不同浓度Cr3+和Cd2+在单一和复合胁迫下对藻细胞浓度、叶绿素a及相关抗氧化酶活性的影响。[结果] 随着Cr3+和Cd2+浓度不断增加,藻细胞浓度呈先增长后下降趋势;叶绿素a含量呈现先下降后升高再下降的现象,浓度为1 mg/L的单一和复合胁迫下有最大值,且毒性作用表现为Cr3+ < Cd2+ < Cr3++Cd2+;与藻细胞膜相关的丙二醛(MDA)和过氧化氢(H2O2)含量随着重金属离子浓度的增大而增长;重金属离子浓度低于10 mg/L时对藻细胞内抗氧化酶系统中的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)表现为促进作用,而大于10 mg/L时具有抑制作用。[结论] 结果表明在单一或复合重金属胁迫下,普通小球藻会充分调动与抗逆性相关的酶来维持自身的正常生长。 相似文献
7.
BackgroundCathepsin H (E.C.3.4.22.16) belongs to a family of lysosomal cysteine protease which regulates diverse normal biological processes mainly in intracellular proteolysis.MethodsPurification of cathepsin H from an unstudied system i.e. buffalo lung has been achieved by a simple process developed after incorporating appropriate alteration in the available methods for isolation of the enzyme from other sources. The use of DEAE-Cellulose and SP-Sephadex C-50 helped in better and simultaneous separation of cathepsin B and H up to homogeneity.ResultsThe SDS-PAGE result showed buffalo cathepsin H to be a single-chain molecule having MW, NH2- and COOH- terminal residues of 25.4 kDa, Lys and Val respectively. The enzyme was a glycoprotein with pI of 6.2; it hydrolyzed Leu-NA (Vmax/Km = 301.6) as the most efficient substrate followed by Arg-NA, Arg-Arg-NA and BANA. Buffalo enzyme showed maximum activity at 36 °C, pH 6.75 and at a buffer concentration of 2 × 10?3 M.ConclusionCatheptic activity was found to be quite stable at least for 20–30 min between pH 4.5–7.0, buffer concentration of 1 × 10?2 to 4 × 10?2 M and the temperature resistance up to 36 °C. The effects of various substances present in the buffers routinely used for the assay of catheptic activity revealed that the activity of buffalo lung cathepsin H depends not only qualitatively but also quantitatively on the constituents of assay buffer.General significanceThis study seems to provide valuable information regarding the biochemistry of cathepsin H in general as well as influence of buffer constituents on enzyme activity and physiological role in particular. 相似文献
8.
Daniel A. Converso Marcelo E. Fernández 《Journal of enzyme inhibition and medicinal chemistry》2013,28(2):171-183
The major peroxidase from 15-day-old wheat plants was purified to homogeneity by FPLC ion exchange and molecular exclusion chromatography. It consists of a single polypeptide of M1 37, 500 according to gel filtration and SDS-PAGE and has a pI of 7.0. Kinetics of pyrogallol peroxidation showed that the enzyme follows the accepted mechanism for peroxidase, with kinetic constants k1= 4.4 × 106 M-1 s-1 and k3 = 8.6 × 105 M-1s-1. The effect of different metal ions was assayed on peroxidase activity. None of the ions used had any effect on enzyme activity, except for Cd(II), which was an inhibitor. This was an unexpected and novel finding for a peroxidase. The kinetics of pyrogallol peroxidation at different concentrations of Cd(II) have been studied and a mechanism for Cd(II) inhibition proposed. The results obtained could explain, in part, cadmium-induced oxidative stress. 相似文献
9.
Junichi Sato Kouji Takeda Rika Nishiyama Toshihiro Watanabe Mitsuru Abo Etsuro Yoshimura Junichi Nakagawa Akira Abe Shinji Kawasaki Youichi Niimura 《Biometals》2011,24(2):311-321
We purified free flavin-independent NADPH oxidoreductase from Synechocystis sp. PCC6803 based on NADPH oxidation activity elicited during reduction of t-butyl hydroperoxide in the presence of Fe(III)-EDTA. The N-terminal sequencing of the purified enzyme revealed it to be ferredoxin-NADP+ oxidoreductase (FNR
S
). The purified enzyme reacted with cytochrome c, ferricyanide and 2,6-dichloroindophenol (DCIP). The substrate specificity of the enzyme was similar to the known FNR. DNA
degradation occurring in the presence of NADPH, Fe(III)-EDTA and hydrogen peroxide was potently enhanced by the purified enzyme,
indicating that Synechocystis FNR
S
may drive the Fenton reaction. The Fenton reaction by Synechocystis FNR
S
in the presence of natural chelate iron compounds tended to be considerably lower than that in the presence of synthetic
chelate iron compounds. The Synechocystis FNR
S
is considered to reduce ferric iron to ferrous iron when it evokes the Fenton reaction. Although Synechocystis FNR
S
was able to reduce iron compounds in the absence of free flavin, the ferric reduction by the enzyme was enhanced by the addition
of free flavin. The enhancement was detected not only in the presence of natural chelate iron compounds but also synthetic
chelate iron compounds. 相似文献
10.
Abstract A 8-glucosidase was isolated from Candida guilliermondii, a yeast capable of growth on cellobiose. The enzyme was partially purified by treatment with polyethylcneimine and ammonium sulfate precipitation. Further purification was achieved by affinity chromatography using a Sepharose 4B matrix to which oxidized salicin was coupled through adipic dihydrazide. The final product was a 12.5-fold purification of the crude extract with a recovery of 27% of the initial enzyme activity. Polyacryl-amide disc electrophoresis of the purified enzyme gave a single band. A Km of 1.25 × 10?4M was obtained using p_-nitrophenyl-β-D_-glucopyranoside as the substrate. The optimum pH for enzyme activity was 6.8. Maximum activity was observed at a temperature of 37°C. Enzyme activity was completely inhibited by Hg++, Pb++, and Zn++ ions. The molecular weight of the enzyme is 48, 000 as estimated by sucrose density gradient centri-fugation. 相似文献
11.
Saied Ghadimi Ali Asghar Ebrahimi Valmoozi Mehrdad Pourayoubi Keyvan Asad Samani 《Journal of enzyme inhibition and medicinal chemistry》2013,28(4):556-561
Phosphoramido acid esters (CH3)2NP(O)X(p-OC6H4-CH3) (containing P-Cl (1), P-O (2), P-F (3), P-CN (5), and P-N (4,6) bonds, X for 2, 4 and 6 is OCH3, (C2H5)2N and morpholin) have been synthesized to investigate the structure-activity study of AChE enzyme inhibition, through the parameters logP, δ31P and IC50. After their characterization by 31P, 31P{1H}, 13C, 1H NMR, IR and mass spectroscopy, the parameters logP and δ31P (31P chemical shift in NMR) were used to evaluated the lipophilicity and electronical properties. The ability of compounds to inhibit human AChE was predicted by PASS software (version 1.193), and experimentally evaluated by a modified Ellman's assay. 相似文献
12.
【背景】碱性蛋白酶是工业用酶中占比最大的酶类,广泛应用于清洁、食品、医疗等行业。近期研究发现碱性蛋白酶在生产生物活性肽方面有巨大潜力,这将进一步拓宽其在保健食品领域中的应用。【目的】利用枯草芽孢杆菌异源表达地衣芽孢杆菌来源的碱性蛋白酶SubC。【方法】通过筛选3种枯草芽孢杆菌宿主菌株(Bacillus subtilis 1A751、MA07、MA08)和6种信号肽(AmyE、AprE、NprE、Pel、YddT、YoqM),同时优化诱导剂浓度、发酵培养基和发酵时长,最终得到最优重组菌株MA08-AmyE-subCopt。【结果】重组菌株MA08-AmyE-subCopt的胞外酶活力为3.33×103 AU/mL,胞外蛋白分泌量为胞内可溶蛋白表达量的4倍,与携带野生型信号肽的对照组菌株WT相比,酶活提高了73.4%。【结论】异源碱性蛋白酶SubC在枯草芽孢杆菌中成功表达,为碱性蛋白酶SubC的表达和在保健食品领域的工业化应用提供了理论基础。 相似文献
13.
F.L. González Flecha P.R. Castello J.J. Gagliardino J.P.F.C. Rossi 《The Journal of membrane biology》1999,171(1):25-34
We have previously demonstrated (Diabetes
39:707–711, 1990) that in vitro glycation of the red cell Ca2+ pump diminishes the Ca2+-ATPase activity of the enzyme up to 50%. Such effect is due to the reaction of glucose with lysine residues of the Ca2+ pump (Biochem. J.
293:369–375, 1993). The aim of this work was to determine whether the effect of glucose is due to a full inactivation of a fraction
of the total population of Ca2+ pump, or to a partial inactivation of all the molecules. Glycation decreased the V
max
for the ATPase activity leaving unaffected the apparent affinities for Ca2+, calmodulin or ATP. The apparent turnover was identical in both, the glycated and the native enzyme. Glycation decreased
the V
max
for the ATP-dependent but not for the calmodulin-activated phosphatase activities. Concomitantly with the inhibition, up
to 6.5% of the lysine residues were randomly glycated. The probabilistic analysis of the relation between the enzyme activity
and the fraction of nonmodified residues indicates that only one Lys residue is responsible for the inhibition. We suggest
that glucose decreases the Ca2+-ATPase activity by reacting with one essential Lys residue probably located in the vicinity of the catalytic site, which
results in the full inactivation of the enzyme. Thus, Ca2+-ATPase activity measured in erythrocyte membranes or purified enzyme preparations preincubated with glucose depends on the
remaining enzyme molecules in which the essential Lys residue stays unglycated.
Received: 9 March 1999/Revised: 11 May 1999 相似文献
14.
Yaşar Demir Azize Alayli Safinur Yildirim 《Preparative biochemistry & biotechnology》2013,43(4):291-299
Abstract In this research, protease enzyme was purified and characterized from milk of Euphorbia amygdaloides. (NH4)2SO4 fractionation and CM‐cellulose ion exchange chromatography methods were used for purification of the enzyme. The optimum pH value was determined to be 5, and the optimum temperature was determined to be 60°C. The Vmax and KM values at optimum pH and 25°C were calculated by means of Linewearver‐Burk graphs as 0.27 mg/L min?1 and 16 mM, respectively. The purification degree was controlled by using SDS‐PAGE and molecular weight was found to be 26 kD. The molecular weight of the enzyme was determined as 54 kD by gel filtration chromatography. These results show that the enzyme has two subunits. In the study, it was also researched whether purified and characterized protease can be collapsed to milk. It was determined that protease enzyme can collapse milk and it can be used to produce cheese. 相似文献
15.
《Redox report : communications in free radical research》2013,18(4):179-184
AbstractThe neutrophil enzyme myeloperoxidase catalyzes the oxidation of tyrosine to tyrosyl radicals, which cross-link to proteins and initiate lipid peroxidation. Tryptophan is present in plasma at about the same concentration as tyrosine and has a similar one-electron reduction potential. In this investigation, we have determined the ability of myeloperoxidase to catalyze the oxidation of tryptophan to assess whether or not this reaction may contribute to oxidative stress at sites of inflammation. We show that tryptophan is a poor substrate for myeloperoxidase because, even though it reacts rapidly with compound I (kI 2.1×106 M-1s-1), it reacts sluggishly with compound II (kII 7 M-1s-1). Tryptophan reversibly inhibited production of hypochlorous acid by purified myeloperoxidase by converting the enzyme to a mixture of compound II and compound III. It gave 50% inhibition (I50) at a concentration of 2 µM. In contrast, it was an ineffective inhibitor of hypochlorous acid production by human neutrophils (I50 80 µM) unless superoxide dismutase was present (I50 5 µM). We propose that compound I of myeloperoxidase will oxidize tryptophan at sites of inflammation. Enzyme turnover will result from the reaction of superoxide or tyrosine with compound II. Thus, tryptophan radicals are potential candidates for exacerbating oxidative stress during inflammation. 相似文献
16.
《Biochimica et Biophysica Acta (BBA)/General Subjects》2022,1866(2):130044
BackgroundSkeletal muscle AMP deaminase (AMPD1) regulates the concentration of adenine nucleotides during muscle contraction. We previously provided evidence that rabbit AMPD1 is composed by two HPRG 73 kDa subunits and two 85 kDa catalytic subunits with a dinuclear zinc site with an average of two histidine residues at each metal site. AMPD1 is mainly expressed in fast twitching fibers and is inhibited by ATP. The limited trypsinization of the 95-residue N-terminal domain of rabbit AMPD1 desensitizes the enzyme towards ATP inhibition at the optimal pH 6.5, but not at pH 7.1.MethodsThe modified residues of rabbit AMPD1 after incubation with radioactive diethyl pyrocarbonate ([14C]DEP) causing the desensitization to inhibition by ATP at pH 7.1 have been identified by sequence analysis and MS analysis of the radioactive peptides liberated from the carbethoxylated enzyme by limited proteolysis with trypsin.ResultsThe study confirms the presence of a dinuclear zinc site in rabbit AMPD1 and shows that carbethoxylation of His-51 at the N-terminus of the catalytic subunit removes the inhibition of the enzyme by ATP at pH 7.1.ConclusionsThe desensitization to ATP is due to the modification of His-51 of the Zn2 coordination sphere which is transduced in a conformational change of the enzyme C-terminus, where an ATP-binding site has been localized.General significanceThe progress in the study of the complex regulation of rabbit AMPD1 that shares an identical amino acid sequence with the human enzyme is important in relation to the role of the enzyme during mammalian evolution. 相似文献
17.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(5):487-496
AbstractThe interaction of yeast alcohol dehydrogenase (ADH) with the reactive chlorotriazine dye Vilmafix Blue A-R (VBAR) was studied. VBAR was purified to homogeneity on lipophilic Sephadex LH-20 and characterised by reverse phase HPLC and analytical TLC. Incubation of ADH with purified VBAR at pH 8.0 and 37°C resulted in a time-dependent inactivation of the enzyme. The observed rate of enzyme inactivation (kobs) exhibited a non-linear dependence on VBAR concentration from 22 to 106nmol, with a maximum rate of inactivation (k3) of 0.134min?1 and kD of 141.7 μM. The inhibition was irreversible and activity could not be recovered by gel-filtration chromatography. The inactivation of ADH by VBAR was competitively inhibited by the nucleotides NADH and NAD+. These results suggest that VBAR acts as an affinity label at the nucleotide binding site of yeast ADH. 相似文献
18.
Hong-In Lee Jin-Won Lee Tran-Chin Yang Sa-Ouk Kang Brian M. Hoffman 《Journal of biological inorganic chemistry》2010,15(2):175-182
Superoxide dismutases (SODs) protect cells against oxidative stress by disproportionating O2
− to H2O2 and O2. The recent finding of a nickel-containing SOD (Ni-SOD) has widened the diversity of SODs in terms of metal contents and
SOD catalytic mechanisms. The coordination and geometrical structure of the metal site and the related electronic structure
are the keys to understanding the dismutase mechanism of the enzyme. We performed Q-band 14N,1/2H continuous wave (CW) and pulsed electron–nuclear double resonance (ENDOR) and X-band 14N electron spin echo envelope modulation (ESEEM) on the resting-state Ni-SOD extracted from Streptomyces seoulensis. In-depth analysis of the data obtained from the multifrequency advanced electron paramagnetic resonance techniques detailed
the electronic structure of the active site of Ni-SOD. The analysis of the field-dependent Q-band 14N CW ENDOR yielded the nuclear hyperfine and quadrupole coupling tensors of the axial Nδ of the His-1 imidazole ligand. The tensors are coaxial with the g-tensor frame, implying the g-tensor direction is modulated by the imidazole plane. X-band 14N ESEEM characterized the hyperfine coupling of Nε of His-1 imidazole. The nuclear quadrupole coupling constant of the nitrogen suggests that the hydrogen-bonding between Nε–H and OGlu-17 present for the reduced-state Ni-SOD is weakened or broken upon oxidizing the enzyme. Q-band 1H CW ENDOR and pulsed 2H Mims ENDOR showed a strong hyperfine coupling to the protons(s) of the equatorially coordinated His-1 amine and a weak hyperfine
coupling to either the proton(s) of a water in the pocket at the side opposite the axial Nδ or the proton of a water hydrogen-bonded to the equatorial thiolate ligand. 相似文献
19.
[Fe]-Hydrogenase catalyzes the reversible activation of H2. CO and CN− inhibit this enzyme with low affinity (Ki ≅ 0.1 mM) by binding to the iron site of the bound iron-guanyrylpyridinol cofactor. We report here that isocyanides, which are formally isoelectronic with CO and CN−, strongly inhibit [Fe]-hydrogenase (Ki as low as 1 nM). The [NiFe]- and [FeFe]-hydrogenases tested were not inhibited by isocyanides. UV–Vis and infrared spectra revealed that the isocyanides bind to the iron center of [Fe]-hydrogenase. The inhibition kinetics are in agreement with the proposed catalytic mechanism, including the open/closed conformational change of the enzyme. 相似文献
20.
SUMMARYUVA-induced lipid peroxidation in cultured human skin fibroblasts, as measured by the release in the supernatant of thiobarbituric acid-reactive substances, is found to be linear with increasing irradiation dose (up to about 250 kJ m?2). Concomitantly, within this dose range catalase is strongly inactivated by UVA radiation according to an exponential process (k≈0.01 kJ?1 m2). This suggests that catalase is not involved in modulating the peroxidation process. Inactivation of catalase by 3-amino-1,2,4-triazole can be efficiently achieved prior to irradiation. This inactivation has no consequence on the extent of peroxidation triggered by subsequent exposure to UVA radiation. It may be therefore strongly suggested that catalase is not, via H2O2 removal, a key enzyme in the cellular defence equipment towards UV A-peroxidative stress. An alternative interpretation may be formulated which supports the view that H2O2 produced upon exposure to UVA has no or very little role in triggering the lipid peroxidation process. 相似文献