两种新呼吸链抑制剂对心肌制剂抑制作用的比较

用抑制剂作为分子工具研究呼吸链的电子传递机制已有相当长的历史,电子传递链各个区段均有酶专一的抑制剂被发现和使用.鉴于呼吸链中三个与泛醌反应相关的酶[还原辅酶Ⅰ:泛醌还原酶(NADH-Q reductase NQR)、琥珀酸:泛醌还原酶(succinate-Q reductase SQR)和泛醌:细胞色素 c 还原酶(QH₂-cytochrome c re-ductase QCR)]均催化同一底物反应,从酶学角度看应存在一类抑制剂能对三个催化泛醌反应的酶兼有抑制作用.经合成和筛选发现3-硝基-N-十二烷基水杨酰胺和2-羟基-3-N-十二烷基酰胺吡啶具备这类性质,它们对催化泛醌反应的三个酶都有抑制作用,而对与泛醌无关的末端氧化酶(cytochrome c oxidase)无任何作用.3-硝 基-N-十二烷基水杨酰胺对检测的心肌制剂各段酶活性的抑制能力均强于2-羟基-3-N-十二烷基酰胺吡啶.     

3-硝基-N-甲基水杨酰胺对琥珀酸细胞色素c还原酶的抑制作用

 我们发现3-硝基-N-甲基水杨酰胺对猪心线粒体呼吸链的琥珀酸细胞色素c还原酶活力有抑制作用。抑制部位在呼吸链中PMS和DCPIP的电子给体之间。抑制性质表现为可逆非竞争性。     

3-硝基-N-甲基水杨酰胺抑制琥珀酸细胞色素c还原酶的动力学特征

 用可逆非竞争性抑制的动力学方程分析3-硝基-N-甲基水杨酰胺对琥珀酸细胞色素c还原酶的抑制作用,发现在高浓度抑制剂条件下实验数据偏离动力学方程。以大豆磷脂取代酶中的天然磷脂后,不再出现这种偏离。这一现象可能与呼吸链酶的双体特征有关。     

苯并噻唑醌类化合物对呼吸链酶系的抑制作用

合成了2-氯-5-正十二硫烷基-6-甲基-4,7-苯并噻唑醌(2-Cl-DMMDBT)和2-氯-5-正丁烷氨基-6-甲基-4,7-苯并噻唑醌(2-Cl-BAMDBT)两种化合物,研究了它们对线粒体呼吸链酶系的抑制作用.结果表明:2-Cl-DMMDBT和2-C1-BAMDBT对琥珀酸氧化酶及泛醌氧化酶的电子传递活性均表现一定的抑制作用,而对细胞色素氧化酶无作用,说明二者的抑制作用发生在泛醌反应区.二者对NADH氧化酶的抑制行为略有不同,2-Cl-DMMDBT是一个逐渐加强的过程,最终可致酶活性完全抑制,而2-Cl-BAMDBT则表现为瞬间抑制.比较了2-Cl-DMMDBT和2-Cl-BAMDBT对琥珀酸氧化酶的抑制能力,长侧链的2-Cl-DMMDBT比短侧链的2-Cl-BAMDBT抑制能力强很多.     

N-糖链合成抑制剂对NIH3T3细胞粘附作用和整合蛋白表达的影响

研究了Ｎ－糖链合成抑制剂——Ｄｅｏｘｙｍａｎｎｏｊｉｒｉｍｙｃｉｎ（ＤＭＭ）和衣霉素（ＴＭ）对ＮＩＨ３Ｔ３细胞粘附作用和细胞表面α５β１整合蛋白含量的影响．研究结果发现甘露糖苷酶Ⅰ抑制剂－ＤＭＭ处理ＮＩＨ３Ｔ３细胞后,３Ｈ－甘露糖（３Ｈ－Ｍａｎ）参入ＮＩＨ３Ｔ３细胞较对照细胞增加一倍,多天线复杂型糖链增加１８％,而细胞表面α５β１整合蛋白与纤连蛋白粘附能力却下降１７％,但对膜整合蛋白α５和β１亚基表达量无明显影响,提示不成熟的糖链对整合蛋白参与的细胞粘附功能有一定影响,但不影响糖蛋白运输及整合到膜上．十四糖二磷酸长萜醇合成抑制剂——ＴＭ为０．５μｇ／ｍｌ时,Ｎ－糖链合成显著抑制,３Ｈ－Ｍａｎ参入减少了５２％,细胞粘附能力下降了３７％,细胞表面膜整合蛋白α５亚基下降了２２％,而β１亚基无明显变化,提示ＴＭ的脱糖基化作用可引起α５亚基转运至细胞膜表面下降,以至影响了细胞的粘附能力．此外,脱糖整合蛋白与纤连蛋白（ｆｉｂｒｏｎｅｃｔｉｎ,Ｆｎ）的结合力下降也是原因之一．     

鸡心脱辅基细胞色素c自发折叠的进一步研究

将野生型鸡心脱辅基细胞色素ｃ及其突变体Ｖ９２Ａ的１７位半胱氨酸残基突变为丝氨酸,再将表达纯化的Ｖ９２Ａ／Ｃ１７Ｓ和Ｗ／Ｃ１７Ｓ用荧光探针ＩＡＥＤＡＮＳ标记．通过测量ＡＥＤＡＮＳ－Ｃｙｓ－１４的荧光光谱、荧光寿命以及ＡＥＤＡＮＳ与Ｔｒｐ－５９之间的荧光共振能量转移效率、比较了Ｖ９２Ａ与野生型鸡心脱辅基细胞色素ｃ因折叠状态不同引起的Ｎ端构象状态及肽链间相互作用的差异．结果显示Ａｐｏ．ｃ无论在低盐浓度下的无规卷曲状态还是高盐浓度下的融球态,Ｖ９２Ａ都较野生型处于更松散的折叠状态,此外,在鸡心脱辅基细胞色素ｃ的自发折叠中Ｎ端肽段并不起主要作用     

重组人粒系集落刺激因子（rhG—CSF）对白血病细胞HL—60生长 …

本文研究了ｒｈＧ－ＣＳＦ对人白血病细胞系ＨＬ－６０的作用。结果表明：ｒｈＧ－ＣＳＦ能够显著抑制ＨＬ－６０细胞生长和Ｃ－ｍｙｃ基因的表达,降低＾３Ｈ－ＴｄＲ的摄入。在含ｒｈＧ－ＣＳＦ的培养液中经过２－５天的培养,部分ＨＬ－６０细胞具备ＮＢＴ还原能力。这或许说明ｒｈＧ－ＣＳＦ能导致ＨＬ－６０细胞向成熟方向分化的结果。     

一组丝瓜籽小分子核糖体失活蛋白LuffinS的分离、纯化和性质

通过硫酸铵分级沉淀、ＣＭ－５２阳离子交换层析、ＨＲＬＣ分子排阻层析及ＦＰＬＣＭｏｎｏＳ离子交换层析等步骤,从丝瓜籽中分离到一组分子量为８ｋＤ左右的小分子核糖体失活蛋白——ＬｕｆｉｎＳ１、ＬｕｆｉｎＳ２、ＬｕｆｉｎＳ３。末端分析结果表明,它们的Ｎ端氨基酸分别为Ａｌａ、Ｐｒｏ和Ｔｈｒ。氨基酸序列分析确定了ＬｕｆｉｎＳ２的Ｎ端９个氨基酸的序列是Ｐｒｏ－Ａｒｇ－Ａｒｇ－Ｇｌｙ－Ｇｌｎ－Ｇｌｕ－Ａｌａ－Ｐｈｅ－Ａｓｐ。ＬｕｆｉｎＳｓ对核糖体的失活机制与天花粉蛋白（ＴＣＳ）一致,是ＲＮＡＮ－糖苷酶催化型的。它们对无细胞蛋白质生物合成的抑制活性较ＴＣＳ略强,ＩＣ５０分别为１．３×１０－１１、１．０×１０－１０和６．３×１０－１１ｍｏｌ／Ｌ左右。因此ＬｕｆｉｎＳｓ有可能开发成免疫毒素的高效“弹头”。     

人胎肝中低分子抑瘤物的分离纯化、结构鉴定及其对肿瘤细胞生长的选择性抑制作用

在胎儿组织中存在一类低分子肿瘤抑制物。胎肝细胞的甲醇－丙酮提取物中保留有大部分抑瘤活性。用体外液体培养条件下对人急性粒系白血病细胞系（ＨＬ－６０）的抑制作用为指标,跟踪指导分离过程。提取物经反相Ｃ１８中压液相色谱、ＳｅｐｈａｄｅｘＬＨ－２０凝胶色谱及氨基键合相高效液相色谱分离得到一纯活性物质,经ＮＭＲ和ＭＳ鉴定为７－酮基胆固醇（７－ｋｅｔｏｃｂｌｅｓｔｅｒｏｌ,７－ＫＣ）。体外琼脂培养条件下７－ＫＣ对小鼠ＷＥＨ１－３和Ｓ－１８０细胞较对正常小鼠骨髓粒一巨噬系祖细胞有更强的抑制增殖及集落生成作用。７－ＫＣ对ＨＬ－６０细胞增殖较对正常人骨髓ＣＦＵ－ＧＭ有更强的抑制作用。     

HPC脂质体的制备及抗HL-60细胞增殖作用的初探

采用十六烷基磷酸胆碱（ＨＰＣ）作为脂质体膜材,配以胆固醇和双十六烷基磷酸盐,反相蒸发法制备出ＨＰＣ脂质体,连续５周每周测定一次它对ＣＦ（ｃａｒｂｏｘｙｆｌｕｏｒｅｓｃｅｉｎ,羧基荧光素）的包封率,可知制备的脂质体在前２周内相当稳定,５周后包封率仅减少２４％,可满足实际应用的需要．冰冻蚀刻法测定脂质体的平均直径在５００ｎｍ左右,该直径的脂质体较适于和细胞发生相互作用且稳定性比小单层脂质体好．四氮唑（ｄｉｍｅｔｈｙｌｔｈｉａｚｏｌｄｉｐｈｅｈｙｌｔｅｔｒａｚｏｌｉｕｍｂｒｏ－ｍｉｄｅ,ＭＴＴ）分析可知,在脂质体浓度达１５μｍｏｌ／Ｌ,对ＨＬ－６０细胞的增殖具有抑制作用．在相同的脂浓度下,ＨＰＣ脂质体抑制ＨＬ－６０细胞生长比游离ＨＰＣ有较强的抑制细胞增殖作用,当ＨＰＣ浓度低于５μｍｏｌ／Ｌ时,细胞生长不受抑制．当ＨＰＣ浓度在１０μｍｏｌ／Ｌ时,ＨＰＣ脂质体表现出对细胞生长的抑制作用,而游离ＨＰＣ在此浓度下的抑制作用较低     

Effect of substituents of the benzoquinone ring on electron-transfer activities of ubiquinone derivatives

The effect of substituents on the 1,4-benzoquinone ring of ubiquinone on its electron-transfer activity in the bovine heart mitochondrial succinate-cytochrome c reductase region is studied by using synthetic ubiquinone derivatives that have a decyl (or geranyl) side-chain at the 6-position and various arrangements of methyl, methoxy and hydrogen in the 2, 3 and 5 positions of the benzoquinone ring. The reduction of quinone derivatives by succinate is measured with succinate-ubiquinone reductase and with succinate-cytochrome c reductase. Oxidation of quinol derivatives is measured with ubiquinol-cytochrome c reductase. The electron-transfer efficacy of quinone derivatives is compared to that of 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone. When quinone derivatives are used as the electron acceptor for succinate-ubiquinone reductase, the methyl group at the 5-position is less important than are the methoxy groups at the 2- and 3-positions. Replacing the 5-methyl group with hydrogen causes a slight increase in activity. However, replacing one or both of 2- and 3-methoxy groups with a methyl completely abolishes electron-acceptor activity. Replacing the 3-methoxy group with hydrogen results in a complete loss of electron-acceptor activity, while replacing the 2-methoxy with hydrogen results in an activity decrease by 70%, suggesting that the methoxy group at the 3-position is more specific than that at the 2-position. The structural requirements for quinol derivatives to be oxidized by ubiquinol-cytochrome c reductase are less strict. All 1,4-benzoquinol derivatives examined show partial activity when used as electron donors for ubiquinol-cytochrome c reductase. Derivatives that possess one unsubstituted position at 2, 3 or 5, with a decyl group at the 6-position, show substrate inhibition at high concentrations. Such substrate inhibition is not observed when fully substituted derivatives are used. The structural requirements for quinone derivatives to be reduced by succinate-cytochrome c reductase are less specific than those for succinate-ubiquinone reductase. Replacing one or both of the 2- and 3-methoxy groups with a methyl and keeping the 5-position unsubstituted (plastoquinone derivatives) yields derivatives with no acceptor activity for succinate-Q reductase. However, these derivatives are reducible by succinate in the presence of succinate-cytochrome c reductase. This reduction is antimycin-sensitive and requires endogenous ubiquinone, suggesting that these (plastoquinone) derivatives can only accept electrons from the ubisemiquinone radical at the Qi site of ubiquinol-cytochrome c reductase, and cannot accept electrons from the QPs of succinate-ubiquinone reductase.     

The role of Sdh4p Tyr-89 in ubiquinone reduction by the Saccharomyces cerevisiae succinate dehydrogenase

Succinate dehydrogenase (complex II or succinate:ubiquinone oxidoreductase) is a tetrameric, membrane-bound enzyme that catalyzes the oxidation of succinate and the reduction of ubiquinone in the mitochondrial respiratory chain. Two electrons from succinate are transferred one at a time through a flavin cofactor and a chain of iron-sulfur clusters to reduce ubiquinone to an ubisemiquinone intermediate and to ubiquinol. Residues that form the proximal quinone-binding site (Q(P)) must recognize ubiquinone, stabilize the ubisemiquinone intermediate, and protonate the ubiquinone to ubiquinol, while minimizing the production of reactive oxygen species. We have investigated the role of the yeast Sdh4p Tyr-89, which forms a hydrogen bond with ubiquinone in the Q(P) site. This tyrosine residue is conserved in all succinate:ubiquinone oxidoreductases studied to date. In the human SDH, mutation of this tyrosine to cysteine results in paraganglioma, tumors of the parasympathetic ganglia in the head and neck. We demonstrate that Tyr-89 is essential for ubiquinone reductase activity and that mutation of Tyr-89 to other residues does not increase the production of reactive oxygen species. Our results support a role for Tyr-89 in the protonation of ubiquinone and argue that the generation of reactive oxygen species is not causative of tumor formation.     

The role of Sdh4p Tyr-89 in ubiquinone reduction by the Saccharomyces cerevisiae succinate dehydrogenase

Succinate dehydrogenase (complex II or succinate:ubiquinone oxidoreductase) is a tetrameric, membrane-bound enzyme that catalyzes the oxidation of succinate and the reduction of ubiquinone in the mitochondrial respiratory chain. Two electrons from succinate are transferred one at a time through a flavin cofactor and a chain of iron-sulfur clusters to reduce ubiquinone to an ubisemiquinone intermediate and to ubiquinol. Residues that form the proximal quinone-binding site (Q_P) must recognize ubiquinone, stabilize the ubisemiquinone intermediate, and protonate the ubiquinone to ubiquinol, while minimizing the production of reactive oxygen species. We have investigated the role of the yeast Sdh4p Tyr-89, which forms a hydrogen bond with ubiquinone in the Q_P site. This tyrosine residue is conserved in all succinate:ubiquinone oxidoreductases studied to date. In the human SDH, mutation of this tyrosine to cysteine results in paraganglioma, tumors of the parasympathetic ganglia in the head and neck. We demonstrate that Tyr-89 is essential for ubiquinone reductase activity and that mutation of Tyr-89 to other residues does not increase the production of reactive oxygen species. Our results support a role for Tyr-89 in the protonation of ubiquinone and argue that the generation of reactive oxygen species is not causative of tumor formation.     

Electron nuclear double resonance (ENDOR) of the Qc.- ubisemiquinone radical in the mitochondrial electron transport chain

We present an electron nuclear double resonance (ENDOR) study of the bound Qc.- ubisemiquinone in the mitochondrial quinol cytochrome c reductase complex. An ENDOR probe specifically modified for insertion into our electron paramagnetic resonance cavity was used for this study. We observed strongly hyperfine-coupled protons whose exchangeable nature indicated they were hydrogen-bonded to the quinone oxygen(s). It is thought that such hydrogen bonds are critical in binding the ubiquinone to protein, in stabilizing its semiquinone form, and in modulating the thermodynamic properties of the bound ubiquinone in the mitochondrial quinol cytochrome c reductase complex. Additional ENDOR features were assigned to protons of the quinone ring itself and to weakly coupled protons that may be associated with nearby amino acids. From very weakly hyperfine-coupled, distant, exchangeable protons there was also ENDOR evidence to suggest proximity and accessibility of the ubiquinone site to the solvent.     

Inhibitory effect of α-tocopherol and its derivatives on bovine heart succinate-cytochrome c reductase

α-Tocopherol and its derivatives inhibit succinate-cytochrome c reductase activity at a concentration of 0.5 μmol/mg protein in 50 mM phosphate buffer, pH 7.4, containing 0.4 % sodium cholate when α-tocopherol is predispersed in sodium cholate solution. The inhibitory site is located at the cytochrome b-c₁ region. Succinate-ubiquinone reductase activity of succinate-cytochrome c reductase was not impaired by treatment with α-tocopherol. The α-tocopherol-inhibited succinate-cytochrome c reductase activity can be reversed by the addition of ubiquinone and its analogs. When ubiquinone- and phospholipid-depleted succinate-cytochrome c reductase was treated with α-tocopherol followed by reaction with a fixed amount of 2,3-dimethoxy-6-methyl-5-(10-bromodecyl)-1,4-benzoquinone and phospholipid, the amount of α-tocopherol needed to express the maximal inhibition was only 0.3 μmol/mg protein. When ubiquinone- and phospholipid-depleted enzyme was treated with a given amount of α-tocopherol and followed by titration with 2,3-dimethoxy-6-methyl-5-(10-bromodecyl)-1,4-benzoquinone, restoration of activity was enhanced at low concentrations of ubiquinone analog, indicating that α-tocopherol can serve as an effector for ubiquinone. The maximal binding capacity of α-[¹⁴C]tocopherol, dispersed in 50 mM phosphate buffer containing 0.25% sodium cholate, pH 7.4, to succinate-cytochrome c reductase was shown to be 0.68 μmol/mg protein. A similar binding capacity, based on cytochrome b content, was observed in submitochondrial particles. Binding of α-tocopherol to succinate-cytochrome c reductase not only caused an inhibition of enzymatic activity but also caused a reduction of cytochrome c₁ in the absence of substrate, a phenomenon analogous to the removal of phospholipids from the enzyme preparation. Furthermore, binding of α-tocopherol to succinate-cytochrome c reductase decreased the rate of reduction of cytochrome b by succinate. Since electron transfer from succinate to ubiquinone was not affected by α-tocopherol treatment, the decrease in reduction rate of cytochrome b by succinate must be due to a change in environment around cytochrome b. These results as well as the fact that reactivation of α-tocopherol-inhibited enzyme requires only low concentrations of ubiquinone were used to explain the inhibitory effect as a result of a change in protein conformation and protein-phospholipid interaction rather than the direct displacement of ubiquinone by α-tocopherol. This deduction was further supported by the fact that no ubiquinone was released from succinate-cytochrome c reductase upon treatment with α-tocopherol.     

5-Membered NH...N hydrogen bonded molecular scaffold in a model dipeptide containing 3-aminophenylacetic acid: Crystal and solution conformations

Summary Crystallographic and spectroscopic studies of a model dipeptide containing unusual amino acid residues establish the presence of an intramolecular, 5-membered NH…N hydrogen bond involving an amide NH (from 3-amino phenyl acetic acid residue) and an amide N atom from an adjacent amino acid residue in solid state and in solution. The dipeptide also forms an infinite β-sheet ribbon structure in crystals.     

硝基水杨酰胺抑制泛醌-细胞色素c还原酶的作用对氢醌的敏感性

泛醌－细胞色素ｃ还原酶（ＱＣＲ）是线粒体呼吸链的三个能量偶联部位之一,它起着将电子从还原型泛醌传递给细胞色素ｃ（Ｃｙｔ．ｃ）的作用,根据Ｋｉｎｇ和Ｙｕ提出的泛醌结合蛋白理论［１］,泛醌－细胞色素ｃ还原酶中含有泛醌结合蛋白ＱＰｃ．研究表明,泛醌－细胞色...     

Evidence of ubisemiquinone radicals in electron transfer at the cytochromes b and c1 region of the cardiac respiratory chain

A highly purified reduced ubiquinone-cytochrome c reductase preparation (the b-c₁III complex) has been made. The b-c₁III complex is not reconstitutively active with succinate dehydrogenase. When the complex at about 10 mg/ml is reduced by succinate in the presence of catalytic (nanomolar) amounts of SDH and a ubiquinone protein (required in the succinate dehydrogenase region i.e, OP-S), a ubisemiquinone radical(s) has been detected using EPR measurements. The formation of the radical(s) is concurrent with the reduction of cytochrome b after the complete reduction of cytochrome c₁. All these rates are dependent on the amounts of succinate dehydrogenase and QP-S used. The maximal concentration of the radical formed is independent of the amounts of succinate dehydrogenase and QP-S added but dependent on the amount of succinate present. The formation of the radical and the reduction of b and c₁ by succinate requires the presence of phospholipids. Addition of thenoyltrifluoroacetone not only prevents the formation of the ubisemiquinone but also abolishes the prior formed radical and causes the reoxidation of b. Antimycin A also diminishes the radical intensity but causes only slight reoxidation of prior reduced cytochrome b. Treatment of the b-c₁III complex with α-chymotrypsin results in the diminishing of the radical formation. Consideration of all these results presented collectively indicates the existence of a ubiquinone binding protein in the b-c₁III complex preparation.     

Stabilized ubisemiquinone in reconstituted succinate ubiquinone reductase

QP-S, a ubiquinone (Q) protein, accepts electrons from succinate through succinate dehydrogenase (SDH). A new method has produced a preparation of QP-S which has a different amino acid composition and SDS gel electrophoretic pattern from that of the old preparation (Biochemistry 19, 3579-3585 (1980)). The new preparation contains less than 1 nmol heme/mg protein; the activity of the preparation was not proportional to its heme content. A thenoyltrifluoroacetone sensitive free radical signal was detected by EPR spectroscopy in succinate-Q reductase reconstituted from this QP-S and SDH; the characteristics of this species identify it as ubisemiquinone. At pH 7.4, the Em of the two electron step was about 70 mV with E1 = 5 mV and E2 = 125 mV. The properties of the radical differed slightly from those of "Qs" radical in more intact preparations (e.g. submitochondrial particles). The present is the simplest system in which such a succinate reducible ubisemiquinone free radical has been demonstrated.     

Isolation and characterization of complex I, rotenone-sensitive NADH: ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei.

Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-beta-D-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine-SDS/PAGE including the 51 kDa, PSST and TYKY subunits.