兔小肠刷状缘膜蔗糖酶—异麦芽糖酶复合体的圆二色光谱研究

在磷酸钾缓冲液中,pH6.8,25℃时,S-I酶复合体的近紫外CD谱显示280～281nm的负峰,293～294nm的正峰。在288,269,和263nm处有3个肩膀。溶液pH值变化和对此酶的热失活处理都能使280～281nm负峰缩小,可能表示酪氨酸残基微环境和酶活力有关。S-I酶复合体的远紫外CD谱显示较大的215nm负峰和较小的192nm正峰,表示其骨架构象以β-折迭和无规卷曲为主。此远紫外CD谱在pH2.3～11范围里维持不变,也不受55℃热失活处理的影响。Na~+和tris对此酶CD谱不产生影响。高浓度盐酸胍能改变其近紫外和远紫外CD谱形。     

猪心线粒体F_1-ATP酶的水解活性与色、酪氨酸残基相关的构象的关系

线粒体H~ -ATP酶具有双向功能,即ATP的合成与ATP的水解。H~ -ATP酶的头部F_1具有催化这二种功能的活性部位,F_1的活性表现位与它结合核苷酸的类型、数目以及亲和力有关。至于F_1分子活性部位中什么结构与这两种功能有关,目前尚不肯定。自1960年以来关于线粒体F_1的水解功能与其结构中氨基酸残基关系的研究,多采用特异的化学试剂去修饰某些氨基酸残基,然后测其水解活力被抑制的情况。Senior认为不是含—SH基的半胱氨酸而是酪氨酸残基,还可能包括色氨酸残基与ATP的水解有关。也有报道认为是与组氨酸、谷氨酸或精氨酸残基有关。现在蛋白质结构     

生物膜能量偶联ATP酶的比较研究——Ⅰ.鼠肝线粒体内膜与ATP酶(F_1)的人工重组及ANS萤光探针与ATP酶的结合

本文报导了大鼠肝线粒体内膜ATP酶的析离和重组,以及膜对ATP酶的结构和功能的影响。用(1)胰蛋白酶-尿素、(2)硅钨酸盐和(3)枯草杆菌蛋白酶三种方法分别制备的去ATP酶(F_1)的线粒体内膜与可溶性的F_1重组后,完全恢复或部分恢复到天然线粒体内膜的ATP酶活力水平。寡霉素敏感性测定、ANS结合的发射萤光光谱测定、电镜负染标本观察和低温处理试验等都一致证明,ATP酶与膜重组后表现了一系列与天然膜相似的性质。ANS萤光探针与线粒体内膜结合的萤光增强效应主要在于ANS与ATP酶(F_1)的结合并与F_1的分子构象有密切关系。经胰蛋白酶-尿素处理去掉F_1的线粒体内膜基本上丧失了ANS的萤光增强效应。可溶性F_1经0℃处理2小时后,丧失酶水解活力的84%和ANS萤光增强效应的96.4%。F_1与膜结合后,则表现了对0℃低温的稳定性。结果提示,ANS可能与ATP酶分子的疏水微区相结合;ATP酶分子疏水结构的存在对于表现酶的水解活力和ANS的萤光增强效应是必要的条件;低温处理破坏了酶分子内的疏水结构;膜与ATP酶结合则有稳定酶分子的疏水结构和分子构象的作用。     

生物膜能量偶联 ATP 酶的研究:鼠肝线粒体 ATP 酶(F_1)的解离和重组

纯化的鼠肝线粒体ATP 酶(F_1)在1MKCl-TEA 缓冲液(Tris-SO_4~-,50mM;EDTA,1mM;ATP,2mM;pH=7.6)中,2～3℃下处理40～90分钟丧失ATP 水解活力,比活力从70～100下降到0.5～1.0。7%聚丙烯酰胺凝胶电泳证明,随着酶活力的下降和F_1的电泳酶带的消失,在凝胶柱上出现三条F_1的解离蛋白带。解离后的F_1于30℃对TEA(pH5.7)介质透析脱盐能重新出现有活力的F_1,活力恢复到原来酶活力的19～22%,此种重组的F_1在pH 中性偏碱条件下保持稳定。重组过程不需外加Mg~( )的促进和SH 基的保护。重组的F_1与去F_1的Tu 膜可重新结合,完全恢复原来线粒体内膜的ATP 酶水解活力和对DCCD 的敏感性。     

生物膜能量偶联ATP酶的研究:鼠肝线粒体ATP酶(F_1)~*的解离和重组

纯化的鼠肝线粒体ATP酶(F_1)在1MKCl-TEA缓冲液(Tris-So_4~-,50mM;EDTA,1mM;ATP,2 mM;pH=7.6)中,2～3℃下处理40～90分钟丧失ATP水解活力,比活力从70～100下降到0.5～1.0。7%聚丙烯酰胺凝胶电泳证明,随着酶活力的下降和F_1的电泳酶带的消失,在凝胶柱上出现三条F_1的解离蛋白带。解离后的F_1于30℃对TEA(pH5.7)介质透析脱盐能重新出现有活力的F_1,活力恢复到原来酶活力的19～22%,此种重组的F_1在pH中性偏碱条件下保持稳定。重组过程不需外加Mg~( )的促进和SH基的保护。重组的F_1与去F_1的Tu膜可重新结合,完全恢复原来线粒体内膜的ATP酶水解活力和对DCCD的敏感性。     

Mg~(2+)对线粒体H~+-ATP酶的F_O在脂质体重建时的影响

线粒体ATP合成酶是由具有H~+转运活性的F_0亚基,可溶性的催化中心F_1和连接二者的致寡霉素敏感蛋白(OSCP)所组成. 将纯化的猪心线粒体H—ATP酶复合体的F_0亚基,用胆酸盐透析法在有Mg~(2+)和无Mg~(2+)条件下在大豆磷脂脂质体上重建得脂酶体(L·F_0).用探剂9-AA荧光淬灭法和电权法测定了两种脂酶体的质子转运活力.由两种方法所得的实验结果均表明,在透返介质中加入1mmolmg~(2+)条件下形成的脂酶体(L·F_0)+Mg~(2+)较无Mg~(2+)者的质子转运活性明显增加.前者的荧光强度变化较后者增加约30%;由电极法测得的质子转运的初速度,前者为5nmolH~+′sF_0,后者为3nmolH~+′s·nmolF_O,质子转运活性高约一倍.这进一步支持Mg~(2+)通过调节脂的物理状态而诱导F_O具有较适合的构象,并进而将这一影响传递至F_1,使整个H~+—AhP酶具有较高活性的假设.     

Mg~(2+)对线粒体H~+-ATP酶的F_O在脂质体重建时的影响

线粒体ATP合成酶是由具有H~+转运活性的F_0亚基,可溶性的催化中心F_1和连接二者的致寡霉素敏感蛋白(OSCP)所组成. 将纯化的猪心线粒体H—ATP酶复合体的F_0亚基,用胆酸盐透析法在有Mg~(2+)和无Mg~(2+)条件下在大豆磷脂脂质体上重建得脂酶体(L·F_0).用探剂9-AA荧光淬灭法和电权法测定了两种脂酶体的质子转运活力.由两种方法所得的实验结果均表明,在透返介质中加入1mmolmg~(2+)条件下形成的脂酶体(L·F_0)+Mg~(2+)较无Mg~(2+)者的质子转运活性明显增加.前者的荧光强度变化较后者增加约30%;由电极法测得的质子转运的初速度,前者为5nmolH~+′sF_0,后者为3nmolH~+′s·nmolF_O,质子转运活性高约一倍.这进一步支持Mg~(2+)通过调节脂的物理状态而诱导F_O具有较适合的构象,并进而将这一影响传递至F_1,使整个H~+—AhP酶具有较高活性的假设.     

乙醇对F_1-ATP酶和H~+-ATP酶的抑制与其核苷酸结合位点状态的关系

 本文利用动力学方法研究了乙醇对F_1-ATP酶和H~(+)-ATP酶复合体的抑制与其结合核苷酸位点状态的关系,结果表明天然情况下乙醇对F_1呈现反竞争性抑制类型,对H~(+)-ATP酶呈现非竞争性抑制类型,且乙醇对F_1和H~(+)-ATP酶的抑制与核苷酸结合位点的构象密切相关。游离状态下和膜结合状态下的F_1在部分结合的核苷酸被洗脱前后动力学行为的不同,反映了二种状态下的F_1具有不同的构象,且F_0和膜脂对F_1起着一定的调控作用。     

脲对果菠萝蛋白酶活力与构象的影响

本文用荧光光谱,紫外差示光谱和CD谱研究果菠萝蛋白酶在不同浓度的脲溶液中的构象及酶活力的变化情况。酶的荧光强度随脲浓度增大而明显增加,8mol/L脲使荧光强度增强65％,发射峰出现红移。差示谱表明在232nm和288nm出现二个正峰,它们均随脲浓度增大而加剧,前者与主链构象变化有关,而后者与生色基团(Trp、Tyr)的微环境变化相关。CD谱表明:天然酶在208nm和225nm处有二个负峰,脲变性后,225nm的负峰基本上不随脲浓度增大而变化,但208nm峰则明显发生变化并逐渐出现红移,6mol/L以上此峰则完全消失。     

生物膜能量偶联ATP酶的比较研究——Ⅱ.鼠肝线粒体ATP酶(F_1)与人原发性肝癌线粒体内膜的杂交重组及其对肝癌发生的遗传控制的初步分析

1974到1975年,我们用人原发性肝癌细胞的线粒体内膜进行ATP酶活力测定,结果证明人肝癌线粒体ATP酶活力极低(0.04～0.1微克分子/分/毫克蛋白)只相当于正常大鼠线粒体的1/10～1/25(0.49～1.07微克分子/分/毫克蛋白)。Walker肉瘤和人肝硬变组织的线粒体与人肝癌的酶活力相近。电镜负染标本观察证明肝癌线粒体内膜大部分失去特征性的直径为90(?)的ATP酶颗粒,表现为光滑膜。ANS萤光探针的发射萤光光谱测定和2,4-二硝基酚的激活试验均证明人肝癌细胞线粒体内膜的ATP酶大量消失是肝癌细胞的特征之一。用提取的大鼠肝线粒体ATP酶(F_1)与人肝癌线粒体内膜进行人工杂交重组,结果证明,重组后的杂交膜的ATP酶活力比人肝癌线粒体内膜高6～11倍;寡霉素敏感性也显著提高。电镜负染标本观察表明杂交膜出现了典型的直径为90(?)的ATP酶的颗粒形态;ANS萤光增强效应测定证明杂交膜的萤光强度比肝癌膜高276%(相对单位);0℃低温处理2小时,ANS萤光强度不变;酶活力在0℃2小时后,仍相当于原来活力的90%。此项试验结果证明杂交重组获得成功。鼠肝线粒体ATP酶与人肝癌线粒体内膜杂交后的特性表现了与天然线粒体内膜的ATP酶的一系列相似的特性。讨论了ATP酶复合体杂交重组试验在探索肝癌发生与细胞中两个遗传系统控制的可能关系问题。     

Effects of some alcohols on the conformation of mitochondrial H+-ATPase complex and F1-ATPase from pig heart

The conformations of the H+-ATPase complex and F1-ATPase in low concentrations of methanol, ethanol, n-propanol, iso-propanol and t-butanol were studied by circular dichroism. For F1-ATPase, all but methanol first increased and then decreased the circular dichroism magnitude of helical bands as the alcohol concentration was increased. With ethanol, n-propanol, iso-propanol and t-butanol, the alpha-helix content reached a maximum at about 5% alcohol and began to decrease at 10%. The content of beta-sheet showed the opposite effect, reaching a minimum at 5% and increasing slightly at higher concentrations. None of the alcohols studied had a significant effect on the conformation of the H+-ATPase complex. This difference implies that the alcohols had a greater effect on free F1-ATPase than on the membrane-bound F1-ATPase. The hydrophobic protein F0 and the membrane lipids in the H+-ATPase complex may stabilize and protect F1 from the effects of the alcohols.     

A kinetic study on the hydrolytic activity of mitochondrial ATPase (F0-F1 complex) from pig heart

A kinetic study of mitochondrial ATPase (F0-F1 complex) from pig heart reported in this paper shows that when it was incubated with free Mg2+ (0-2mM), the hydrolytic activity of the ATPase was competitively activated by the Mg2+ and revealed no cooperativity. In the case of incubation with free ATP the hydrolytic activity was competitively inhibited and revealed positive cooperativity. These results are quite different from those of free F1 as obtained by Gautheron and coworkers (1). This indicates that either Mg2+ or ATP produces different effects on F1 when it is in different states, i.e., free state and membrane bound state. This may be considered to mean that the conformation of F1 in membrane bound state, which is influenced by F0 and membrane lipids is different from that of F1 in free state, thus exhibiting different catalytic site cooperativity between subunits, which is the fundamental feature of the mechanism of the enzyme action.     

N,N'-dicyclohexylcarbodiimide-sensitive K+-dependent ATPase activity in Escherichia coli

ATPase activity sensitive to N,N'-dicyclohexylcarbodiimide and dependent on K+ content in medium is observed only in anaerobically grown Escherichia coli and as the analysis of mutants with defects in different subunits of (F0F1) H+-ATPase and in potassium transport shows only under the structural integrity of both F0F1 and K+-ionophore (the Trk system). The obtained results confirm the data on the H+/K+-exchange and indicate that the F0F1 and Trk systems in anaerobically grown bacteria unite into the same membrane supercomplex inside which the direct energy transfer occurs without a mediation of delta-mu H+.     

Effect of 2-hydroxy-5-nitrobenzyl bromide on proton translocation by the mitochondrial H+-ATPase

2-Hydroxy-5-nitrobenzyl bromide, a highly reactive reagent towards tryptophan residues in proteins, is shown to activate the passive proton flux through the inner mitochondrial membrane of bovine heart submitochondrial particles (ETPH). When added at low concentrations, the reagent increased both the ATPase activity of the particles and the passive proton transport rate through the membrane. The presence of oligomycin reduced the extent of the 2-Hydroxy-5-nitrobenzyl bromide action on the proton conductivity suggesting that it acted primarily on the H+-ATPase complex. Similar effects were observed on F1-depleted particles, whilst no effect was observed on the isolated F1-ATPase activity. The results suggest that polypeptides bearing tryptophan residues may be involved in the gating function of proton channels of the mitochondrial membrane and this is particularly evident for the F0F1-ATPase complex.     

Catalytic sites of mitochondrial ATPase with different properties. Effect of citrate, free ATP and ADP in the presence of dithionite

The extent of stimulation of the hydrolytic activity of mitochondrial ATPase by the reducing agent dithionite has been found to depend on substrate concentration both for the membrane bound enzyme and for the isolated and purified F1ATPase. The results suggest the existence of three catalytic sites differing in their standard reduction potential. The activating effect of free ATP on the hydrolytic activity of rat liver F1-ATPase has been found to be more pronounced on the reduced form of the enzyme. On the contrary, the inhibitory effect of ADP was higher on the oxidized form of F1-ATPase. Citrate has also been found to be an inhibitor of F1-ATPase; its effect was more pronounced on the reduced form of the enzyme, and exhibited a competitive pattern of inhibition with respect to free ATP. The results obtained have been interpreted in the sense that free ATP and ADP may be modifying the standard reduction potential of the enzyme, and suggest the existence of three independent redox cycles in ATPase governed by the exchange of ADP and Pi for the newly synthesized ATP.     

Diethylstilbestrol. A novel F0-directed probe of the mitochondrial proton ATPase

At low concentrations, diethylstilbestrol (DES) is shown to be a potent F0-directed inhibitor of the F0F1-ATPase of rat liver mitochondria. In analogy to other F0-directed inhibitors, DES inhibits both the ATPase and ATP-dependent proton-translocation activities of the purified and membrane bound enzyme. When added at low concentrations with dicyclohexylcarbodiimide (DCCD), a covalent inhibitor, DES acts synergistically to inhibit ATPase activity of the complex. At higher concentrations, DES restores DCCD-inhibited ATPase activity. However, there is no restoration of ATP-dependent proton translocation. Under these conditions DCCD remains covalently bound to the F0F1-ATPase complex and F1 remains bound to Fo. Significantly, when the F0F1-ATPase is inhibited by the Fo-directed inhibitor venturicidin rather than DCCD, DES is also able to restore ATPase activity. In contrast, DES is unable to restore ATPase activity to F0F1 preparations inhibited by the Fo-directed inhibitors oligomycin or tricyclohexyltin. However, combinations of [DES + DCCD] or [DES + venturicidin] can restore ATPase activity to F0F1 preparations inhibited by either oligomycin or tricyclohexyltin. Results presented here indicate that the F0 moiety of the rat liver mitochondrial proton ATPase contains a distinct binding site for DES. In addition, they suggest that at saturating concentrations simultaneous occupancy of the DES binding site and sites for either DCCD or venturicidin promote "uncoupled" ATP hydrolysis.     

Purification and characterization of the F1-ATPase from Clostridium thermoaceticum.

The F1 portion of the H+-ATPase from Clostridium thermoaceticum was purified to homogeneity by solubilization at low ionic strength, ion-exchange chromatography, and gel filtration. The last indicated the Mr to be 370,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the pure enzyme revealed four bands with Mr corresponding to 60,000, 55,000, 37,000, and 17,000 in an apparent molar ratio of 3:3:1:1. The purified enzyme would bind to stripped membranes to reconstitute dicyclohexylcarbodiimide-sensitive ATPase activity. Phosphohydrolase activity, measured at 58 degrees C, was optimal at pH 8.5. In the presence of a 1 mM excess of Mg2+ over the concentration of ATP, the Km for ATP was 0.4 mM, and the Vmax was 6.7 mumol min-1 mg-1. Unlike the membrane-bound F1F0 complex, the F1-ATPase was relatively insensitive to the inhibitors dicyclohexylcarbodiimide and tributyltin chloride. Both the complex and the F1-ATPase were inhibited by quercetin, azide, 7-chloro-4-nitro-benz-2-oxa-1,3-diazole, and free magnesium, and both were stimulated by primary alcohols and sulfite. In whole cells, the F1F0-ATPase catalyzed the synthesis of ATP in response to a pH gradient.     

Nucleotide effects on kinetic properties of mitochondrial ATPase

The hydrolytic activity of mitochondrial ATPase, both in its soluble form as F1-ATPase, or as membrane bound in whole mitochondria, was affected by the presence of free nucleoside di- or triphosphates; these effects were largely depending not only on their concentration but also on the substrate concentration. The existence of a regulatory site or sites is proposed; these sites would have a higher affinity for the free nucleoside triphosphates than for the diphosphates, and the interaction of any of these nucleotides with the proposed regulatory site or sites would lead to an activation. The nucleotide regulatory site or sites seem to be different from the anion binding sites since neither free ATP nor free GTP compete with activating or inhibitory anions.