跨膜Ca~(2+)梯度对重组腺苷酸环化酶活力及构象的影响

本文将分离并部分纯化的腺苷酸环化酶催化亚基(ACc)重组于具有或不具有跨膜Ca~(2+)梯度的大豆磷脂脂质体上.酶活性的测试结果表明,酶活性中心一侧Ca~(2+)浓度低于另一侧(500倍)的脂酶体(类似生理条件)酶活性最高;在Ca~(2+)浓度梯度相反的情况下酶活性最低.外加Ca~(2+)载体A23187消除脂酶体两侧的Ca~(2+)梯度可导致前者酶活力降低而后者酶活力升高.不具有Ca~(2+)浓度梯度的脂酶体酶活性介于上述两种脂酶体之间. 蛋白内源荧光及KI对其淬灭效率的测试结果均反映上述几种脂酶体中ACc构象的差异.     

STUDY ON THE INTERACTION OF Se AND ERYTHROCYTE MEMBRANE——EFFECT OF Se ON THE Na, K-ATPase ACTIVITY AND FLUIDITY OF ERYTHROCYTE MEMBRANES

The content of binding-Se in erythrocyte membrane may affect its structure and function. Decrease in So content will lead to a lower activity of Na, K-ATPase and lipid fluidity in ghosts, and a marked increase in Na, K-ATPase activity as well as lipid fluidity of erythrocyte membrane was observed by supplementation of low concentration of Na_2SeO_3 (0.03—0.3 ppm Se), in vitro. This effect was obvious only when the ghosts wore incubated with Se at 0—4°C for at least half an hour prior to assay.     

膜蛋白嵌入脂质体的研究——金属离子对猪心线粒体H+-ATP酶在脂质体重组的影响

用胆酸盐透析法将猪心线粒体H⁺-ATP酶在脂质体重组过程中,1mM Mg²⁺能显著提高重组酶的活性(³2P₁-ATP交换,酶水解活力及其对寡霉素、二环己基碳二亚胺DCCD的敏感性和ANS荧光变化),而且重复性较好.除Mg²⁺外,对其它二价金属离子和精脒计影响重组的作用也进行了比较。对³2P_i-ATP交换活性的影响大小为Mg^a+>Ca²⁺>Mn²⁺>sr^a+;对抑制剂敏感性的影响程度为Ca²⁺>Mg²⁺>Mn²⁺>Sr²⁺.cd²⁺,Zn²⁺和精脒³⁺对重组酶的二种活性均无明显影响.对金属离子影响重组H⁺-ATP酶活性的机理也进行了讨论.     

玉米线粒体抗氰化物氧化的研究——α-酮戊二酸和琥珀酸氧化对氰化物敏感性的比较

1 mM KCN完全抑制玉米线粒体末端氧化酶的活性和琥珀酸氧化,但а-酮戊二酸的氧化仍止常进行.当同时加入KCN和KSCN时,α-酮戊二酸的氧化才被抑制. 当琥珀酸氧化被1 mM KCN完全抑制后,加入α-酮戊二酸呼吸恢复,但此时细胞色素c和a仍处于还原状态. 与KCN不同,抗霉素A对玉米线粒体α-酮戊二酸氧化具有显著的抑制效应. 根据上述结果,可以认为,玉米线粒体抗氰化物氧化途径的组份不包含细胞色素c和a,它与主链的分叉点似在细胞色素b之后.     

硒与红细胞膜的研究——Ⅰ.硒对红细胞膜Na,K-ATP酶活性和流动性的影响

人红细胞膜结合硒含量能够影响膜的结构与功能。结合硒含量减少会导致Na,K-ATP酶活性和膜脂流动性的降低。加入微量Na_2SeO_3(0.03—0.3ppm Se)能使Na,K-ATP酶活性和流动性显著增加。人红细胞膜必须与硒经半小时以上预处理才能显示出明显的效应。     

EFFECT OF TRANSMEMBRANE Ca~(2+) GRADIENT ON CONFORMATION AND ENZYME ACTIVITY OF RECONSTITUTED ADENYLATE CYCLASE

Adenylate cyclase from bovine brain cortex was reconstituted into asolectin liposomes with (500-fold) or without transmembrane Ca~(2+) gradient. The enzyme activity of four types of proteoliposomes (the active center of enzyme exposing outside) was compared. The highest adenylate cyclase activity was observed in the vesicles with outside lower Ca~(2+)concentration (≈10~(-6) mol/L, similar to thephysiological condition). If the transmembrane Ca~(2+) gradient was in the inverse direction (i.e. outside higher Ca~(2+) concentration, 0.5 mmol/L), a lowest enzymatic activity would appear. The difference in enzymatic activity between the two types of proteoliposomes could be diminished following the addition of Ca~(2+) ionophore A23187. Proteoliposomes without transmembrane Ca~(2+) gradient exhibited intermediate activities.The conformation difference of adenylatecyclases in the above-mentioned proteoliposomes was also detected by measuring intrinsic fluorescence and fluorescence quenching with KI.     

STUDY ON THE INTERACTION OF Se AND ERYTHROCYTE MEMBRANE——PROTECTIVE EFFECT OF Se ON THE ERYTHROCYTE MEMBRANES

During the ageing of erythrocyte membrane, the spectrin content, Na, K-ATPase activity as well as the lipid fluidity are obviously decreased. However, supplementation of a trace amount of Na_2SeO_3 in the medium could prevent the dissociation of spectrin from membrane and delay the changes of Na, K-ATPase activity and lipid fluidity. The effectiveness is proportional to Se concentration within the range of 0.1—1.0 ppm.Similar effect of supplementation of Se on the intact erythrocytes during ageing has been also observed.The protective action of Se on biomembranes is generally interpreted in terms of the activity of Se-containing glutathione peroxidase (GSHPx). However, GSHPx mainly distributes in the cytoplasma of erythrocytes, therefore it seems that the protective action of supplemented Se on the isolated erythrocyte membrane might not be related to the activity of GSHPx.     

膜蛋白嵌入脂质体的研究——Mg~(2 )通过改变脂质分子的流动性来影响重建H~ -ATP酶的活性

本文用ANS荧光探剂,自旋标记探剂5NS的实验结果表明,L·(H~ -A7P酶)_( Mg)~(2 )(Mg~(2 )存在下嵌有猪心线粒体H~ -ATP酶的脂酶体)的流动性小于L·(H~ -ATP酶)_(-Mg)~(2 )(无Mg~(2 )条件下组装的脂酶体)。DPH荧光偏振的测定也得到相似的结果。但自旋标记物12NS,16NS的实验结果反映两种脂酶体脂质分子的流动性并没有明显差异。这表明,Mg~(2 )能影响ATP酶重建体系中靠近表层部份脂质分子的流动性,而对较深层的流动性没有明显影响。可以设想,在Mg~(2 )存在下,重建猪心线粒体H~ -ATP酶活性的提高很可能是通过它对脂质分子靠近表层部份的物理状态的改变,继而影响疏水蛋白与F_1的构象,从而使H~ -ATP酶表现较高的活性。     

脱血红素细胞色素c的解折叠状态与跨膜运送能力的相关性

与马心脱血红素细胞色素c(Apocytochrome c,简称apocyt.c)相比,鸡心apocyt.c在透析复性过程中存在着显著的由无规到部分折叠的自发构象变化。用处于不同复性阶段的马心和鸡心apocyt.c对包有胰蛋白酶的单层大豆磷脂大脂质体的转运实验发现,马心apocyt.c的转运速率变化较小,而鸡心apocyt.c的跨膜能力随着分子折叠程度的增加而发生明显的下降。用圆二色技术测定马心和鸡心apocyt.c与大豆磷脂结合后的二级结构提示:apocyt.c的有效的跨膜运送依赖于其在脂双层中处于较松散的折叠结构。     

硒与红细胞膜的研究——Ⅱ.硒对红细胞膜的保护作用

人红细胞膜老化(4℃,1—3周)过程中,收缩蛋白(spectrin)合量减少,Na,K-ATP酶活性和流动性都有明显降低。如果在老化介质中加入微量Na_2SeO_3(0.1—1.0ppm Se)可防止收缩蛋白从膜上脱落并延缓Na,K-ATP酶活性和膜流动性的变化。在0.1—1.0ppm Se范围内,加入硒量愈多,效应也愈明显。当完整红细胞老化时,外加硒对膜也有相似的保护作用。硒对膜的保护作用一般用含硒的谷胱甘肽过氧化物酶(GSHPx)的活性来解释,但该酶主要分布在红细胞细胞质内,因此人红细胞膜结合硒对膜具有明显保护作用似乎很难归结于GSHPx的作用。