山莨若碱通过膜脂影响兔肾(Na~++K~+)-ATP酶的构象及活性

研究了山莨菪碱对处于不同脂双层的兔肾外髓质(Na~++K~+)-ATP酶活性的影响,结果表明山莨菪碱对(Na~++K~+)-ATP酶的抑制作用与该酶所处的脂环境密切相关,如对去脂后的酶活性无明显影响,而对重组于酸性磷脂脂质体的酶比对重组于中性磷脂脂质体的酶有更大的抑制作用。园二色性实验表明,山莨菪碱使带349个界面脂分子的(Na~++K~+)-ATP酶二级结构发生明显变化,而对带189个界面脂分子的酶无明显作用。另外利用差示量热扫描研究表明山莨菪碱对酸性磷脂和中性磷脂脂质体或脂酶体相变行为有不同的影响。     

山莨菪碱对重组的大鼠脑(Na~++K~+)-ATP酶活力的影响

本文研究了山莨菪碱对经胆酸盐透析重组的鼠脑(Na~++K~+)-ATP酶活性的影响.结果表明.用大豆磷脂重组的(Na~++K~+)-ATP酶活力恢复最大;酸性磷脂PG次之;中性磷脂DPPC最低.对用DPPC和大豆磷脂重建的(Na~++K~+)-ATP酶,山莨菪碱降低酶的水解活性,而对PG重组的脂酶体,山莨菪碱则提高(Na~++K~+)-ATP酶的活力.     

山莨菪碱对重组鼠脑(Na~++K~+)—ATP酶旋转运动的影响

本文采用饱和转移顺磁共振技术研究了山莨菪碱对重组鼠脑(Ba~++K~+)-ATP酶旋转运动的影响.从鼠脑纯化的(Na~++K~+)-ATP酶经马来酰亚胺自旋探针标记,再重组于大豆磷脂脂质体.结果表明,(Na~++K~+)-ATP酶旋转相关时间为6—12微秒左右,山莨菪碱加速重组后的(Na~++K~+)-ATP酶的旋转运动.Arrhcnius图示出在10℃左右出现—明显折点,关于山莨菪碱抑制重组(Na~++K~+)-ATP酶活性与增加酶蛋白的旋转运动的关系进行了讨论.     

山莨菪碱对重组鼠脑(Na~++K~+)—ATP酶旋转运动的影响

本文采用饱和转移顺磁共振技术研究了山莨菪碱对重组鼠脑(Ba~++K~+)-ATP酶旋转运动的影响.从鼠脑纯化的(Na~++K~+)-ATP酶经马来酰亚胺自旋探针标记,再重组于大豆磷脂脂质体.结果表明,(Na~++K~+)-ATP酶旋转相关时间为6—12微秒左右,山莨菪碱加速重组后的(Na~++K~+)-ATP酶的旋转运动.Arrhcnius图示出在10℃左右出现—明显折点,关于山莨菪碱抑制重组(Na~++K~+)-ATP酶活性与增加酶蛋白的旋转运动的关系进行了讨论.     

(Na~++K~+)—ATP酶与磷脂膜的相互作用

本文研究了不同磷脂对兔肾外髓质(Na~++K~+)-ATP酶活性的影响、结果表明,DOPC、PG重组活性最高,用DMPC重组导致酶失活,酸性磷脂有利于维持该酶活性.DSC及自旋标记ESR实验结果示出(Na~++K~+)-ATP酶有选择地与酸性磷脂相互作用.     

(Na~++K~+)—ATP酶与磷脂膜的相互作用

本文研究了不同磷脂对兔肾外髓质(Na~++K~+)-ATP酶活性的影响、结果表明,DOPC、PG重组活性最高,用DMPC重组导致酶失活,酸性磷脂有利于维持该酶活性.DSC及自旋标记ESR实验结果示出(Na~++K~+)-ATP酶有选择地与酸性磷脂相互作用.     

羟基自由基对兔脑微粒体膜脂及膜蛋白的损伤

本文研究了过氧化氢与亚铁离子体系产生的羟基自由基对兔脑微粒体脂质过氧化作用及对膜上(Na~++K~+)-ATP酶活性的影响.结果表明,羟基自由基导致兔脑微粒体脂质过氧化,增加丙二醛的含量.羟基自由基还使微粒体膜巯基数下降,(Na~++K~+)-ATP酶活力受到抑制.阿魏酸钠对抑制微粒体脂质过氧化及对膜巯基和(Na~++K~+)-ATP酶均有保护作用.自旋捕集实验结果进一步证明药物对羟基自由基的猝灭作用.     

菝葜皂甙原对Na~ 、K~ -ATP酶抑制的动力学研究

本文对菝葜皂甙原抑制Na~ 、K~ -ATP酶的动力学与机理进行了研究,并且与哇巴因的抑制作用进行了比较。动力学研究表明菝葜皂甙原抑制Na~ 、K~ -ATP酶,对底物Na~ 、K~ 均为混合性抑制剂;而对ATP则为反竞争性抑制剂。观察了磷脂对哇巴因和菝葜皂甙原抑制Na~ 、K~ -ATP酶的影响。磷脂不影响哇巴因的抑制作用;但多种磷脂都不同程度地降低菝葜皂甙原对Na~ 、K~ -ATP酶的抑制作用。     

山莨菪碱对大鼠脑突触膜(Na~++K~+)-ATP酶抑制动力学的研究

山茛菪碱可以明显抑制大鼠脑突触膜(Na~++K~+)-ATP酶的活性。动力学研究结果表明,这种抑制作用为竞争性。     

关于溴氰菊酯神经毒作用的生物化学研究

 本文利用生物化学的手段,对大鼠进行了急性和亚急性毒性实验,研究溴氰菊酯对动物中枢神经系统离子调节作用的影响。急性实验结果表明:<1>溴氰菊酯能显著抑制脑微粒体上的Ca~(2+)+Mg~(2+)-ATP酶和Na~++K~+-ATP酶活性,但并不降低ecto-Ca~(2+)-ATP酶(细胞表面的Ca~(2+)-ATP酶)的活性;<2>溴氰菊酯对大鼠小脑组织中的环腺苷酸含量无明显影响,但却能显著升高与其作用相反的环鸟苷酸含量。体外实验证明,溴氰菊酯能够减少线粒体对Ca~(2+)的主动摄取。在对大鼠进行的亚急性实验中,发现溴氰菊酯中毒组与对照组大鼠的Ca~(2+)+Mg~(2+)-ATP酶、Na~++K~+-ATP酶和ecto-Ca~(2+)-ATP酶的活性均无显著性差异。根据以上结果推测,在急性中毒的条件下,溴氰萄酯能引起大鼠脑神经细胞内Ca~(2+)和Na~+的浓度增高,致使神经兴奋性发生改变。     

山莨菪碱对肌质网Ca~(2+)-ATPase活力和转运功能的影响

本文研究了山莨菪碱对肌质网Ca~(2 )-ATPase活力及转运功能的影响.对膜结合及分离纯化的Ca~(2 )-ATPase,体系中加入不同量的药物都对酶的活力及转运效率无明显影响.当将药物与肌质网或纯化的Ca~(2 )-ATPase预保温后,山莨菪碱则表现出在低浓度使酶激活,高浓度抑制酶的活力.但都导致SRCa~(2 )转运效率降低.对用保温,超声及去污剂透析三种不同方法重建的脂酶体,结果表明:山莨菪碱通过作用于膜脂后,在低浓度激活Ca~(2 )-ATPase、高浓度抑制酶的活力.比较药物对不同类型纯磷脂重建的脂酶体活性的影响发现:山莨菪碱对含有酸性磷脂的脂酶体Ca~(2 )-ATPase的作用较不含酸性磷脂的要大.     

不同磷脂和糖脂对莱氏衣原体膜上Mg~（2＋）－ATPase活性的影响

莱氏衣原体膜上Ｍｇ~（２＋）－ＡＴＰａｓｅ用ＤＯＣ溶解后,经Ｓｅｐｈａｒｏｓｅ－６Ｂ和ＤＥＡＥ－ＣｅｌｌｕｌｏｓｅＤＥ－５２离子交换柱,得到了部分纯化的Ｍｇ~（２＋）ＡＴＰａｓｅ,并将此ＡＴＰａｓｅ与不同极性头部的磷脂和膜糖脂重组,研究了不同的极性头部的磷脂和膜糖脂对ＡＴＰａｓｅ活性的影响。此酶的活性不依赖酸性磷脂,ＰＧ、ＤＰＧ、大豆磷脂等明显抑制酶活性,中性磷脂ＤＭＰＣ、ＰＥ、ＰＣ则能增加酶活性,其中尤以非双层脂ＰＥ的作用最为明显。从莱氏衣原体膜上提取的糖脂（ＭＧＤＧ,ＤＧＤＧ）单独和ＡＴＰａｓｅ重组时,酶活性增加并不明显,当ＭＧＤＧ和ＤＧＤＧ以等比例混合时,能大大地增加酶活性。这表明Ｍｇ~（２＋）－ＡＴＰａｓｅ的活性很大程度上与磷脂的表面电荷及磷脂的组成相关。     

Alterations in phospholipid-dependent (Na+ +K+)-ATPase activity due to lipid fluidity. Effects of cholesterol and Mg2+.

The (Na+ +K+)-activated, Mg2+-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble form depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na+ +K+)-ATPase in its pH optimum being around 7.0, showing optimal activity at Mg2+:ATP mol ratios of approximately 1 and a Km value for ATP of 0.4 mM. Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg2+: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 degrees C, with activation energy (Ea) values of 13-15 kcal/mol above this temperature and 30-35 kcal below it. A further discontinuity was also found at 8.0 degrees C and the Ea below this was very high (greater than 100 kcal/mol). Increased Mg2+ concentrations at Mg2+:ATP ratios in excess of 1:1 inhibited the (Na+ +K+)-ATPase activity and also abolished the discontinuities in the Arrhenius plots. The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na+ +K+)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20 degrees C and Ea values of 22 and 68 kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg2+-ATPase normally showed a linear Arrhenius plot with an Ea of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg2+-ATPase activity, which now also showed a discontinuity at 20 degrees C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the Km values for ATP. Since both cholesterol and Mg2+ are known to alter the effects of temperature on the fluidity of phospholipids, the above results are discussed in this context.     

Studies on the purified, lipid-reconstituted (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B membranes. Dependence of enzyme activity on lipid headgroup and hydrocarbon chain structure

The purified (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B membranes was successfully reconstituted with a number of different phospho- and glycolipids, and the ability of these lipids to support the function of this enzyme was evaluated by their ability to increase the specific activity of the purified enzyme and by their ability to restore its lipid-phase state-dependent properties which were lost during purification. The incorporation of this ATPase into liposomes composed of the endogenous membrane lipids of the organism, or of zwitterionic phospholipids such as phosphatidylcholine or phosphatidylethanolamine, results in a full reconstitution of its activity and its lipid-phase state-dependent properties. In contrast, anionic phospholipids alone, or in combination with zwitterionic phospholipids at concentrations higher than 10 mol % of the anionic phospholipid, cause an irreversible inhibition of this ATPase. However, when combined with neutral glycolipids, larger amounts of anionic phospholipid can be tolerated without enzyme inhibition. Phosphatidylcholines with acyl chains of 14-24 linear carbon atoms and varying degrees of branching and unsaturation successfully reconstitute the enzyme, in marked contrast to the shorter chain homologues, which were ineffective. Our results indicate that the full expression of the activity of the A. laidlawii B ATPase requires a host lipid bilayer membrane of low to moderate negative surface charge which is predominantly liquid-crystalline and of a minimal bilayer thickness. Once such requirements are met, the enzyme exhibits considerable flexibility regarding the nature of the lipids which can effectively support its function. In particular, the activity of the A. laidlawii B ATPase is not very sensitive to lipid "fluidity" in the liquid-crystalline state.     

Odorous chemical perturbations of (Na+ + K+)-dependent ATPase activities. Effects on native and lipid-substituted preparations from individual turbinals from dog olfactory tissue.

Individual turbinals from the right and left sides of dog olfactory tissue were removed and nerve-ending-particle preparations were prepared. (Na+ + K+)-dependent ATPase activities of the individual preparations, and the effect of several odorous compounds [including (+)- and (-)-carvone] on the (Na+ + K+)-dependent ATPase activities, were determined. The maximally stimulatory odorant concentration in the reaction mixture for the majority of odorants was found to be 1.0 mM. Matched pairs of left/right turbinals showed a lack of bilateral symmetry of response. (Na+ + K+)-dependent ATPase activities of various dog brain nerve-ending particle preparations responded only slightly to 1.0 mM odorants. The role of phospholipids in the (Na+ + K+)-dependent ATPase activity was found to be critical. Partial replacement of endogenous lipid with either synthetic phospholipids or extracted lipids resulted in changes in stimulation obtained with endogenous lipids alone.     

Effects of rubratoxin B on the kinetics of cationic and substrate activation of (Na+-K+)-ATPase and p-nitrophenyl phosphatase.

Rubratoxin B, a lactone-containing bisanhydride metabolite of certain toxigenic molds, inhibited (Na+-K+)-stimulated ATPase activity of mouse brain microsomes in a dose-dependent manner with an estimated IC50 of 6 x 10(-6) M. Hydrolysis of ATP was linear with time and enzyme concentration, with or without rubratoxin in reaction mixtures. Altered pH and activity curves for (Na+-K+)-ATPase demonstrated comparable inhibition by rubratoxin in buffered acidic, neutral, and alkaline pH ranges. Kinetic studies of cationic-substrate activation of (Na+-K+)-ATPase indicated classical competitive inhibition for Na+ and K+. Results also showed competitive inhibition for K+ activated p-nitrophenyl phosphatase as demonstrated by altered binding site parameters without change in the catalytic velocity of dephosphorylation of the enzyme . phosphoryl complex. Noncompetitive inhibition with regards to activation by ATP and p-nitrophenyl phosphate was indicated by altered Vmax values with no change in Km values. Inhibition was partially restored by repeated washings. Preincubation with sulfhydryl agents protected the enzyme from inhibition. Cumulative inhibition studies with rubratoxin and ouabain indicated possible interaction between the two inhibitors of (Na+-K+)-ATPase. Rubratoxin appeared to exert its effects on (Na+-K+)-ATPase by interacting at Na+ and K+ sites.     

Reconstitution of (Na+ + K+)-ATPase proteoliposomes having the same turnover rate as the membranous enzyme

Membranous (Na+ + K+)-ATPase from the electric eel was solubilized with 3-[3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate (Chaps). 50 to 70% of the solubilized enzyme was reconstituted in egg phospholipid liposomes containing cholesterol by using Chaps. The obtained proteoliposomes consisted of large vesicles with a diameter of 134 +/- 24 nm as the major component, and their protein/lipid ratio was 1.25 +/- 0.07 g protein/mol phospholipid. The intravesicular volume of these proteoliposomes is too small to consistently sustain the intravesicular concentrations of ligands, especially K+, during the assay. The decrease in K+ concentration was cancelled by the addition of 20 microM valinomycin in the assay medium. The low value of the protein/lipid ratio suggests that these proteoliposomes contain one Na+/K+-pump particle with a molecular mass of 280 kDa per one vesicle as the major component. In these proteoliposomes, the specific activity of the (Na+ + K+)-ATPase reaction was 10 mumol Pi/mg protein per min, and the turnover rate of the ATP-hydrolysis was 3500 min-1, the same as the original enzyme under the same assay condition. The ratio of transported Na+ to hydrolyzed ATP was 3, the same as that in the red cell. The proteoliposomes could be disintegrated by 40-50 mM Chaps without any significant inactivation. This disintegration of proteoliposomes nearly tripled the ATPase activity compared to the original ones and doubled the specific ATPase activity compared to the membranous enzyme, but the turnover rate was the same as the original proteoliposomes and the membranous enzyme. This disintegration of proteoliposomes by Chaps suggests the selective incorporation of the (Na+ + K+)-ATPase particle into the liposomes and the asymmetric orientation of the (Na+ + K+)-ATPase particle in the vesicle.