脂肪族类两亲物对肌浆网膜蛋白结构的影响

本文观察和比较了六种C_(18)脂肪族类两亲物(fatty amphiphile,FA),包括硬脂酸(stearic acid)、硬脂胺(stearyl amine)、硬脂醇(stearyl alcohol)、油酸(oleic acid)、油胺(oleylamine)和油醇(oleyl alcohol),对肌浆网(sarcoplasmic reticulum,SR)钙泵蛋白结构的影响。当FA∶SR(μmol∶mg)的比例为2.67∶1—21.33∶1时,除油醇(oleyl alcohol)外,其余五种FA引起天然兔骨骼肌肌浆网蛋白内源荧光强度降低。随FA∶SR比例升高,降低幅度加大。五种FA的最大降低幅度在10—32％之间。其中带电荷FA比不带电荷FA的作用强,但均未见峰位位移。当FA∶SR的比例为2.67∶1时,除硬脂醇(stearyl alcohol)外,其余五种FA使N-(3-芘)-马来酰胺(N-(3-pyrene)maleimide,N-(3-p)-M)修饰的SR蛋白巯基荧光强度分别上升9％,40％,150％,193％和5％,但也未见峰位位移。ATP可减弱胺类FA引起的SR蛋白内源荧光降低和巯基荧光升高的幅度。油酸、油胺和硬脂胺既抑制SRCa~(2+),Mg~(2+)-ATPase活力和SR钙蓄积,又使SR钙泵蛋白的巯基修饰荧光显著上升。提示C_(18)脂肪族类两亲物使SR功能受损与其引起钙泵蛋白构象的显著变化有关。     

脂肪族类两亲物对肌浆网膜脂区结构的影响

C18饱和脂肪酸和胺可增加DPH标记肌浆网(SR)的荧光偏振度,而C18单不饱和脂肪酸。胺和醇则使其偏振度下降。加入MgATP,可除去单不饱和脂肪胺引起的DPH标记的荧光偏振度下降,并使之高于未加脂肪胺的对照水平。饱和酸及相应胺可使标记于膜脂中层和深层的TAS和12AS的荧光偏振度上升,不饱和酸及相应胺和醇仅使12AS荧光偏振下降。说明脂肪族类两亲物对SR膜流动性的影响与脂肪链饱和程度有关。饱和者主要使膜中、深层流动性下降.不饱和者主要使膜深层流动性升高。     

H~＋－ATPase单位点水解过程中Fo构象的变化

利用Ｈ＋－ＡＴＰ酶复合体（也称ＡＴＰ合成酶）中的Ｆｏ的色氨酸荧光,观察了复合体中Ｆ１结合ＡＴＰ或ＡＤＰ（酶蛋白与底物分子比为１：１）时,Ｆｏ的荧光猝灭常数的变化（用竹红菌乙作为膜区蛋白荧光的猝灭剂）结果表明Ｆ１结合ＡＴＰ或ＡＤＰ时Ｆｏ可得到不同的猝灭常数（Ｋｓｖ）,也就是Ｆｏ会产生不同的构象变化。加入二价金属离子起动ＡＴＰ水解反应结束后：ＡＴＰ＋Ｈ２Ｏ→ＡＤＰ＋Ｐｉ,这时可以在Ｆｏ观察到与ＡＤＰ加Ｍｇ２＋时相同猝灭常数Ｋｓｖ;用荧光强度随时间进程变化的实验可观察到Ｆ１水解过程中导致Ｆｏ构象变化的动力学过程。这些结果说明了Ｈ＋－ＡＴＰ酶复合体ＡＴＰ合成的过程中Ｆ１与Ｆｏ之间存在着构象之间的通信与传递。     

内源无机磷酸盐对叶绿体Mg^2＋—ATP酶功能的调节

用ＳＴＮ提取的菠菜叶片叶绿体再用ＳＴＮ洗涤后,它的内源无机磷酸盐含量急剧减少,其Ｍｇ＾２＋－ＡＴＰ酶水解ＡＴＰ的能力明显下降。进一步研究表明：叶绿体的内源无机磷酸盐含量减少会使反映叶绿体类囊体膜内外△ＰＨ变化的９－氨基吖啶的荧光猝灭减少,并加速光激活态ＡＴＰ酶的暗失活。     

牛心线粒体ATP酶抑制蛋白对酶的催化部位构象的影响

以标记在ＡＴＰ酶（Ｆ_１）催化部位的ＴＮＰ－ＡＴＰ为荧光探针,比较测定了Ｆ_１与其抑制蛋白（ＩＦ_１）结合前后的ＴＮＰ－ＡＴＰ荧光光谱、荧光寿命和荧光偏振光谱。结果表明在ＩＦ１的作用下,酶分子催化部位的极性下降,ＴＮＰ－ＡＴＰ分子运动的自由度减小,提示ＩＦ_１引起了Ｆ_１催化部位的构象改变。     

牛心线粒体ATP酶抑制蛋白对酶的催化部位构象的影响

以标记在ＡＴＰ酶（Ｆ１）催化部位的ＴＮＰ－ＡＴＰ为荧光探针,比较测定了Ｆ１与其抑制蛋白（ＩＦ１）结合前后的ＴＮＰ－ＡＴＰ荧光光谱,荧光寿命和荧光偏振光谱,结果表明在ＩＦ１的作用下,酶分子催化部位的极性下降,ＴＮＰ－ＡＴＰ分子的自由度减小,提示ＩＦ１引起了Ｆ１催化部位的构象改变。     

牛脑V型质子转运ATP酶复合体依赖于温度的活性变化

在ＫＣｌ介质中牛脑Ｖ－型质子转运ＡＴＰ酶复合体活力温度的Ａｒｒｈｅｎｉｕｓ图在３３℃附近呈现明显的折点,同样做其Ｎ－［１－芘］马来酰亚胺（Ｎ－［１－Ｐ］Ｍ）的荧光－温度的Ａｒｒｈｅｎｉｕｓ图,发现其折点温度也为３３℃,当加入１００μｍｏｌ／ＬＮＥＭ（Ｎ－ｅｔｈｙｌｍａｌｅｉｍｉｄｅ）,ＡＴＰ酶复合体活力部分被抑制后的Ａｒｒｈｅｎｉｕｓ图折点下降为２７℃,加入０．７５－０．８５ｍｏｌ／Ｌ尿素则活力的Ａｒｒｈｅｎｉｕｓ图的折点变为３０℃。加入６％（Ｖ／Ｖ）的乙醇后,活力的Ａｒｒｈｅｎｉｕｓ图的折点上升为３８℃。加入ＮＥＭ,尿素和乙醇的内源荧光和Ｎ－［１－Ｐ］Ｍ标记的荧光测定,表明它们确实引起了牛脑Ｖ－型质子转运复合体构象的改变,这表明引起构象变化配基的加入,可改变牛脑Ｖ－型质子转运ＡＴＰ酶复合体的Ａｒｒｈｅｎｉｕｓ图折点温度,也说明牛脑Ｖ－型质子转运ＡＴＰ酶复合体Ａｒｒｈｅｎｉｕｓ图折点与酶复合体的构象直接相关。     

H~＋－ATPase单位点水解过程中Fo构象的变化

利用Ｈ＾＋－ＡＴＰ酶复合中的Ｆｏ的色氨酸荧光,观察了复合体中Ｆ１结合ＡＴＰ或ＡＤＰ时,Ｆｏ的荧光猝灭常数的变化结果表明Ｆ１结合ＡＴＰ或ＡＤＰ时Ｆｏ可得到不同的猝来常数,也就是Ｆｏ会产生不同的构象变化。这些结果说明了Ｈ＾＋ＡＴＰ酶合ＡＴＰ合成的过程中Ｆ１与Ｆｏ之间存在着构象之间的通信与传递。     

内源无机磷酸盐对叶绿体Mg~（2＋）－ATP酶功能的调节

用ＳＴＮ（含蔗糖,０．４ｍｏｌ／Ｌ;ＮａＣｌ,０．０１ｍｏｌ／Ｌ和Ｔｒｉｓ－ＨＣｌ,０．０２ｍｏｌ／Ｌ,ｐＨ７．４）提取的菠菜叶片叶绿体再用ＳＴＮ洗涤后,它的内源无机磷酸盐含量急剧减少,其Ｍｇ２＋－ＡＴＰ酶水解ＡＴＰ的能力明显下降。进一步研究表明：叶绿体的内源无机磷酸盐含量减少会使反映叶绿体类囊体膜内外△ｐＨ变化的９－氨基吖啶的荧光猝灭减少,并加速光激活态ＡＴＰ酶的暗失活。     

DOPE、DOPC对重组去脂肌质网Ca~(2+)-ATPase活力和构象的影响

经磷脂酶Ａ２ 去脂的肌质网Ｃａ２ ＋ － ＡＴＰａｓｅ 重组于不同比例的二油酰磷脂酰胆碱（Ｄｉｏｌｅｏｙｌｐｈｏｐｈａｔｉｄｙｌｃｈｏｌｉｎｅ,ＤＯＰＣ） 和二油酰磷脂酰乙醇胺（Ｄｉｏｌｅｏｙｌｐｈｏｐｈａｔｉｄｙｌｅｔｈａｎｏｌａｍｉｎｅ,ＤＯＰＥ） 形成脂酶体,研究了不同磷脂环境中Ｃａ２ ＋ － ＡＴＰａｓｅ 的ＡＴＰ 水解和Ｃａ２ ＋ 转运活力。结果表明,ＤＯＰＣ 和ＤＯＰＥ 分别有利于ＡＴＰ 水解和Ｃａ２ ＋ 的转运,ＤＯＰＥ 可以增强Ｃａ２ ＋ － ＡＴＰａｓｅ 的ＡＴＰ水解和Ｃａ２ ＋ 转运之间的偶联效率。利用内源荧光、荧光淬灭及Ｆｏｒｓｔｅｒ 能量转移原理测定Ｃａ２ ＋ －ＡＴＰａｓｅ 相应的构象变化, 发现随着ＤＯＰＥ／ ＤＯＰＣ 比例的改变使Ｃａ２ ＋ － ＡＴＰａｓｅ 构象发生相应的变化。     

Energy-dependent redistribution of a lipophilic anion in sarcoplasmic reticulum vesicles and Ca2-ATPase molecules

The distribution of lipophilic anion of phenyldicarbaundecarborane (PCB-) between water phase and fragments of sarcoplasmic reticulum (SR) from skeletal muscle was studied, using a bilayer lipid membrane (BLM) as a selective electrode. Addition of ATP leads to an increase in PCB- binding to SR vesicles. The ATP effect is totally reversible only in the presence of both EGTA and A23187. Chlorides, in contrast with oxalate and phosphate, do not reduce the ATP-dependent PCB- binding. Oxalate decreases also the energy-dependent extrusion of protons from SR into the medium. Preliminary incubation of SR fragments with calcium gluconate leads to a decrease in PCB- binding. Addition of ATP to purified Ca2+-ATPase is coupled with a release of PCB- and calcium from the enzyme. It is suggested that ATP-dependent binding of PCB- to SR membranes reflects calcium incorporation into the hydrophobic region of Ca2+-ATPase molecules.     

The remotor muscle of the lobster antenna: sarcoplasmic reticulum and skinned fiber experiments

The striated remotor muscle of the lobster antenna has an extraordinarily profuse sarcoplasmic reticulum as shown by electron microscopy. Gel electrophoresis reveals a simple protein composition in which the Ca2+-ATPase predominates. Vesicles of sarcoplasmic reticulum (SR) from this remotor are shown to operate Ca2+ binding, Ca2+ transport, and Ca2+-activated hydrolysis of ATP with an usual efficiency (2 Ca2+ transported per ATP hydrolysed, 4 mumol ATP hydrolysed/mg protein/min). Skinned fiber experiments were performed. They indicate behaviour of the remotor expected from observations by EM and gel electrophoresis: contraction of low maximal intensity under Ca2+ excitation, long internal diffusion time due to the large volume of SR to be crossed, and large Ca2+ content released in a caffeine-sensitive manner.     

Spin-labeling of adenosine triphosphatase in sarcoplasmic reticulum membrane and change in the state of the spin labels induced by deoxycholate.

Fragmented sarcoplasmic reticulum (SR) was reacted with a thiol-directed spin label, N-(1-oxyl-2,2,6,6,-tetramethyl-4-piperidinyl)maleimide, under various conditions. It was found that ATP inhibited the binding of the label to SR protein in the initial phase of the reaction, but as the incubation time was extended up to 18 h, the amount of label bound to SR protein in the control and ATP-containing samples became almost identical. The Ca2+-dependent ATPase control and ATP-containing samples became almost identical The Ca2+-dependent ATPase (ATP phosphohydrolase [EC 3.6.1.3]) of SR was protected by the presence of ATP during incubation with relatively low concentrations of spin label, irrespective of the total amount of label bound, although with increasing concentration of bound label the ATPase activity decreased. Deoxycholate slightly reduced the rotational freedom of the label bound to SR protein and decreased the initial rate of quenching of protein-bound nitroxide by ascorbate. From an analysis of these results, it was concluded that the binding of deoxycholate to protein decreases the accessibility of ascorbate to the protein-bound label.     

Conformational change of the foot protein of sarcoplasmic reticulum as an initial event of calcium release

Heavy sarcoplasmic reticulum vesicles were labeled with the thiol-reacting fluorescent probe N-(7-dimethylamino-4-methyl-4-coumarinyl)maleimide (DACM), and the DACM-labeled foot protein moiety was purified. The fluorescence intensity of the DACM attached to the foot protein decreased by the addition of low (activating) concentrations of ryanodine, while it increased at higher (inhibitory) concentrations, suggesting that the lower fluorescence represents the active state of the foot protein, while the higher fluorescence, its inactive state. Under conditions that induce Ca2+ release from SR (Ca2+ jump, addition of Ca2+ release inducing reagents such as caffeine and polylysine), the fluorescence intensity of the protein-attached DACM decreased rapidly (e.g. k congruent to 70 s-1 under optimum conditions). The initial rate of Ca2+ release from the DACM-labeled SR showed a close correlation with the amplitude of the fluorescence change of the foot protein-attached DACM under variety of conditions; e.g. in the presence of Ca2+, polylysine, ATP, and ruthenium red, etc. The fluorescence change of the foot protein was much faster than Ca2+ release from SR under a variety of conditions of Ca2+ release. We propose that the binding of release triggering reagents to the foot protein induces a rapid conformational change, which in turn regulates Ca2+ release.     

Heparin induces Ca2+ release from the terminal cisterns of skeletal muscle sarcoplasmic reticulum

Using a Ca2+-selective electrode and the chlorotetracycline fluorescence technique, the effects of heparin on Ca2+ transport in the sarcoplasmic reticulum (SR) of skeletal muscles in the absence of oxalate were investigated. It was shown that heparin (0.5-10 micrograms/ml) causes a rapid release of 40-50 nmol Ca2+/mg protein from the terminal cistern SR vesicles bound to 130-150 nmol/mg protein of Ca2+ in the presence of ATP. However, heparin has practically no effect on the longitudinal cistern fraction of SR. The effects of heparin can be prevented by ruthenium red. No influence of heparin is observed in the case of the Ca2+-induced release of Ca2+ from the terminal cisterns. When the Ca2+ release is induced by heparin, no Ca2+-induced release of Ca2+ takes place.     

The interaction between ruthenium red and the isolated sarcoplasmic reticulum

The multivalent anions, ATP and oxalate, present at 5mM concentrations in incubation mixtures with isolated sarcoplasmic reticulum (SR) preparations, reduce the binding of ruthenium red (RR) to the SR. When oxalate is omitted from the incubation mixture and ATP is used at reduced concentrations, it is possible to observe an inhibitory effect of RR on calcium uptake by the SR and on ATPase activity. However, this inhibition is only partial and it remains clear that calcium transport in the SR is much less susceptible to inhibition by RR than is calcium transport in mitochondria. The effect of multivalent anions in suppressing the effect of RR, a hexavalent cation, is probably due to the formation of soluble complexes with RR.     

The interaction of 1-anilino-8-naphthalenesulfonate with the membranes of the sarcoplasmic reticulum and various lipid compounds

Summary (1) The enzymatic removal of lipids from the vesicular membranes of the sarcoplasmic reticulum does not interfere with the fluorescence of the 1-anilino-8-naphthalenesulfonate (ANS) vesicular complex. (2) The fluorescence intensity of the ANS vesicular complex is considerably (50%) reduced by oleic acid (0.5mm) because it displaces ANS from its binding sites. (3) Stearic acid, which also combines with the membranes, interferes neither with ANS binding nor with ANS fluorescence. (4) Of all lipid compounds tested, oleylamine produces the most pronounced fluorescence enhancement of ANS. (5) The complexes formed between oleic acid and cetyltrimethyl ammonium salts or between oleic acid and polylysine produce a much higher fluorescence enhancement than the isolated components. (6) Low concentrations of ether added to ANS-containing vesicular suspensions reduce their fluorescence intensity. It returns to the initial intensity when the ether is removed. (7) A small cyclic change of the fluorescence of the vesicular ANS complex takes place during active calcium uptake.     

Effect of temperature on fluorescence of labels and probes of various localizations incorporated into sarcoplasmic reticulum samples

Studies were carried out of temperature relationship of dansylchloride, N-3-pyrenylmaleinimide fluorescence, SR membranes, self-luminescence caused by Ca-ATPase tryptophane - provided fluorescence and of pyrene excimerization in membrane preparations of sarcoplasmic reticulum (SR) of rabbit skeletal muscles. Temperature relationship of fluorescence intensity of dansylchloride and N-3-pyrenylmaleimide in Arrhenius coordinates has bends at 15 and 35 degrees. Selffluorescence of protein samples linearly depends on temperature. Temperature relationship of the ratio between the intensities of exsimeric and monomeric forms of pyrene Fa/Fm in Arrhenius coordinates has the bend at 20-22 degrees. Hence only the latter relationship coincides with the shape of Arrhenius graph for enzymatic activity of SR Ca-ATPase.