低氧、血管紧张素Ⅱ对肺内小动脉平滑肌细胞膜Ca2 ┐ATPase活力的影响

本课题观察了低氧及血管紧张素Ⅱ（ａｎｇｉｏｔｅｎｓｉｎⅡ,ＡｎｇⅡ）对分离培养家兔肺内小动脉平滑肌细胞（ＰＡＳＭ－Ｃｓ）膜Ｃａ２＋－ＡＴＰａｓｅ活力的影响,同时用钙通道阻断剂维拉帕米（ｖｅｒａｐａｍｉｌ,ＶＰ）进行干预,进一步了解细胞内钙与Ｃａ２＋－ＡＴＰａｓｅ活力的关系。结果表明：ＰＡＳＭＣｓ膜Ｃａ２＋－ＡＴＰａｓｅ活力对低氧具有短暂的耐受性,随低氧时间延长,Ｃａ２＋－ＡＴＰａｓｅ活力呈时间依赖性抑制;低氧、ＡＮＧⅡ均能抑制Ｃａ２＋－ＡＴＰａｓｅ活力（Ｐ＜０．０１）低氧＋ＡⅡ对Ｃａ２＋－ＡＴＰａｓｅ活力的抑制具叠加效应（Ｐ＜０．０５）;ＶＰ可逆转低氧、ＡｎｇⅡ、低氧＋ＡｎｇⅡ对Ｃａ２＋－ＡＴＰａｓｅ活力的抑制（Ｐ＜０．０１）。结果提示：低氧,ＡＮＧⅡ可通过抑制肺血管平滑肌细胞膜Ｃａ２＋－ＡＴＰａｓｅ活力而可能削弱肺血管平滑肌舒张功能也可能是低氧性肺动脉高压（ＨＰＨ）形成的原因之一。     

神经节苷脂（Gangliosides）对人红细胞膜Ca~（2＋）－Mg~（2＋）ATPase的影响

神经节苷脂（Ｇａｎｇｌｉｏｓｉｄｅｓ）是红细胞膜Ｃａ~（２＋）－Ｍｇ~（２＋）ＡＴＰａｓｅ的一种激活剂,这种激活作用也是依赖于Ｃａ~（２＋）存在。在２００μｍｏｌ／ＬＣａ~（２＋）存在的反应体系中,１００μｇ／ｍＬＧａｎｇｌｉｏｓｉｄｅｓ对Ｃａ~（２＋）－Ｍｇ~（２＋）ＡＴＰａｓｅ的激活作用最大,为基本酶活性的１５０％以上。实验还发现ＣａＭ拮抗剂三氟拉嗪（ＴＦＰ）、粉防已碱（Ｔｅｔ）等也同样抑制Ｇａｎｇｌｉｏｓｉｄｅｓ的这种激活作用。其抑制的ＩＣ_（５０）值为２５μｍｏｌ／Ｌ和３０μｍｏ１／Ｌ;而此浓度下抑制剂存在的反应体系中,对Ｃａ~（２＋）－Ｍｇ~（２＋）ＡＴＰａｓｅ的基本活性影响不大。     

PAF对肺内小动脉平滑肌细胞TXA_2，PGI_2乃Ca~（2＋）－ATPase的影响

本工作采用分离培养家兔肺内小动脉平滑肌细胞（ＰＡＳＭＣｓ），观察了外源性血小板活化因子（ｐｌａｔｅｌｅｔａｃｔｉｖａｔｉｎｇｆａｃｔｏｒ，ＰＡＦ）、ＢＮ５２０２１（ＰＡＦ受体拮抗剂）、吲哚美辛、维拉帕米对ＰＡＳＭＣｓ产生血栓素Ａ_２（ＴｘＡ_２）、前列环素（ＰＧＩ_２）及对细胞膜Ｃａ~（２＋）－ＡＴＰａｓｅ活力的影响。结果表明：（１）基础状态下ＰＡＳＭＣｓ存在花生四烯酸（ＡＡ）代谢。（２）外源性ＰＡＦ通过受体后途径激活环加氧酶促进ＡＡ代谢致ＴＸＡ_２及ＰＧＩ－２增加，ＴＸＡ_２／ＰＧＩ_２比值无明显变化。（３）外源性ＰＡＦ能直接抑制Ｃａ~（２＋）－ＡＴＰａｓｅ活力。（４）维拉帕米可逆转ＰＡＦ抑制ＰＡＳＭＣｓ膜Ｃａ~（２＋）－ＡＴＰａｓｅ活力的效应。     

花生幼苗下胚轴质膜Ca2+-ATP酶及其对低温胁迫的反应

经６％－１２％ＤｅｘｔｒａｎＴ７０密度梯度离心,获得了纯度较高的７ｄ龄花生幼苗下胚轴质膜制剂,质膜Ｃａ＾２＋－ＡＴＰａｓｅ在反应系统不存在Ｍｇ＾２＋时,可正常表现水解ＡＴＰ的活性,但此活性明显低于Ｍｇ＾２＋激活的ＡＴＰａｓｅ,Ｃａ＾２＋－ＡＴＰａｓｅ不受Ｎａ３ＶＯ４抑制,不被Ｋ＾＋激活,而被Ｃｌ＾－抑制,Ｃａ＾２＋－ＡＴＰａｓｅ的最适,ｐＨ不同于Ｍｇ＾２＋激活的ＡＴＰａｓｅ,低温胁迫显著提高质膜Ｃ     

玉米素对叶绿体耦联因子Mg~（2＋）－ATPase活力的调节

用２μｇ／ｍｌ玉米素溶液预处理叶绿体或在光活化前于活化液中加入２μｇ／ｍｌ玉米素溶液,观察到玉米素能促进叶绿体膜上耦联因子ＤＴＴ光活化Ｍｇ２＋－ＡＴＰａｓｅ及Ｍｇ２＋ＧＴＰａｓｅ的活力．且对ＧＴＰａｓｅ的促进比例常较ＡＴＰａｓｅ的大些。王米素对ＯＧ活化可溶性ＣＦ１Ｍｇ２＋－ＡＴＰａｓｅ活力同样表现出促进作用。用玉米素预处理ＣＦ１－β亚基（含微量ＣＦ１－α亚基）也观察到它能促进ＣＦ１－β亚基催化的Ｍｇ２＋－ＡＴＰａｓｅ活力。这些结果表明,玉米素在ＣＦ１上的作用部位至少有一个在β亚基或α．β亚基交界处调节其催化功能的。     

稀土离子对烟草RuBPcase的激活作用及EXFAS研究

研究了稀土离子（Ｌｎ３ ＋） 对烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ）１ ,５ － 二磷酸核酮糖羧化酶（ＲｕＢＰｃａｓｅ）活力的影响。结果表明,在该酶的反应体系中,用Ｌｎ３ ＋ 替代Ｍｇ２ ＋ ,烟草ＲｕＢＰｃａｓｅ 的活力随Ｌｎ３ ＋ 浓度的变化曲线呈双相效应, 即在高浓度时, Ｌｎ３ ＋ 抑制该酶活性; 低浓度的Ｌｎ３ ＋ 提高ＲｕＢＰｃａｓｅ 活性。其活化效应为轻稀土离子大于重稀土离子,但Ｌｎ３ ＋ 的活化效应低于Ｍｇ２ ＋ 。在有Ｍｇ２ ＋ 的反应体系中,Ｌｎ３ ＋ 在低浓度时也有提高ＲｕＢＰｃａｓｅ 活性的能力,提高幅度较低;而高浓度的Ｌｎ３ ＋ 显著地抑制酶活性。进一步对ＲｕＢＰｃａｓｅ － Ｌａ 二元复合物的ＥＸＦＡＳ 研究,证实Ｌａ３ ＋ 与ＲｕＢＰｃａｓｅ 氨基酸残基的Ｏ 原子键合,键长为２ ．５１?;Ｌａ３ ＋ 还与Ｓ 原子结合。最后对Ｌｎ３ ＋ 和ＲｕＢＰｃａｓｅ 相互作用的分子机制进行讨论     

轻稀土离子对钙调蛋白激活的磷酸二酯酶活力作用的影响

研究了轻稀土离子（Ｌｎ３＋）对钙调蛋白（ＣａＭ）调控的磷酸二酯酶（ＰＤＥ）活力的影响。结果表明,在无Ｃａ２＋的ＣａＭ（Ａｐｏ—ＣａＭ）体系中,由ＣａＭ调节的ＰＤＥ的活力随Ｌｎ３＋浓度的变化曲线是双相效应,即在高浓度时,Ｌｎ３＋具有抑制ＣａＭ调节ＰＤＥ活力的能力;低浓度的Ｌｎ３＋可以提高ＣａＭ调节ＰＤＥ活力的能力。在Ｃａ２＋４－ＣａＭ－ＰＤＥ体系中,高浓度的Ｌｎ３＋的加入能抑制ＣｈＭ调节ＰＤＥ活力的能力,其抑制程度因其离子不同而异。ＣａＭ的两类拮抗剂ＪｕＡ（非竞争性抑制剂）和ＴＦＰ（竞争性抑制剂）都能抑制ＣａＭ－Ｌｎ３＋－ＰＤＥ系统的活性。最后对Ｌｎ３＋和ＣａＭ相互作用的分子机制进行了初步的讨论。     

抗寒锻炼对冬小麦幼苗质膜Ca^2＋—ATPase的稳定作用

通过氯化铈（ＣｅＣｌ３）沉淀的电镜细胞化学方法,观察了抗寒锻炼对冬小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）幼苗质膜Ｃａ２＋ＡＴＰａｓｅ的稳定作用,主要结果是：（１）正常温度（２０℃）下生长的冬小麦幼苗（未经抗寒锻炼）,其质膜上有很强的Ｃａ２＋ＡＴＰａｓｅ活性反应;当经过－９℃３ｈ的低温处理后,质膜的Ｃａ２＋ＡＴＰａｓｅ活性明显降低;在处理１２ｈ后,质膜的Ｃａ２＋ＡＴＰａｓｅ活性进一步降低;当处理时间延长到２４ｈ,质膜的Ｃａ２＋ＡＴＰａｓｅ完全失活,同时细胞的超微结构受到破坏。（２）冬小麦幼苗在２℃低温下锻炼１５ｄ后,其质膜的Ｃａ２＋ＡＴＰａｓｅ活性高于未经抗寒锻炼的小麦幼苗。抗寒锻炼后的小麦幼苗在－９℃处理３ｈ后,质膜的Ｃａ２＋ＡＴＰａｓｅ活性与低温处理前相比无明显降低;经低温处理１２ｈ,质膜仍保持较高的Ｃａ２＋ＡＴＰａｓｅ活性,较同样低温处理（－９℃,１２ｈ）但未经抗寒锻炼的幼苗高;当－９℃低温处理２４ｈ后,质膜上仍可观察到Ｃａ２＋ＡＴＰａｓｅ的活性反应,而且细胞的超微结构也未受到破坏。结果表明,抗寒锻炼可提高冬小麦幼苗质膜Ｃａ２＋ＡＴＰａｓｅ在低温下的稳定性     

Ca2+与CaM对大白菜离体叶圆片Ca2+-ATPase和β-1,3-葡聚糖合成酶活性的影响（简报)

经 Ｃａ~（２＋）与 ＣａＭ促进剂和抑制剂处理的大白菜离休叶圆片,其细胞膜 Ｃａ~(２＋)ＡＴＰａｓｅ活性同时受Ｃａ~（２＋）与ＣａＭ的调节,而β－１,３－葡聚糖合成酶活性仅受 Ｃａ~（２＋）的调节,与 ＣａＭ无关。     

跨膜Ca~(2+)梯差对大豆下胚轴质膜H~+-ATPase活力的影响

采用两相法得到高纯度封闭的大豆下胚轴质膜微囊,研究了跨膜Ｃａ２＋梯差对质膜Ｈ＋－ＡＴＰａｓｅ质子转运和ＡＴＰ水解活力的影响。结果表明,在１０００：０．１,１０００：０．５,１０００：１及１０００：１０（μｍｏｌ／Ｌ：μｍｏｌ／Ｌ）几种梯差下,随着跨膜钙梯差的减小,质膜Ｈ＋－ＡＴＰａｓｅ质子转运活力逐步降低。然而,上述几种梯差对Ｈ＋－ＡＴＰａｓｅ水解活力的影响却很小。进一步研究发现,１０００：０．１及１０００：１（μｍｏｌ／Ｌ：μｍｏｌ／Ｌ）两种梯差对Ｋｍ值没有影响,但Ｋ＋对Ｈ＋－ＡＴＰａｓｅ的激活作用在两种梯差下存在显著差别。ＭＣ５４０荧光、ＤＰＨ荧光偏振结果表明,跨膜钙梯差影响着膜脂的聚集状态和流动性。本文对跨膜Ｃａ２＋梯差对于大豆下胚轴质膜Ｈ＋－ＡＴＰａｓｅ水解与质子转运活力影响的可能机制进行了讨论。     

粉防已碱与红细胞膜ATPase相互作用的研究

粉防已碱是一种新的钙调蛋白拮抗剂,专一性抑制人红细胞膜上依赖CaM的Ca~(2+)-Mg~(2+)-ATPase。在较高浓度下,它也不同程度地抑制Ca~(2+)-Mg~(2+)-ATPase基本活性、Na~+-K~+-ATPase和Mg~(2+)-ATPase的活性。 除CaM外,不饱和脂肪酸和有限水解均导致膜Ca~(2+)-Mg~(2+)-ATPase的活化,所有这些活化作用被Tet在大约相同的浓度范围内抑制,表明Tet除与CaM结合外,也与膜Ca~(2+)-Mg~(2+)-ATPase结合。 Tet具有抗抵渗溶血的性能,反映了拮抗CaM与药物的膜稳定性间存在相关性。     

重金属离子对猪红细胞膜Ca~(2+)-ATP酶的作用

猪红细胞膜Ca~(2+)-ATP酶是一种钙调蛋白(CaM)依赖酶,其活力又依赖巯基的完整性。实验应用Ca~(2+)-ATP酶这一模型体系观察到重金属离子,Pb~(2+)、Cd~(2+)和Hg~(2+)都能替代Ca~(2+),激活CaM,从而激活Ca~(2+)-ATP酶;其最大刺激活力分别为85％、80％和30％,半刺激浓度分别为32、27和0.7μmol/L。当三种重金属离子的浓度增加时,则与Ca~(2+)-ATP酶的巯基结合,抑制酶的活力,Pb2~(2+)、Cd~(2+)和Hg~(2+)的半抑制浓度分别为370、440和2μmol/L。抑制作用为渐进性过程,而刺激作用为即时效应。抑制作用可为巯基化物,特别是二巯基化物所逆转。研究结果提示,CaM可能是重金属中毒最初作用的靶分子,而重金属中毒不仅使CaM“开关”失灵,还可能导致细胞内Ca~(2+)的调节全面失控。     

A derivative of bisbenzylisoquinoline alkaloid is a new and potential calmodulin antagonist

A new derivative of bisbenzylisoquinoline (berbamine type): 0-(4-ethoxylbutyl) berbamine (EBB) was found to possess powerful and specific calmodulin (CaM) inhibitory properties. It inhibited CaM-stimulated Ca2+-Mg2+-ATPase in human erythrocyte membrane with IC50 value of 0.35 microM compared to that of 60 microM of berbamine. CaM-independent basal Ca2+-Mg2+-ATPase, Na+-K+-ATPase and Mg2+-ATPase were not effect at 1.0 microM of EBB at which CaM-dependent Ca2+-Mg2+-ATPase was already potently inhibited. The inhibition of CaM-dependent Ca2+-Mg2+-ATPase was competitive with respect to CaM. Higher amount of CaM reversed the inhibition caused by higher concentration of EBB. Using dansyl-CaM (D-CaM), it was shown that EBB binds directly to CaM and caused a conformational change of CaM polypeptide chain. From fluorescence titration curve we obtained evidence that in the presence of Ca2+, CaM has two specific binding sites for EBB and additional unspecific binding sites. The Ca2+-dependent binding sites of EBB on CaM were novel region different from the binding sites for TFP.     

Effect of tricyclohexylhydroxytin on synaptosomal Ca2+-dependent ATP hydrolysis and rat brain subcellular calmodulin

Effect of tricyclohexylhydroxytin (plictran) on Ca2+-ATPase activity was studied in rat brain synaptosomes under in vitro and in vivo conditions. Plictran inhibited basal Ca2+-ATPase activity with an IC50 value of 6 nM suggesting its interaction with calcium transport phenomenon. Plictran inhibited calmodulin (CaM) activated Ca2+-ATPase in a concentration-dependent manner. A complete reversal of calmodulin activation of Ca2+-ATPase was observed with 2-3 nM plictran. A 50 per cent decrease of CaM activated Ca2+-ATPase was observed with 0.5 nM plictran, a concentration at which no significant effect was observed on basal enzyme activity. Of all the brain fractions studied, calmodulin levels in P2 fractions alone were reduced significantly to about 75 per cent of control values in plictran treated rats. The synaptosomal Ca2+-ATPase was also decreased by 35 per cent, 42 per cent and 65 per cent in 10, 20 and 40 mg plictran kg-1 day-1 treated rats for 3 days respectively. The activity levels of Ca2+-ATPase in 10 and 20 mg plictran kg-1 day-1 treated rats were restored to normal level by exogenously added calmodulin. These results suggest that plictran may disrupt synaptic function by altering calcium and calmodulin regulated processes in the central nervous system.     

The high affinity (Ca2+-Mg2+)-ATPase in liver plasma membranes is a Ca2+ pump. Reconstitution of the purified enzyme into phospholipid vesicles

The purified (Ca2+-Mg2+)-ATPase from rat liver plasma membranes (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215) was incorporated into soybean phospholipid vesicles, together with its activator. In the presence of millimolar concentrations of Mg2+, the reconstituted proteoliposomes displayed a rapid, saturable, ATP-dependent Ca2+ uptake. Half-maximal Ca2+ uptake activity was observed at 13 +/- 3 nM free Ca2+, and the apparent Km for ATP was 16 +/- 6 microM. Ca2+ accumulated into proteoliposomes (2.8 +/- 0.2 nmol of Ca2+/mg of protein/90 s) was totally released upon addition of the Ca2+ ionophore A-23187. Ca2+ uptake into vesicles reconstituted with enzyme alone was stimulated 2-2.5-fold by the (Ca2+-Mg2+)-ATPase activator, added exogenously. The (Ca2+-Mg2+)-ATPase activity of the reconstituted vesicles, measured using the same assay conditions as for ATP-dependent Ca2+ uptake activity (e.g. in the presence of millimolar concentrations of Mg2+), was maximally activated by 20 nM free Ca2+, half-maximal activation occurring at 13 nM free Ca2+. The stoichiometry of Ca2+ transport versus ATP hydrolysis approximated 0.3. These results provide a direct demonstration that the high affinity (Ca2+-Mg2+)-ATPase identified in liver plasma membranes is responsible for Ca2+ transport.     

Fe2+ and other divalent metal ions uncouple Ca2+ transport from (Ca2+-Mg2+)-ATPase in rat liver plasma membranes

The addition of nanomolar concentrations of free Fe2+, Mn2+, or Co2+ to rat liver plasma membranes resulted in an activation of ATP hydrolysis by these membranes which was not additive with the Ca2+-stimulated ATPase activity coupled to the Ca2+ pump. Detailed analysis showed that, if fact, (i) as for the stimulation of (Ca2+-Mg2+)-ATPase by Ca2+, activation of ATP hydrolysis by Fe2+, Mn3+, or Co2+ followed a cooperative mechanism involving two ions; (ii) two interacting sites for ATP were involved in the activation of both Fe2+- and Ca2+-stimulated ATPase activities; (iii) micromolar concentrations of magnesium caused the same dramatic inhibition of both activities; and (iv) the subcellular distribution of Fe2+-activated ATP hydrolysis activity corresponded to that of plasma membrane markers. This suggests that the (Ca2+-Mg2+)-ATPase might be stimulated not only by Ca2+, but also by Fe2+, Mn2+, or Co2+. However, interaction of (Ca2+-Mg2+)-ATPase with Fe2+, Mn2+, or Co2+ inhibited the Ca2+ pump activity. Furthermore, neither the formation of the phosphorylated intermediate of (Ca2+-Mg2+)-ATPase, nor ATP-dependent (59Fe) uptake could be detected in the presence of Fe2+ concentrations which stimulated ATP hydrolysis. We conclude that: (i) under the influence of certain metal ions, the Ca2+ pump in the liver plasma membrane may be switched to an uncoupled state which displays ATP hydrolysis activity, but does not insure ion transport; (ii) therefore the Ca2+ pump in liver plasma membranes specifically insures Ca2+ transport.     

Conformational changes of (Ca2+-Mg2+)-ATPase of erythrocyte plasma membrane caused by calmodulin and phosphatidylserine as revealed by circular dichroism and fluorescence studies

Two spectroscopic techniques, circular dichroism and steady-state fluorescence, were employed in order to study conformational changes of the purified, detergent-solubilized (Ca2+-Mg2+)-ATPase of porcine erythrocyte ghost membranes. Circular dichroism (CD) spectra in the peptide region were obtained from the purified (Ca2+-Mg2+)-ATPase of porcine erythrocyte ghost membranes with the aim to investigate the secondary structure of the enzyme in the presence of calmodulin (CaM) or phosphatidylserine (PS), as well as in the E1 and E2 states. The E1 conformation was stabilized by 10 microM free Ca2+, while the E2 conformation was stabilized by 0.1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). It was found that the E1 and E2 states of the enzyme strikingly differed in their secondary structure (66% and 46% of calculated alpha-helix content, respectively). In the presence of Ca2+, PS decreased the helical content of the ATPase to 61%, while CaM to 55%. Quenching of intrinsic fluorescence of (Ca2+-Mg2+)-ATPase by acrylamide, performed in the presence of Ca2+, gave evidence for a single class of tryptophan residues with Stern-Volmer constant (KSV) of 10 M-1. Accessibility of tryptophan residues varied depending on the conformational status of the enzyme. Addition of PS and CaM decreased the KSV value to 7.6 M-1 and 8.5 M-1, respectively. In the absence of Ca2+, KSV was 7.0 M-1. KI and CsCl were less effective as quenchers. The fluorescence energy transfer between (Ca2+-Mg2+)-ATPase tryptophan residues and dansyl derivative of covalently labeled CaM occurred in the presence of EGTA, but was further promoted by Ca2+. It is concluded that the interaction of CaM and PS with (Ca2+-Mg2+)-ATPase results in different conformational states of the enzyme. CD and fluorescence spectroscopy allowed to distinguish these states from the E1 and E2 conformational forms of the ATPase.     

Two Ca2+-dependent ATPases in rat liver plasma membrane. The previously purified (Ca2+-Mg2+)-ATPase is not a Ca2+-pump but an ecto-ATPase

We have shown that the rat liver plasma membrane has at least two (Ca2+-Mg2+)-ATPases. One of them has the properties of a plasma membrane Ca2+-pump (Lin, S.-H. (1985) J. Biol. Chem. 260, 7850-7856); the other one, which we have purified (Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020) and characterized (Lin, S.-H. (1985) J. Biol. Chem. 260, 10976-10980) has no established function. In this study we present evidence that the purified (Ca2+-Mg2+)-ATPase is a plasma membrane ecto-ATPase. In hepatocytes in primary culture, we can detect Ca2+-ATPase and Mg2+-ATPase activities by addition of ATP to the intact cells. The external localization of the active site of the ATPase was confirmed by the observation that the Ca2+-ATPase and Mg2+-ATPase activities were the same for intact cells, saponin-treated cells, and cell homogenates. Less than 14% of total intracellular lactate dehydrogenase, a cytosolic enzyme, was released during a 30-min incubation of the hepatocytes with 2 mM ATP. This indicates that the hepatocytes maintained cytoplasmic membrane integrity during the 30-min incubation with ATP, and the Ca2+-ATPase and Mg2+-ATPase activity measured in the intact cell preparation was due to cell surface ATPase activity. The possibility that the ecto-Ca2+-ATPase and Mg2+-ATPase may be the same protein as the previously purified (Ca2+-Mg2+)-ATPase was tested by comparing the properties of the ecto-ATPase with those of (Ca2+-Mg2+)-ATPase. Both the ecto-ATPase and the (Ca2+-Mg2+)-ATPase have broad nucleotide-hydrolyzing activity, i.e. they both hydrolyze ATP, GTP, UTP, CTP, ADP, and GDP to a similar extent. The effect of Ca2+ and Mg2+ on the ecto-ATPase activity is not additive indicating that both Ca2+- and Mg2+-ATPase activities are part of the same enzyme. The ecto-ATPase activity, like the (Ca2+-Mg2+)-ATPase, is not sensitive to oligomycin, vanadate, N-ethylmaleimide and p-chloromercuribenzoate; and both the ecto-ATPase and purified (Ca2+-Mg2+)-ATPase activities are insensitive to protease treatments. These properties indicate that the previously purified (Ca2+-Mg2+)-ATPase is an ecto-ATPase and may function in regulating the effect of ATP and ADP on hepatocyte Ca2+ mobilization (Charest, R., Blackmore, P.F., and Exton, J.H. (1985) J. Biol. Chem. 260, 15789-15794).     

Cholera toxin blocks glucagon-mediated inhibition of the liver plasma membrane (Ca2+-Mg2+)-ATPase

We have previously shown that liver plasma membrane (Ca2+-Mg2+)-ATPase activity is inhibited by glucagon. To investigate the possible involvement of a GTP-binding (G) protein in this regulation, we have examined the effects of pertussis toxin and cholera toxin on inhibition of (Ca2+-Mg2+)-ATPase by glucagon. Treatment of liver plasma membranes with pertussis toxin did not affect the sensitivity of (Ca2+-Mg2+)-ATPase to the hormone. In contrast, treatment of plasma membranes or prior injection of animals with cholera toxin prevented inhibition of the (Ca2+-Mg2+)-ATPase by glucagon. Even though adenylate cyclase activity was increased by cholera toxin treatment, addition of cyclic AMP did not mimic the effect of cholera toxin in blocking glucagon-mediated inhibition of (Ca2+-Mg2+)-ATPase activity. These data suggest that a cholera toxin-sensitive protein, perhaps Gs or a Gs-like protein, is involved in the regulation of liver (Ca2+-Mg2+)-ATPase activity. The results emphasize the possible role of Gs-like proteins in regulation of enzymes other than adenylate cyclase and suggest that the study of (Ca2+-Mg2+)-ATPase may provide a useful enzymatic system to examine such regulation.