共查询到20条相似文献,搜索用时 578 毫秒
1.
大麦根细胞质膜Ca~(2+)-ATP酶和Ca~(2+)转运系统的特性 总被引:1,自引:0,他引:1
用大麦质膜微囊研究细胞质膜 Ca~(2+)转运过程,发现质膜 Ca~(2+)—ATP酶在反应系统中不存在Mg~(2+)时可正常表现活性。跨膜Ca~(2+)转运按其对Mg~(2+)的需求可分为两个过程,一个是不需Mg~(2+)的、具高Ca~(2+)亲和力和较低的转运能力;另一个则是需Mg~(2+)的、具低Ca~(2+)亲和力和较高的转运能力。前者的动力学特征与Ca~(2+)—ATP酶相近,而后者则相差很大。据此推测,大麦根细胞质膜上除Ca~(2+)—ATP酶外,还存在另一个不同的Ca~(2+)转运系统。由两者分别承担的Ca~(2+)转运过程在细胞钙信使系统中可能起着不同的作用。 相似文献
2.
本文以ts-RSV LA90细胞为模型,用放射性同位素示踪技术测定了通过细胞质膜的~(45)Ca~(2+)流水平;同时用钙指示剂Indo-1 AM和光学多道分析仪测定了胞内[Ca~(2+)]_i,初步研究了Ca~(2+)流和[Ca~(2+)]_i在v-src基因引起细胞转化过程中的动态变化。结果表明LA90细胞质膜上~(45)Ca~(2+)流的改变是细胞转化过程中可以检测到的早期事件之一,转化状态(33℃)细胞的~(45)Ca~(2+)流大于正常状态(40℃)的,细胞从正常到转化(40℃→33℃)的25分钟内~(45)Ca~(2+)流就有明显增大。TMB-8可以抑制转化引起的~(45)Ca~(2+)流出的增大,小牛血清可以刺激正常状态细胞的~(45)Ca~(2+)流出增大,~(45)Ca~(2+)流出与温度有一定依赖关系;细胞转化引起的~(45)Ca~(2+)流入增大,可被异博定抑制,~(45)Ca~(2+)流入不受温度的影响。LA90细胞[Ca~(2+)]_i在转化早期有明显升高,并维持在较正常细胞高2—3倍的水平,A23187-Br可提高正常LA90细胞[Ca~(2+)]_i,[Ca~(2+)]_i不受温度的影响。从质膜上~(45)Ca~(2+)流和[Ca~(2+)]_i的增大说明转化细胞虽然对胞外Ca~(2+)浓度依赖性下降,但维持增殖及转化状态仍然需要一定的胞外Ca~(2+),并通过提高质膜Ca~(2+)流入和释放内源性Ca~(2+),使转化细胞[Ca~(2+)]_i维持在较高水平上。LA90细膜质膜上~(45)Ca~(2+)流和[Ca~(2+)]_i的增大在细胞转化中起着重大作用。 相似文献
3.
我们曾报道跨膜Ca~(2+)梯度可通过膜脂影响肌质网Ca~(2+)-ATP 酶的构象和活性。本文就跨膜Ca~(2+)梯度对肌质网Ca~(2+)-ATP 酶的调节是否具有特异性作进一步研究。结果表明这种特异性表现在两方面:一是跨膜Ca~(2+)梯度对肌质网Ca~(2+)-ATP 酶功能的调节不能归结于跨膜Ca~(2+)浓度梯度所导致的膜电位的作用,离子载体FCCP 可消除跨膜电位但并不影响肌质网Ca~(2+)-ATP 酶的活力;二是其它二价金属离子如Sr~(2+)的跨膜梯度对肌质网Ca~(2+)-ATP 酶活力基本无影响。荧光偏振系列探剂n-AS 测定的结果表明跨膜Ca~(2+)与Sr~(2+)梯度对嵌有Ca~(2+)-ATP 酶的脂酶体的中部流动性的影响有较大差异。而Ca~(2+)-ATP 酶的Ca~(2+)结合位点正处于脂双层中部,这进一步提示膜脂参与了跨膜Ca~(2+)梯度对Ca~(2+)-ATP 酶的调节作用。 相似文献
4.
徐友涵 《中国生物化学与分子生物学报》1986,2(5):15-21
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。 相似文献
5.
从猪脑中提取钙调蛋白和突触质膜,我们研究了山莨菪碱对经有限蛋白水解和磷脂酶A_2处理后的突触膜Ca~(2+)-ATPase活性影响。发现药物对不同预处理后的Ca~(2+)-ATPase表现出不同影响并调节钙调蛋白对它的激活作用。 相似文献
6.
本文介绍用二相分配法制备蚕豆叶片原生质膜上的Ca~(2+)·Mg~(2+)-ATPase,用以研究镧系,稀土离子对此酶活性的影响。初步证实Pr~(3+)、Nd~(3+)对依赖于CaM的以及不依赖于CaM的蚕豆叶片原生质膜上Ca~(2+)·Mg~(2+)-ATPase活性的抑制不是CaM专一的。 相似文献
7.
本文应用荧光钙测定技术观察了血管紧张素Ⅱ(AⅡ)对新生Wistar鼠脑细胞胞浆Ca~(2+)浓度([Ca~(2+)]_i)的影响。结果表明:血管紧张素Ⅱ在1nmol/L—1μmol/L浓度下可诱导新生鼠脑细胞[Ca~(2+)]_i增加,具量效关系。在无外Ca~(2+)存在对,其增加幅度有所减少。上述效应可被血管紧张素Ⅱ拮抗剂Saralasin所阻断,并呈剂量依赖关系。上述结果提示,血管紧张素Ⅱ可激活血管紧张素AⅡ受体,增加脑细胞[Ca~(2+)]_i,该效应通过细胞内Ca~(2+)释放和细胞外Ca~(2+)内流两条适径实现,前者的作用是主要的。 相似文献
8.
本文测定了数种蝙蝠葛碱衍生物对钙调素(CaM)激活的人红细胞膜Ca~(2+)-Mg~(2+)-ATPase活力的影响。结果表明,这些化合物对该酶都有不同程度的抑制作用,其机制表现为竞争性抑制,过量的CaM能完全逆转这些化合物所引起的抑制。当Ca~(2+)-Mg~(2+)-ATPase被胰蛋白酶(trypsin)限制性酶解完全活化后,其活力不再受CaM激活,但仍被这些化合物所抑制。 相似文献
9.
本文以豚鼠和大白鼠心肌肌浆网膜(SR)Ca~(2+)-ATPase的活力,心肌质膜(SL)(Ca~(2+)Mg~(2+))-ATPase的活力和电子显微镜的方法探索克山病病区粮中低硒与心肌细胞钙转运调控的共系,实验结果为硒对克山病有预防作用的观点提供了新的理论依据,并进一步支持了“克山病是一种心肌线粒体病”的观点。 相似文献
10.
根据Cd~(2+)、Pb~(2+)、Hg~(2+)和Al~(3+)对丹磺酰标记钙调蛋白(D-CaM)的荧先强度、最大发射波长及偏振度的影响来研究它们对CaM及Ca~(2+)-Mg~(2+)-ATPase构象变化的影响.研究发现,无论溶液中是否存在Ca~(2+)-Mg~(2+)-ATPase,Cd~(2+)、Pb~(2+)和Hg~(2+)对D-CaM的荧光最大发射波长、偏振度的影响以Cd~(2+)的最大,Pb~(2+)次之,Hg~(2+)最小,Al~(3+)对D-CaM产生的影响与这三种二价金属离子的并不相同.这证明这几种离子与CaM和Ca~(2+)-Mg~(2+)-ATPase的作用并不遵循同样的机理. 相似文献
11.
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。 相似文献
12.
The high affinity (Ca2+-Mg2+)-ATPase in liver plasma membranes is a Ca2+ pump. Reconstitution of the purified enzyme into phospholipid vesicles 总被引:2,自引:0,他引:2
C Pavoine S Lotersztajn A Mallat F Pecker 《The Journal of biological chemistry》1987,262(11):5113-5117
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. 相似文献
13.
A high-affinity Mg2+-independent Ca2+-ATPase (Ca2+-ATPase) has been differentiated from the Mg2+-dependent, Ca2+-stimulated ATPase (Ca2+,Mg2+-ATPase) in rat brain synaptosomal membranes. Using ATP as a substrate, the K0.5 of Ca2+ for Ca2+-ATPase was found to be 1.33 microM with a Km for ATP of 19 microM and a Vmax of 33 nmol/mg/min. Using Ca-ATP as a substrate, the Km for Ca-ATP was found to be 0.22 microM. Unlike Ca2+,Mg2+-ATPase, Ca2+-ATPase was not inhibited by N-ethylmaleimide, trifluoperazine, lanthanum, zinc, or vanadate. La3+ and Zn2+, in contrast, stimulated the enzyme activity. Unlike Ca2+, Mg2+-ATPase activity, ATP-dependent Ca2+ uptake was negligible in the absence of added Mg2+, indicating that the Ca2+ transport into synaptosomal endoplasmic reticulum may not be a function of the Ca2+-ATPase described. Ca2+-ATPase activity was not stimulated by the monovalent cations Na+ or K+. Ca2+, Mg2+-ATPase demonstrated a substrate preference for ATP and ADP, but not GTP, whereas Ca2+-ATPase hydrolyzed ATP and GTP, and to a lesser extent ADP. The results presented here suggest the high-affinity Mg2+-independent Ca2+-ATPase may be a separate form from Ca2+,Mg2+-ATPase. The capacity of Mg2+-independent Ca2+-ATPase to hydrolyze GTP suggests this protein may be involved in GTP-dependent activities within the cell. 相似文献
14.
The effect of regucalcin, a calcium-binding protein isolated from rat liver cytoplasm, on ATP-dependent calcium transport in the plasma membrane vesicles of rat liver was investigated. (Ca2+-Mg2+)-ATPase activity in the liver plasma membranes was significantly increased by the presence of regucalcin (0.1-0.5 \sgmaelig;M) in the enzyme reaction mixture. This increase was completely inhibited by the presence of sulfhydryl group modifying reagent Nethylmaleimide (5.0 mM NEM) or digitonin (0.04%), which can solubilize the membranous lipids. When ATP-dependent calcium uptake by liver plasma membrane vesicles was measured by using 45CaCl2, the presence of regucalcin (0.1-0.5 \sgmaelig;M) in the reaction mixture caused a significant increase in the 45Ca2+ uptake. This increase was about 2-fold with 0.5 \sgmaelig;M regucalcin addition. An appreciable increase was seen by 5 min incubation with regucalcin addition. The regucalcin-enhanced ATP-dependent 45Ca2+ uptake by the plasma membrane vesicles was completely inhibited by the presence of NEM (5.0 mM) or digitonin (0.04%). These results demonstrate that regucalcin activates (Ca2+-Mg2+)-ATPase in the liver plasma membranes and that it can stimulate ATP-dependent calcium transport across the plasma membranes. 相似文献
15.
Protein kinase-dependent phosphorylation of cardiac sarcolemmal Ca2(+)-ATPase, as studied with a specific monoclonal antibody 总被引:1,自引:0,他引:1
T Ogurusu S Wakabayashi K Furukawa Y Tawada-Iwata T Imagawa M Shigekawa 《Journal of biochemistry》1990,108(2):222-229
Phosphorylation of the Ca2(+)-pump ATPase of cardiac sarcolemmal vesicles by exogenously added protein kinases was examined to elucidate the molecular basis for its regulation. The Ca2(+)-pump ATPase was isolated from protein kinase-treated sarcolemmal vesicles using a monoclonal antibody raised against the erythrocyte Ca2(+)-ATPase. Protein kinase C (C-kinase) was found to phosphorylate the Ca2(+)-ATPase. The stoichiometry of this phosphorylation was about 1 mol per mol of the ATPase molecule. The C-kinase activation resulted in up to twofold acceleration of Ca2+ uptake by sarcolemmal vesicles due to its effect on the affinity of the Ca2+ pump for Ca2+ in both the presence and absence of calmodulin. Both the phosphorylation and stimulation of ATPase activity by C kinase were also observed with a highly-purified Ca2(+)-ATPase preparation isolated from cardiac sarcolemma with calmodulin-Sepharose and a high salt-washing procedure. Thus, C-kinase appears to stimulate the activity of the sarcolemmal Ca2(+)-pump through its direct phosphorylation. In contrast to these results, neither cAMP-dependent protein kinase, cGMP-dependent protein kinase nor Ca2+/calmodulin-dependent protein kinase II phosphorylated the Ca2(+)-ATPase in the sarcolemmal membrane or the purified enzyme preparation, and also they exerted virtually no effect on Ca2+ uptake by sarcolemmal vesicles. 相似文献
16.
邓瑞春 《氨基酸和生物资源》1998,20(3):5-8
蟾蜍血清梭曼水解酶已被纯化。该酶有两个温度作用高峰,最适为pH10,其次为pH7。最适温度为30℃。以梭曼为底物,在pH7.73条件下测定其米氏常数为5.864mmol/L,最大反应速度为692μmol/L。过高的底物浓度对酶活性有抑制作用。Mg++、Ca++对酶有激活作用,而Ni++、Cu++、Fe++、Na+、K+对酶活性无明显影响。 相似文献
17.
Regulation of erythrocyte Ca2+ pump activity by protein kinase C 总被引:8,自引:0,他引:8
Using either inside-out vesicles (IOV) prepared from human erythrocytes or purified Ca2+-ATPase from the same source, the effects of protein kinase C (Ca2+/phospholipid-dependent enzyme) on Ca2+ transport and Ca2+-ATPase activity were measured. Incubation of IOV with protein kinase C in the presence, but not absence, of either 12-O-tetradecanoylphorbol-13-acetate or diolein led to a Ca2+-dependent stimulation of ATP-dependent calcium uptake. The effect was a 5-7-fold increase of Vmax without a significant change in the apparent Km for Ca2+. By comparison, the effect of calmodulin was a 14-fold stimulation of Vmax and a 4-fold reduction in apparent Km. The effect of protein kinase C and calmodulin on Ca2+ uptake were nearly additive. Stimulation of IOV Ca2+ transport by protein kinase C was entirely reversible by treatment of activated IOV with alkaline phosphatase. Incubation of purified Ca2+-ATPase with protein kinase C in the presence of 12-O-tetradecanoylphorbol-13-acetate or diolein led to a stimulation of Ca2+-dependent ATPase activity. These results indicate that protein kinase C stimulates the activity of the plasma membrane Ca2+ pump by a direct effect on the pump protein. 相似文献
18.
Using plasma membrane-enriched vesicles, the properties of the H+-ATPase (EC 3.6.1.35) from the rust fungus Uromyces viciae-fabae were studied. The enzyme is strictly Mg2+-dependent and is inhibited by vanadate. The pH-optimum is at 6.7. By Western blot analysis using a monoclonal antibody against corn plasma membrane H+-ATPase a polypeptide of approximately 104 kDa could be detected. The vanadate-sensitive H+-ATPase activity of microsomal vesicles obtained from different stages of rust development was determined. Uredospores had only a very low enzyme activity (1.9 μmol Pi x mg-1 protein x h-1). In germ tubes the ATPase activity was about twofold higher (4.0 μmol Pi x mg-1 protein x h-1). An eightfold higher ATPase activity (16.1 μmol Pi x mg-1 protein x h-1) was found in microsomal vesicles from haustoria which had been isolated from rust-infected Vicia faba leaves. These results suggest, that the electrochemical gradient generated by the H+-ATPase of haustoria plays an important role for their function, possibly by promoting nutrient uptake from host cells. 相似文献
19.
肾上腺髓质素对大鼠损伤性心肌肌浆网功能的改善 总被引:3,自引:0,他引:3
通过观察下述五个指标,评价肾上腺髓质素(adrenomedullin,Adm)对大鼠损伤性心肌肌浆网功能的改善程度左心室压力最大变化速率(±dp/dtmax)、肌浆网钙摄取和释放及钙泵活性.皮下注射异丙肾上腺素(isoproterenol,ISO,69μmol/kg体重)制备大鼠心肌损伤坏死模型.摘取心脏后用Adm灌流,观察左心室压力最大变化速率(±dp/dtmax);制备并提纯心肌肌浆网(sarcoplasmicreticulum,SR)膜,测定SRCa2+摄取和释放速率、SR钙泵活性和钙通道蛋白~3H-ryanodine受体的最大结合量.结果发现,5×10-5mol/LAdm灌流能使ISO损伤的大鼠心脏左室±dp/dtmax分别增加16.9%(2?135±281vs1?980±302)和29.2%(1?375±267vs1?064±355,均P<0.05);SRCa2+摄取和释放率分别增加23.0%(15.0±1.4vs12.2±1.2)和43.5%(6.6±1.0vs4.6±0.6,均P<0.01);SRCa2+-ATPase活性和~3H-ryanodine受体最大结合量(Bmax)分别增加24.2%(P<0.01)和42.2%(P<0.05).提示Adm对ISO诱导的大鼠心肌损伤具有保护作用,其机制可能与Adm增加SRCa2+-ATPase活性、增加~3H-ryanodine所致SRCa2+摄取和释放升高有关.外源性给予Adm对损伤心肌可能具有临床治疗作用. 相似文献
20.
An antibody prepared against highly purified rabbit muscle Ca2+-ATPase from sarcoplasmic reticulum has been observed to cross-react with proteins in human platelet membrane vesicles. The antibody specifically precipitated Ca2+-ATPase activity from solubilized human platelet membranes and recognized two platelet polypeptides denatured in sodium dodecyl sulfate with Mr = 107,000 and 101,000. Ca2+-ATPase activity from Brij 78-solubilized platelet membranes was purified up to 10-fold. The purified preparation consisted mainly of two polypeptides with Mr approximately 100,000, and 40,000. The lower molecular weight protein appeared unrelated to Ca2+-ATPase activity. The Ca2+-ATPase in human platelet membrane vesicles exhibited "negative cooperativity" with respect to the kinetics of ATP hydrolysis. The apparent Km for Ca2+ activation of ATPase activity was 0.1 microM. Ca2+-dependent phosphorylation of platelet vesicles by [gamma-32P]ATP at 0 degrees C yielded a maximum of 0.2-0.4 nmol of PO4/mg of protein that was labile at pH 7.0 and 20 degrees C. This result suggests that only about 2-4% of the total protein in platelet membrane vesicles is the Ca2+-ATPase, which agrees with an estimate based on the specific activity of the Ca2+-ATPase in platelet membranes (20-50 nmol of ATP hydrolyzed/min/mg of protein at 30 degrees C). Calmodulin resulted in only a 1.6-fold stimulation of Ca2+-ATPase activity even after extensive washing of membranes with a calcium chelator or chlorpromazine. It is concluded that human platelets contain a Ca2+-ATPase immunochemically related to the Ca2+ pump from rabbit sarcoplasmic reticulum and that the enzymatic characteristics and molecular weight of the platelet ATPase are quite similar to those of the muscle ATPase. 相似文献