来自大鼠脾脏线粒体外膜部分的镁离子和钙离子刺激的三磷酸腺苷酶的动力学特性

Kinetic properties of a magnesium ion- and calcium ion-stimulated adenosine triphosphatase from the outer-membrane fraction of rat spleen mitochondria.

作者信息

Vijayakumar E K, Weidemann M J

出版信息

Biochem J. 1977 Aug 1;165(2):355-65. doi: 10.1042/bj1650355.

DOI:10.1042/bj1650355

PMID:21656

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1164908/

Abstract

Isolated outer membranes from rat spleen mitochondria can be stored in liquid N(2) for several weeks without significant loss of ATPase (adenosine triphosphatase) activity. 2. The ATPase reaction has a broad pH optimum centering on neutral pH, with little significant activity above pH9.0 or below pH5.5. 3. A sigmoidal response of the ATPase activity to temperature is observed between 0 and 55 degrees C, with complete inactivation at 60 degrees C. The Arrhenius plot shows that the activation energy above the transition temperature (22 degrees C) (E(a)=144kJ/mol) is one-third of that calculated for below the transition temperature (E'(a)=408kJ/mol). 4. The outer-membrane ATPase (K(m) for MgATP=50mum) is inactive unless Mg(2+) is added, whereas the inner-membrane ATPase (K(m) for ATP=11mum) is active without added Mg(2+) unless the mitochondria have been depleted of all endogenous Mg(2+) (by using ionophore A23187). 5. The substrate for the outer-membrane ATPase is a bivalent metal ion-nucleoside triphosphate complex in which Mg(2+) (K(m)=50mum) can be replaced effectively by Ca(2+) (K(m)=6.7mum) or Mn(2+), and ATP by ITP. Cu(2+), Co(2+), Sr(2+), Ba(2+), Ni(2+), Cd(2+) and Zn(2+) support very little ATP hydrolysis. 6. Univalent metal ions (Na(+), K(+), Rb(+), Cs(+) and NH(4) (+), but not Li(+)) stimulate the MgATPase activity (<10%) at low concentrations (50mm), but, except for K(+), are slightly inhibitory (20-30%) at higher concentrations (500mm). 7. The Mg(2+)-stimulated ATPase activity is significantly inhibited by Cu(2+) (K(i)=90mum), Ni(2+) (K(i)=510mum), Zn(2+) (K(i)=680mum) and Co(2+) (K(i)=1020mum), but not by Mg(2+), Ca(2+), Ba(2+) or Sr(2+). 8. The outer-membrane ATPase is insensitive to the inhibitors oligomycin, NN'-dicyclohexylcarbodiimide, NaN(3), ouabain and thiol-specific reagents. A significant inhibition is observed at high concentrations of AgNO(3) (0.5mm) and NaF (10mm). 9. The activity towards MgATP is competitively inhibited by the product MgADP (K(i)=0.7mm) but not by the second product P(i) or by 5'-AMP.

摘要

从大鼠脾脏线粒体分离得到的外膜可在液氮中保存数周，而三磷酸腺苷酶（ATPase）活性不会有显著损失。2. ATPase反应在中性pH附近有较宽的pH最适范围，在pH9.0以上或pH5.5以下几乎没有显著活性。3. 在0至55摄氏度之间观察到ATPase活性对温度呈S形响应，60摄氏度时完全失活。阿累尼乌斯曲线表明，转变温度（22摄氏度）以上的活化能（E(a)=144kJ/mol）是转变温度以下计算值（E'(a)=408kJ/mol）的三分之一。4. 外膜ATPase（MgATP的K(m)=50μm）无活性，除非添加Mg(2+)，而内膜ATPase（ATP的K(m)=11μm）在不添加Mg(2+)时仍有活性，除非线粒体已耗尽所有内源性Mg(2+)（通过使用离子载体A23187）。5. 外膜ATPase的底物是二价金属离子 - 核苷三磷酸复合物，其中Mg(2+)（K(m)=50μm）可被Ca(+)（K(m)=6.7μm）或Mn(2+)有效替代，ATP可被ITP替代。Cu(2+)、Co(2+)、Sr(2+)、Ba(2+)、Ni(2+)、Cd(2+)和Zn(2+)几乎不支持ATP水解。6. 单价金属离子（Na(+)、K(+)、Rb(+)、Cs(+)和NH(4)(+)，但不包括Li(+)）在低浓度（50mm）时刺激MgATPase活性（<10%），但除K(+)外，在高浓度（500mm）时略有抑制（20 - 30%）。7. Mg(2+)刺激的ATPase活性受到Cu(2+)（K(i)=90μm）、Ni(2+)（K(i)=510μm）、Zn(2+)（K(i)=680μm）和Co(2+)（K(i)=1020μm）的显著抑制，但不受Mg(2+)、Ca(2+)、Ba(2+)或Sr(2+)的抑制。8. 外膜ATPase对抑制剂寡霉素、N,N'-二环己基碳二亚胺、NaN(3)、哇巴因和硫醇特异性试剂不敏感。在高浓度的AgNO(3)（0.5mm）和NaF（10mm）时观察到显著抑制。9. 对MgATP的活性受到产物MgADP（K(i)=0.7mm）的竞争性抑制，但不受第二种产物P(i)或5'-AMP的抑制。