Barbour R L, Ribaudo J, Chan S H
J Biol Chem. 1984 Jul 10;259(13):8246-51.
Mitochondrial adenine nucleotide translocation was measured in the presence of creatine or creatine phosphate to test the proposed functional couple between the nucleotide translocase and creatine kinase. Rat heart mitochondria were preloaded with the corresponding nucleotide which reacts with creatine kinase only after it is transported across the mitochondrial membrane; namely, radioactive ATP-preloaded mitochondria were assayed in the presence of 10 mM creatine plus ADP, and radioactive ADP-preloaded mitochondria in the presence of 10 mM creatine phosphate plus ATP. Results showed that forward creatine kinase reaction (in the direction of creatine phosphate synthesis) inhibits translocation of external ADP into mitochondrial matrix and backward reaction (cleavage of creatine phosphate to creatine) likewise inhibits translocation of ATP across the mitochondrial membrane. Control experiments showed that without the kinase activity when Mg2+ was omitted from the assay medium, the presence of creatine or creatine phosphate had no effect on ADP or ATP transport, respectively. Therefore, the observed inhibition of nucleotide exchange by these compounds is due to creatine kinase activity which upon reacting with the newly exported nucleotide can effectively compete for transport back into the mitochondrial matrix over nucleotides added in the assay medium. Kinetic analysis also indicated that the forward creatine kinase reaction inhibits external ADP uptake competitively. These results are interpreted to support the proposal that a functional interaction exists between the mitochondrial bound creatine kinase and the adenine nucleotide translocase.
在有肌酸或磷酸肌酸存在的情况下测定线粒体腺嘌呤核苷酸转运，以检验所提出的核苷酸转运酶与肌酸激酶之间的功能偶联。用相应的核苷酸预加载大鼠心脏线粒体，该核苷酸只有在穿过线粒体膜后才与肌酸激酶反应；即，在10 mM肌酸加ADP存在的情况下测定放射性ATP预加载的线粒体，在10 mM磷酸肌酸加ATP存在的情况下测定放射性ADP预加载的线粒体。结果表明，正向肌酸激酶反应（向磷酸肌酸合成方向）抑制外部ADP向线粒体基质的转运，反向反应（磷酸肌酸裂解为肌酸）同样抑制ATP穿过线粒体膜的转运。对照实验表明，当测定介质中省略Mg2+从而没有激酶活性时，肌酸或磷酸肌酸的存在分别对ADP或ATP转运没有影响。因此，观察到这些化合物对核苷酸交换的抑制作用是由于肌酸激酶活性，该活性在与新输出的核苷酸反应后，能够有效地与测定介质中添加的核苷酸竞争转运回线粒体基质。动力学分析还表明，正向肌酸激酶反应竞争性抑制外部ADP摄取。这些结果被解释为支持线粒体结合的肌酸激酶与腺嘌呤核苷酸转运酶之间存在功能相互作用这一观点。
