Hagen T, Joyal J L, Henke W, Aprille J R
Research Division, Faculty of Medicine (Charité), Humboldt University, Berlin, Federal Republic of Germany.
Arch Biochem Biophys. 1993 Jun;303(2):195-207. doi: 10.1006/abbi.1993.1273.
This study investigated the hypothesis that changes in the adenine nucleotide (ATP + ADP + AMP) content of kidney mitochondria can occur by a transport mechanism that catalyzes net transfer of adenine nucleotides across the inner mitochondrial membrane. The adenine nucleotide content of isolated kidney mitochondria was 8.23 +/- 0.85 nmol/mg mitochondrial protein. This amount increased or decreased as a function of the external [ATP-Mg] when mitochondria were incubated in phosphate-containing medium. The increases and decreases were inhibited to different extents by 100 microM EGTA (ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid) or 5 microM carboxyatractyloside (CAT), suggesting two transport mechanisms. The unidirectional components (influx and efflux) of net flux were examined separately for the CAT-insensitive (EGTA-sensitive) and CAT-sensitive (EGTA-insensitive) mechanisms. CAT-insensitive adenine nucleotide influx and efflux were stimulated by [Ca2+]free up to 2 microM; for ATP influx, Km was 1.7 mM, Vmax was 3.5 nmol/min/mg protein, and Mg2+ was required. Efflux varied as a function of both the external and matrix [ATP] and was completely inhibited by mersalyl. ATP was a better substrate than ADP, and ADP transport did not require Mg2+. The CAT-sensitive mechanism was characterized by studying phosphate-induced adenine nucleotide efflux. Efflux varied with external [Pi] and with matrix [ATP] and was not inhibited by cyclosporin. The amount of CAT required for maximal inhibition was 800 pmol/mg protein. In contrast to CAT-insensitive efflux, this pathway was only partially inhibited by mersalyl and showed no preference for ATP vs ADP. In conclusion, two distinct mechanisms for net adenine nucleotide transport were demonstrated. Both exchange adenine nucleotides (ATP-Mg or ADP) for Pi. One mechanism is identical to the CAT-insensitive ATP-Mg/Pi carrier known in liver mitochondria; the other is a CAT-sensitive mechanism that is not present in liver and may represent a novel function of the ADP/ATP translocase or another CAT-sensitive carrier.
本研究探讨了一种假说，即肾脏线粒体中腺嘌呤核苷酸（ATP + ADP + AMP）含量的变化可能通过一种催化腺嘌呤核苷酸跨线粒体内膜净转运的转运机制发生。分离的肾脏线粒体中腺嘌呤核苷酸含量为8.23±0.85 nmol/mg线粒体蛋白。当线粒体在含磷酸盐的培养基中孵育时，该含量随外部[ATP-Mg]的变化而增加或减少。100 microM EGTA（乙二醇双（β-氨基乙醚）N，N'-四乙酸）或5 microM羧基苍术苷（CAT）对增加和减少有不同程度的抑制作用，提示存在两种转运机制。分别研究了CAT不敏感（EGTA敏感）和CAT敏感（EGTA不敏感）机制的净通量单向成分（流入和流出）。CAT不敏感的腺嘌呤核苷酸流入和流出在游离[Ca2+]高达2 microM时受到刺激；对于ATP流入，Km为1.7 mM，Vmax为3.5 nmol/min/mg蛋白，且需要Mg2+。流出随外部和基质[ATP]的变化而变化，并被汞撒利完全抑制。ATP是比ADP更好的底物，且ADP转运不需要Mg2+。通过研究磷酸盐诱导的腺嘌呤核苷酸流出对CAT敏感机制进行了表征。流出随外部[Pi]和基质[ATP]的变化而变化，且不受环孢素抑制。最大抑制所需的CAT量为800 pmol/mg蛋白。与CAT不敏感流出相反，该途径仅被汞撒利部分抑制，且对ATP与ADP无偏好。总之，证明了两种不同的腺嘌呤核苷酸净转运机制。两者都将腺嘌呤核苷酸（ATP-Mg或ADP）与Pi进行交换。一种机制与肝脏线粒体中已知的CAT不敏感的ATP-Mg/Pi载体相同；另一种是肝脏中不存在的CAT敏感机制，可能代表ADP/ATP转位酶或另一种CAT敏感载体的新功能。
