RESUMO
The response of mitochondrial oxygen consumption to ADP in saponin-skinned cardiac fibre bundles has an apparent Km an order of magnitude higher than that in isolated mitochondria. Here we report that incubating skinned cardiac fibre bundles from wild-type mice or double-knockout mice lacking both cytosolic and mitochondrial creatine kinase (CK) with CK and creatine or with yeast hexokinase and glucose as extramitochondrial ADP-producing systems decreases the apparent Km of the bundles for ADP severalfold. We conclude that the affinity of mitochondria for ADP in mouse heart is of the same order of magnitude as that of isolated mitochondria, while the high apparent Km of the bundles is caused by diffusion gradients outside the mitochondria.
Assuntos
Difosfato de Adenosina/fisiologia , Membranas Intracelulares/fisiologia , Mitocôndrias Cardíacas/metabolismo , Miocárdio/metabolismo , Animais , Creatina Quinase/deficiência , Creatina Quinase/genética , Creatina Quinase Mitocondrial , Difusão , Relação Dose-Resposta a Droga , Membranas Intracelulares/enzimologia , Membranas Intracelulares/metabolismo , Isoenzimas/deficiência , Isoenzimas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/genéticaRESUMO
Mitochondria in saponin-skinned cardiac fiber bundles were reported to have an order of magnitude lower apparent affinity to ADP than isolated mitochondria. Although ADP was measured outside the bundles, it was thought that the low affinity was not caused by diffusion gradients because of relatively short diffusion distances. Here we test the hypothesis that considerable ADP diffusion gradients exist and can be diminished by increasing the intrafiber ADP production rate. We increased the ADP-producing activity in rat heart skinned fiber bundles by incubating with 100 IU/ml yeast hexokinase and glucose. Consequently, we observed a significant decrease of the apparent Michaelis constant (K(m)) to ADP of the respiration rate of bundles from 216 +/- 59 to 50 +/- 9 microM. Fitting the results with a mathematical model, we estimated the K(m) of mitochondria in the bundles to be 25 microM. We conclude that the affinity to ADP of in situ mitochondria in heart is of the same order of magnitude as that of isolated mitochondria.