In vitro and in vivo studies of F0F1ATP synthase regulation by inhibitor protein IF1 in goat heart

A method has been developed to allow the level of F(0)F(1)ATP synthase capacity and the quantity of IF1 bound to this enzyme be measured in single biopsy samples of goat heart. ATP synthase capacity was determined from the maximal mitochondrial ATP hydrolysis rate and IF1 content was determined by detergent extraction followed by blue native gel electrophoresis, two-dimensional SDS-PAGE and immunoblotting with anti-IF1 antibodies. Anaesthetized open-chest goats were subjected to ischemic preconditioning and/or sudden increases of coronary blood flow (CBF) (reactive hyperemia). When hyperemia was induced before ischemic preconditioning, a steep increase in synthase capacity, followed by a deep decrease, was observed. In contrast, hyperemia did not affect synthase capacity when applied after ischemic preconditioning. Similar effects could be produced in vitro by treatment of heart biopsy samples with anoxia (down-regulation of the ATP synthase) or high-salt or high-pH buffers (up-regulation). We show that both in vitro and in vivo the same close inverse correlation exists between enzyme activity and IF1 content, demonstrating that under all conditions tested the only significant modulator of the enzyme activity was IF1. In addition, both in vivo and in vitro, 1.3 - 1.4 mol of IF1 was predicted to fully inactivate 1 mol of synthase, thus excluding the existence of significant numbers of non-inhibitory binding sites for IF1 in the F-0 sector. (C) 2004 Elsevier B.V. All rights reserved.