title: In vitro and in vivo studies of F(0)F(1)ATP synthase regulation by inhibitor protein IF(1) in goat heart.
abstract: A method has been developed to allow the level of F(0)F(1)ATP synthase capacity and the quantity of IF(1) 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 IF(1) content was determined by detergent extraction followed by blue native gel electrophoresis, two-dimensional SDS-PAGE and immunoblotting with anti-IF(1) 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 IF(1) content, demonstrating that under all conditions tested the only significant modulator of the enzyme activity was IF(1). In addition, both in vivo and in vitro, 1.3-1.4 mol of IF(1) was predicted to fully inactivate 1 mol of synthase, thus excluding the existence of significant numbers of non-inhibitory binding sites for IF(1) in the F(0) sector.
authors.affiliation: Department of Biomedical Sciences and Technologies and M.A.T.I. Center of Excellence, University of Udine, p.le Kolbe 4, 33100 Udine, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Di Pancrazio
authors.forename: Irene
authors.initials: I
authors.lastname: Mavelli
authors.forename: Miriam
authors.initials: M
authors.lastname: Isola
authors.forename: Gianni
authors.initials: G
authors.lastname: Losano
authors.forename: Pasquale
authors.initials: P
authors.lastname: Pagliaro
authors.forename: David A
authors.initials: DA
authors.lastname: Harris
authors.forename: Giovanna
authors.initials: G
authors.lastname: Lippe
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006041:Goats; D006706:Homeostasis; D066298:In Vitro Techniques; D008297:Male; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism*; D017202:Myocardial Ischemia / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D011506:Proteins / Q000494:pharmacology*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000097795:ATPase Inhibitory Protein
chemical_list: D011506:Proteins; D006180:Proton-Translocating ATPases; D000097795:ATPase Inhibitory Protein