ADP- and oligomycin-sensitive redox behavior of
F_0 b thiol in ATPsynthase depends on neighbored primary
structure: Investigations using 14-C-labeled alpha lipoic acid
Issue title: Proceedings of the Second International
Symposium on Antioxidants in Nutrition and Therapy, Bali, Indonesia, 2–4
October 2002
Affiliations: Allgemein- und Abdominalchirurgie,
Universitätsklinik Mainz, Germany | Biochemie I, Molekulare Bioenergetik, Universität
Frankfurt a.M., Germany | Abteilung Biomathematik, Universität Frankfurt a.M.,
Germany | Medizinische Klinik II, Universität Frankfurt a.M.,
Germany
Note: [] Address for correspondence: Dr Guido Zimmer, Medizinische Klinik
II, Haus 11, Universität, Theodor-Stern.Kai 7, 60590 Frankfurt am Main,
Germany. Tel.: +49 6109 61572; Fax: +49 69 6301 6467; E-mail:
zimmer@zbc.klinik.uni-frankfurt.de
Abstract: Purified ATPsynthase of bovine heart mitochondria has been analyzed
for its mobility and reactivity of oligomycin-sensitive sulfhydryl regions in
presence of the substrate ADP and oligomycin. Labeling of thiol groups at the
hydrophobic F_0 region of the ATPsynthase was increased in
the enzyme initially treated with SDS, N-ethylmaleimide and dithiothreitol
(modified enzyme). After dialysis or gel permeation the ATPsynthase was treated
with [^{14}C] alpha lipoic acid at a molar ratio of 35–85/1
(lipoic acid/ATPsynthase) corresponding to 4–8.6 nmol/mg protein. Under these
conditions, ATPase activity of the native enzyme was significantly decreased.
After preincubation with ADP, PAGE of the native, [^{14}C]
labeled enzyme revealed an increase of radioactivity at a region of 25 kDa
deduced to Cys 197 of subunit b. In the modified enzyme the increase in
radioactivity was found at 10 kDa. In this context, the sequence Lys-Cys-Ile
around Cys 197 of subunit b suggests excessive reactivity of this thiol, as
well as ready reversibility by –SH-S-S- interchange. Therefore, previously
observed reaction by thiol reagents and antioxidants from outside the
mitochondrion can be interpreted with Cys 197 of F_0 b. It
accounts for sulfhydryl unmasked by binding of ADP at F1.
Keywords: oligomycin-sensitive thiols, F[TeX:] _0F[TeX:] _1 ATPsynthase, bovine, Labeling by [TeX:] ^{14}C-alpha lipoic acid, ATPase activity, PAGE, thiol-containing subunits, primary protein structure and thiol reactivity
Journal: BioFactors, vol. 19, no. 1-2, pp. 19-32, 2003
