(24) LOCALIZATION IN MITOCHONDRIA AND CALCIUM-BINDING OF A CHANNEL-FORMING HYDROPHOBIC COMPONENT

O. Gateau-Roesch, E. Pavlov, A.V. Lazareva, E.A. Limarenko, C. Levrat, N.E. Saris, P. Louisot and G.D. Mironova
Department of Biochemistry, INSERM 189, Lyon-Sud Medical School, BP12 69921 OULLINS Cedex FRANCE
Institute of Theoretical and Experimental Biophysics and Institute of Cell Biophysics, Academy of Sciences of Russia, Puschino, Russia
Department of Medical Chemistry, Institute of Biomedicine, University of Helsinki, Finland.

Recently we found that a low molecular weight, hydrophobic Ca-binding component isolated from rat liver mitoplasts by an ethanolic extraction formate a Ca2+-activated channel after reconstitution into BLM. The modified membrane showed a self-sustained oscillation at the membrane electrical current on concentration gradient of calcium accross the membrane (1). In the present work, the localization of this component was investigated after sub-fractionation of purified rat mitochondria. Hypotonic swelling of mitochondria and further separation of membranous fractions on sucrose density gradient resulted in the isolation of a contact sites enriched fraction and an inner membrane fraction which were characterized by marker enzyme activities. It was found that contact sites enriched fraction showed a high recovery of this Ca-activated channel.

The studied component has a high affinity binding site at alkaline pH but at neutral pH, affinity to calcium decreases. At pH 8.5, Kd of the component is equal to 8 10-6 M as at pH 7.4, Kd value is 9 10-5 M. The analysis of calcium binding sites amount showed that each 6-10 molecules of the component bind one calcium ion. As it is wellknown that one molecule of calcium can form 6-8 coordinative bounds and according to IR analysis data, one can suggest that the carbonyl function entering in hydrogen bounded carboxyl group participate in coordination linkage formation.

Study of the functional role of this calcium-binding component is now in progress and we suggest that this channel could take part in formation of mitochondrial pore transition system. At present time, we succed in raising an antibody against this compound, which will be a precious tool for stuying the physiological functioning of the calcium-binding in mitochondria.

(1) Mironova G, Lazareva A, Gateau-Roesch O, Tynela J, Zakirova R, Pavlov Y, Vanier MT, Saris NE (1997) J. Bioenerg. Biomem. 29:561-569.