Febs Letters Volume 65, Number 1-3./Volume 66, Number 1-2. [antikvár]

Volume 65, number 1 FEBS LETTERS May 1976 PROTEINS DEALING WITH Ai-ETHYLMALEIMIDE INHIBITION OE PIG HEART MITOCHONDRIAL PHOSPHATE TRANSPORT SYSTEM Yves BRIAND, Sylvie TOURAILLE, Roger DEBISE and Roger DURAND* Laboratoire de Biochimic, Université de Clermont-Ferrand, B.P. 45, 63170 Auhiere, France Received 12 January 1976 Revised version rece'ved 12 March 1976 I. Introduction Phosphate transport system of pig heart mitochondria was found to be inhibited by mercurial, maleimide [1] or CPDS [2] like rat liver mitochondria [3-8]. A more intensive protective effect of phosphate against maleimide inhibition was observed in pig heart [1] than in rat liver mitochondria [9]. As described by Fonyo [10] mersalyl can protect against NEM reaction in rat liver mitochondria. A similar result has been observed by Coty and Pedersen [11] with p-MB in rat liver mitochondria allowing them to detect a major protein component (mol. wt. 32 000) of the NEM-sensitive phosphate transport system. Consequently NEM-sensitive protein components can be obtained by first protecting either by phosphate or by mersalyl and then by irreversibly blocking non-protected SH groups by addition of radioactive NEM. However one may ask a question: does the protective effect of phosphate against ['H]NEM incorporation reflect the polypeptides linked to the Pj transport and especially does it act at the level of the same proteins as mersalyl. It has been previously demonstrated [12,13] that * To whom reprint requests should be addressed. Abbreviations: CPDS, 6,6'-dithionicotinic acid; NEM, N-ethylmeleimide;p-MB; parachloromercuribenzoate; Tris, Tris (hydroxymethyl) aminomethane; TCA, Trichloracetic acid; SDS, sodium dodecyl sulfate; NIG, nigericin; TEMED, A',Af,A^,A'',.tetramethylenediamine. North-Holland Publishing Company - Amsterdam protein fractions with the specific effect of phosphate on [^H] NEM incorporation were obtained from whole pig heart mitochondria after hypotonic treatment and sonication. Moreover this fraction was found to be valinomycin NEM-sensitive and grisorixin (a new ionophore of nigericin's group). We report here data regarding the comparative protective effects of phosphate, nigericm, and mersalyl against NEM inhibition of pig heart mitochondria phosphate transport system. Electrophoresis analysis of [^H] NEM-labeled proteins is given in each case. 2. Material and methods Pig heart mitochondria were prepared according to Crane et al. [14] but instead of 10 mM phosphate buffer, 10 mM Tris-HCl buffer was used. Proteins were determined by the biuret method [15]. Mitochondria swelling was followed by optical density change as described by Chappell and Crofts [16]; decreasing optical density was measured in the Eppendorf photometer at 546 nm. Mitochondrial proteins were fractionated according to Briand et al. [13] (see fig.2 for details). For [^H] NEM determination, three samples of each protein fraction were taken off and proteins precipitated by 50% trichloracetic acid in 1.5 ml Eppendorf centrifuge tube. After centrifugation the pellets were dissolved in 0.4 nil formic acid for scintillation counting. Polyacrylamide gel electrophoresis in SDS was carried out according to Weber and Osborn [17] and 1