Reconstituted mitochondrial transhydrogenase is a transmembrane protein.

Bovine heart mitochondrial transhydrogenase, a redox-linked proton pump, can be functionally and asymmetrically inserted into liposomes by a cholate-dialysis procedure such that the active site faces the external medium. N-(4-Azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine), a membrane-impermeant photoprobe, when encapsulated in the vesicles, covalently modified the enzyme and inhibited transhydrogenation between NADPH and the 3-acetylpyridine analog of NAD+ (AcPyAD+) in a light-dependent manner. External AcPyAD+ increased the rate of inactivation several fold, whereas NADPH, NADP+, and NADH were without effect. Labeling of the enzyme by intravesicular [35S]NAP-taurine was enhanced by AcPyAD+ and NADP+, decreased by NADH, and not significantly affected by NADPH. These results indicate that transhydrogenase spans the membrane and that substrate binding alters the conformation of that domain of the enzyme protruding from the inner surface of the membrane.

An improved method for the purification of bovine heart mitochondrial transhydrogenase.

An improved procedure for the purification of bovine heart mitochondrial pyridine dinucleotide transhydrogenase is described. The enzyme is purified over 100-fold from submitochondrial particles with a 47.4% yield and a specific activity of 62.3 mumol/min/mg. Heart submitochondrial particle membranes were washed with 2 M NaCl to remove peripheral proteins. This was followed by a 1.5% Triton X-100 extraction of the membranes. The extract was then applied to an affinity column of NAD immobilized to agarose and the enzyme eluted with NADH. This step yielded homogeneous enzyme when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The preparation, when reconstituted into phosphatidylcholine liposomes, coupled proton uptake by the vesicles to the reduction of 3-acetylpyridine adenine dinucleotide by NADPH. The polarity characteristics of transhydrogenase were evaluated by the phase separation technique of Bordier (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607); 24% of the enzyme partitioned into the aqueous phase and 76% partitioned into the detergent phase. The amino acid composition was determined and polarity index was calculated to be 40%. These data indicate that the enzyme has hydrophilic as well as hydrophobic characteristics.

Dicyclohexylcarbodiimide modification of bovine heart mitochondrial transhydrogenase.

Dicyclohexylcarbodiimide (DCCD) inhibits the reduction of oxidized 3-acetylpyridine adenine dinucleotide (AcPyAD+) by NADPH catalyzed by purified and bovine heart submitochondrial particle transhydrogenase. Kinetic studies demonstrate that the modification of 1 residue results in complete inactivation. Both transhydrogenase preparations were labeled with [14C]DCCD. Labeling of the purified enzyme was time-dependent and paralleled the extent of inhibition. The incorporation of approximately 1 mol of [14C]DCCD/monomer resulted in complete inactivation of the enzyme. At longer preincubation times or at higher DCCD concentrations, more than 1 mol of DCCD reacted and cross-linked dimers of transhydrogenase were formed. The effect of substrates on DCCD inactivation was investigated. AcPyAD+ provided no protection, and NADH gave partial protection in submitochondrial particles, but not of the purified enzyme. NADPH and NADP+ stimulated inhibition. These results indicate that DCCD modification occurs outside the active site. In experiments with transhydrogenase reconstituted into K+-loaded phosphatidylcholine liposomes, DCCD inhibited the rate of H+ uptake into the vesicles to a significantly greater extent than transhydrogenation. The incorporation of approximately 1 mol of DCCD/mol of transhydrogenase monomer completely inhibited H+ translocation, whereas complete inactivation of hydride ion transfer accompanied the incorporation of approximately 2 mol of DCCD. These results indicate that DCCD modified transhydrogenase outside the active site, possibly in a putative H+-binding domain that functions to translocate protons across the membrane by a pump rather than by a loop mechanism.