Mitochondria isolated from potato (Solanum tuberosum L.) tuber were investigated for the presence of a nicotinamide nucleotide transhydrogenase activity. Submitochondrial particles derived from these mitochondria by sonication catalyzed a reduction of NAD' or 3-acetylpyridine-NAD' by NADPH, which showed a maximum of about 50 to 150 nanomoles/ minute. milligram protein at pH 5 to 6. The Km values for 3-acetylpyridine-NAD' and NADPH were about 24 and 55 micromolar, respectively. Intact mitochondria showed a negligible activity in the absence of detergents. However, in the presence of detergents the specific activity approached about 30% of that seen with submitochondrial particles. The potato mitochondria transhydrogenase activity was sensitive to trypsin and phenylarsine oxide, both agents that are known to inhibit the mammalian transhydrogenase. Antibodies raised against rat liver transhydrogenase crossreacted with two peptides in potato tuber mitochondrial membranes with a molecular mass of 100 to 115 kilodaltons. The observed transhydrogenase activities may be due to an unspecific activity of dehydrogenases and/or to a genuine transhydrogenase. The activity contributions by NADH dehydrogenases and transhydrogenase to the total transhydrogenase activity were investigated by determining their relative sensitivities to trypsin. It is concluded that, at high or neutral pH, the observed transhydrogenase activity in potato tuber submitochondrial particles is due to the presence of a genuine and specific high molecular weight transhydrogenase. At low pH an unspecific reaction of an NADH dehydrogenase with NADPH contributes to the total transhydrogenase activity.active form of the mammalian transhydrogenase is a dimer. The enzyme from beef heart has been extensively investigated with respect to structure and function, especially its mechanism of proton pumping and interaction with other proton pumps in the native membrane as well as in reconstituted liposomes (for reviews, see Refs. 4,21,22,and 24). Transhydrogenase isolated from Escherichia coli has been sequenced and shown to be composed of two subunits of about 50,000 with altogether 11 possible membrane spanning sequences, but otherwise has properties that resemble those of the mammalian enzyme (2, 3).The presence of a low transhydrogenase activity in plants, approximately 10 nmol/min -mg, has been reported previously in mitochondria from pea stems (8, 20) and mung bean (26). In the latter case, an ATP-driven reduction of NADP+ by NADH was also demonstrated (26). However, these activities have been questioned by others, who have explained them as being due to an unspecific NAD(P)H dehydrogenase or by two separate enzyme systems (14, 16). The main reason for the latter conclusion was that no significant NAD+-dependent formation of NADH from NADPH could be demonstrated (14). One possible complication in the measurements may have been that the assay used was based on the natural substrates, which are rapidly oxidized by various dehydrogenases.In the present s...
Mammalian nicotinamide nucleotide transhydrogenase is translated as a 5000 daltons larger molecular weight precursor in a cell-free system programmed with rat liver polysomes. The mature rat liver enzyme had the same molecular weight as the purified beef heart enzyme, 115 000 daltons. The precursor was not processed in vitro by liver mitochondria or by a rat liver mitochondrial matrix fraction, nor did it appear to bind to mitochondria. In contrast, pre-FeS protein of the cytochrome bc1 complex was processed in the same samples by both mitochondria and matrix, suggesting an important difference in the processing mechanisms or in the efficiency of processing of the two precursors.
It has previously been shown that cobalt accumulates in the myocardium of rats, mainly the sarcoplasmic reticulum (SR) and the mitochondrial inner membrane. In order to investigate the mode of accumulation of cobalt in the SR, rats were given a dietary cobalt supplementation of 40 mg of CoSO4 x 7H2O kg-1 body wt, after which the rats were sacrificed and the sarcoplasmic reticulum was isolated. The SR proteins were subjected to analysis by polyacrylamide gel electrophoresis followed by protein staining and determination of the content of cobalt in each protein band. The major cobalt-binding protein was found to have a molecular weight of about 100,000; a 200,000 molecular weight protein was also found to bind cobalt, although less extensively. These results suggest that cobalt is bound to the monomeric and dimeric forms of Ca(2+)-ATPase in the SR of the myocardium.
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