Enzymatic characteristics and subcellular distribution of a short-chain dehydrogenase/reductase family protein, P26h, in hamster testis and epididymis

A hamster sperm 26 kDa protein (P26h) is strikingly homologous with mouse l ung carbonyl reductase (MLCR) and is highly expressed in the testis, but it s physiological functions in the testis are unknown. We show that recombina nt P26h resembles NADP(H)-dependent MLCR in the tetrameric structure, broad substrate specificity, inhibitor sensitivity, and activation by arachidoni c acid, but differs in a preference for NAD(H) and high efficiency for the oxidoreduction between 5 alpha -androstane-3 alpha, 17 beta -diol (k(cat)/K -M = 243 s(-1) mM(-1)) and 5 alpha -dihydrotestosterone (k(cat)/K-M = 377 s (-1) mM(-1)). The replacement of Ser38-Leu39-Ile40 in P26h with the corresp onding sequence (Thr38-Arg39-Thr40) of MLCR led to a switch in favor of NAD P(H) specificity, suggesting the key role of the residues in the coenzyme s pecificity. While the P26h mRNA was detected only in the testis of the matu re hamster tissues, its enzyme activity was found mainly in the mitochondri al fraction of the testis and in the nuclear fraction of the epididymis on subcellular fractionation, in which a mitochondrial enzyme, isocitrate dehy drogenase, exhibited a similar distribution pattern. The enzyme activity of P26h in the two tissue subcellular fractions was effectively solubilized b y mixing with 1% Triton X-100 and 0.2 M KCl, and enhanced more than 10-fold . The enzymes purified from the two tissue fractions exhibited almost the s ame structural and catalytic properties as those of the recombinant P26h. T hese results suggest that P26h mainly exists as a tetrameric dehydrogenase in mitochondria of testicular cells and plays a role in controlling the int racellular concentration of a potent androgen, 5 alpha -dihydrotestosterone , during spermatogenesis, in which it may be incorporated in mitochondrial sheaths of spermatozoa.