PURIFICATION AND CHARACTERIZATION OF EQUINE TESTICULAR CYTOCHROME-P-450 AROMATASE - COMPARISON WITH THE HUMAN ENZYME

Cytochrome P-450 aromatase was purified by five chromatographic steps from adult stallion testis. It was first separated from NADPH-cytochro me P-450 reductase (reductase) on omega-aminohexyl-Sepharose 4B then p urified to homogeneity on concanavalin A Sepharose 4B, hydroxyapatite- Sepharose 4B, DEAE-Sepharose CL-6B and on a second hydroxyapatite Seph arose 4B. On the other hand, purifications of the equine testicular an d rat liver reductases, which allowed the reconstitution of aromatase activity in vitro, were achieved for each species in one chromatograph ic seep on an adenosine 2',5'-diphosphate-agarose affinity column. Ana lysis on SDS/PAGE indicated single bands with apparent molecular masse s of 53, 82, and 80 kDa for purified equine testicular cytochrome P-45 0 aromatase (eAROM), equine testicular reductase and rat liver reducta se respec tively. eAROM shows a time-and concentration-dependent activ ity that was stable for at least 2 months when stored at -78 degrees C . It is a highly hydrophobic protein composed from 505 residues and di rect sequencing of its N-terminal pare showed good homology when compa red with human aromatase. When deglycosylated by N-glycosidase-F the a pparent molecular mass of eAROM was decreased from 53 to 51 kDa as rev ealed by electrophoresis, its activity, however, was not impaired. eAR OM exhibits much higher affinity for androgens than for 19-norandrogen s, Km values were approximately 3, 16 and 170 nM for androstenedione ( A), testosterone (T) and 19-nortestosterone (19-NT) respectively. Howe ver, it aromatizes 19-norandrostenedione (19-NA) slightly more efficie ntly than A, the estrone (E-1) formed was 4.27 vs 3.54 pmol min(-1) mu g(-1) respectively (P < 0.01). Airer incubation of eAROM with radiola belled A and separation of steroids on HPLC, F-1, 19-hydroxyandrostene dione (19-OHA) and 19-oxoandrostenedione (19-oxoA) were accumulated in the incubation medium in a time-dependent manner. The presence of 4-h ydroxyandrostenedione (4-OHA), a suicide inhibitor of aromatase, cause a time-dependent inactivation of the enzyme. Whereas the activity of eAROM was unchanged in the presence of K+ (up to 250 mM), it was incre ased in the presence of EDTA (up to 50 mM) and decreased in the presen ce of DTT or Mg2+ (from 25 mM). We conclude that: (a) eAROM is a glyco protein, however, deglycosylation by N-glycosidase-F does not appear t o impair its activity, (b) eAROM aromatizes really both androgens and 19-norandrogens having a higher affinity for androgens, (c) the interm ediary compounds of aromatization 19-OHA and 19-oxoA appear to be synt hesized by the same active site that synthesizes E-1 as the final prod uct, (d) the inhibition of eAROM by increasing concentrations of Mg2and the stimulation of its activity by EDTA, taken together, indicate the importance of negatively charged residues in the polypeptide chain of equine aromatase, which play a role in enzymatic activity. (C) 199 7 Elsevier Science Inc.