The x-ray structure of a short-chain dehydrogenase, the bacterial holo 3a,20/3-hydroxysteroid dehydrogenase (EC 1.1.1.53), is described at 2.6 A resolution. This enzyme is active as a tetramer and crystallizes with four identical subunits in the asymmetric unit. It has the a/( fold characteristic ofthe dinucleotide binding region. The fold of the rest of the subunit, the quarternary structure, and the nature of the cofactor-enzyme interactions are, however, significantly different from those observed in the long-chain dehydrogenases. The architecture of the postulated active site is consistent with the observed stereospecificity of the enzyme and the fact that the tetramer is the active form. There is only one cofactor and one substrate-binding site per subunit; the specificity for both 3a-and 2013-ends of the steroid results from the binding of the steroid in two orientations near the same cofactor at the same catalytic site. (1), which includes 11f,-hydroxysteroid (llP-HSD) (2), 7a-hydroxysteroid (3), and 15-hydroxyprostaglandin dehydrogenases (4) from mammals; glucose (5) and ribitol (6) dehydrogenases, as well as a putative nodulation factor (7) from bacteria; and an ADH (8) from insects. Enzymes belonging to this family have -250 amino acid residues, similar coenzyme specificity, and partial sequence homology. Although more than 40 crystal structures of =15 types of NAD(H)-and NADP(H)-linked dehydrogenase enzymes have been determined at medium-to-high resolution (9), to our knowledge no x-ray crystallographic study describing the three-dimensional structure of a dehydrogenase belonging to this short-chain class has been reported. This is only the third structure of an enzyme for which steroids are the substrate to be determined by x-ray diffraction techniques. A lowresolution structure of keto-steroid isomerase (10) and the refined structure of cholesterol oxidase (11) have been published.To account for the ability of 3a,20f3-HSD to transfer a hydride to either end of a steroid molecule, "one steroid-two cofactor sites" and "two steroid orientations-one cofactor site" models (12) have been proposed. When analyzed in conjunction with sequence homology studies, the threedimensional structured especially at the cofactor binding and the substrate binding regions, offers further insight concerning the significance of conserved residues and their possible roles in substrate specificity and overall enzyme function. MATERIALS AND METHODSThe crystals, grown in the presence of 4 mM NADH, belong to the space group P43212 having unit cell dimensions a = 106.2 A and c = 203.8 A and contain one full tetramer (106 kDa) in the asymmetric unit (13 .091], was collected on film from six crystals at the Cornell High Energy Synchrotron Source and processed by using Rossmann's program at Purdue University. The area detector data to 3 A and film data between 3 and 2.6 A were merged into a composite native data set. The Hg derivatives each had a single major binding site per subunit, whereas the Au reagent gave a "multip...

Measurement of progesterone concentration in blubber was developed as a method to detect pregnancy in minke whales. Progesterone was extracted and quantified from blubber samples of minke whale carcasses by radioimmunoassay. Results showed a highly significant difference (almost 60‐fold) between blubber progesterone concentrations of anatomically determined pregnant females versus non‐pregnant female or male carcasses. The results of the study suggest that the blubber progesterone concentrations might be used to determine pregnancy status in free‐ranging whales.

The 3alpha- and 20beta-hydroxysteroid dehydrogenase (HSD) activities of cortisone reductase in Streptomyces hydrogenans have been examined to determine whether both activities are due to one enzyme. This question was raised when changes in the commercial preparations of the enzyme reduced the 3alpha-HSD activity to 5% of its original level while retaining full 20beta-HSD activity. In our experiments, the enzyme was purified to crystallinity and partially characterized. The 3alpha- and 20beta-HSD activities were both coinduced and copurified. The 3alpha- and 20beta-HSD activities were compared using the crystalline enzyme for studies of substrate competition, thermal inactivation at 52 degrees C, loss of activity with three haloacetoxysteroids, and the effects of Me2SO and temperature on the reaction rate. These studies support the conclusion that the 3alpha- and 20beta-HSD activities are due to the same enzyme molecule. In addition, it appears that the binding sites for the two activities do not act independently.

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