Histochemical localization of some hydroxysteroid dehydrogenases in the mouse epididymis.

The mouse epididymis was studied to localize histochemically a number of hydroxysteroid dehydrogenases in the various zones. The epithelium of the posterior half of the initial segment (head) and the anterior half of the middle segment (body) shows a strong reaction for delta5-3beta-, 3alpha,5alpha-, 3alpha,5beta-, 11beta, 16alpha-, 17beta, 20alpha-hydroxysteroid dehydrogenases. This activity attenuates posteriorly. Only the 11beta-hydroxysteroid dehydrogenase is present throughout the length of the epididymis. The luminal contents of the middle segment also show the histochemical utilization of a number of steroids.     

Chromatography of hydroxysteroid dehydrogenases

The structure-function study of hydroxysteroid dehydrogenases has stimulated the development of their chromatography, which in turn reveals more mechanisms of these enzumes. Due to the various membrane associations and mild hydrophobic nature of most of the enzymes studied up to now, hydrophobic interaction chromatography has played a crucial role in their purification, using media such as phenyl-Superose or Sepharose-PEG. At the same time, affinity chromatography, especially the dye-containing columns, proves very efficient for these dehydrogenases, as the latter utilizes adenylyl-containing cofactors. Elution by their specific ligand facilitates their purification. In this paper, the use of detergents in the purification of these enzymes is also reviewed. Hydroxysteroid dehydrogenase preparation is further improved by rapid purification which facilitates the elimination of protein microheterogeneity, caused in vitro by oxidation, reduction or partial proteolysis. This process was shown to increase the crystallizability of the enzymes [Lin et al., J. Cryst. Growth, 122 (1992) 242–245; Zhu et al., J. Mol. Biol., 234 (1993) 242–244]. The fast purification permitted a simpler procedure and better combination of various columns than conventional chromatography. This leads to even higher efficiency, yielding homogeneous and highly active preparations.     

The histochemical distribution of hydroxysteroid dehydrogenases in the human foetal membranes at term

Summary The histochemical distribution of various hydroxysteroid dehydrogenases in human, term, foetal membranes has been investigated using the tetrazolium dye, Nitro-B.T.The trophoblastic layer was the most active, showing 3-, 3-, 11-, 16- and 17-hydroxysteroid dehydrogenase activities, a pattern of activity similar to that of the placental villous trophoblast.The amniotic epithelium showed weak 3-, 3-, 16- and 17-hydroxysteroid dehydrogenase activity; weak 3- and 3-hydroxysteroid dehydrogenase activity was noted in the connective tissue layers.All activity demonstrated was N.A.D.-linked.     

Mechanism based inhibition of hydroxysteroid dehydrogenases.

Steroid hormone action can be regulated not only at the receptor level but also by the enzymes that are responsible for the synthesis and degradation of biologically active steroids. Traditionally the pharmacological intervention of steroid hormone action has focused on the development of steroidal and nonsteroidal hormone receptor agonists and antagonists with appropriate pharmacokinetics. Recently, the development of selective inhibitors/inactivators of steroid metabolizing enzymes has gained momentum. This review will concentrate on the development of mechanism-based inhibitors for one class of steroid hormone transforming enzymes, the hydroxysteroid dehydrogenases.     

Dihydrodiol dehydrogenase activities of rabbit liver are associated with hydroxysteroid dehydrogenases and aldo-keto reductases.

1. Dihydrodiol dehydrogenase activities were investigated in rabbit liver. Using a five-step purification scheme, eight isoenzymes of dihydrodiol dehydrogenase with isoelectric points of 5.55-9.3 and promoter molecular masses of 34-35 kDa were purified to apparent homogeneity and designated CF-1 to CF-6, CM-1 and CM-2. 2. CF-1 and CF-2 had near-neutral isoelectric points of 7.4 and 6.8 and molecular masses of about 125 kDa in the native state. Both enzymes readily accepted NAD+ as well as NADP+ as coenzymes, had relatively low Km values of 0.33 mM and 0.47 mM for benzene dihydrodiol and resembled previously described carbonyl reductases in their substrate specificity towards ketones and quinones. 3. CF-5 and CF-6 had acidic isoelectric points of 5.9 and 5.55 and native molecular masses of approximately 60 kDa. They displayed a strong preference for NADP(H) as coenzyme and had high Km and Vmax with benzene dihydrodiol. Since these enzymes reduced p-nitrobenzaldehyde and glucuronic acid efficiently, they appeared to be closely related to aldehyde reductase. 4. CF-4 had a high 3 alpha-hydroxysteroid dehydrogenase activity for the diagnostic substrate androsterone, a moderate activity for other 3 alpha-hydroxysteroids as well as 17 alpha-hydroxysteroids, and relatively low activities for 3 beta-hydroxysteroids and 17 beta-hydroxysteroids. CF-5 and CM-1 had high 17 beta-hydroxysteroid dehydrogenase activity for the diagnostic substrate 5 alpha-dihydrotestosterone, and low to moderate activities for other 17 beta-hydroxysteroids as well as 3 alpha-hydroxysteroids. 5. The isoenzyme CM-2 had an isoelectric point of 9.3 and was a very active quinone reductase with phenanthrene-9,10-quinone as substrate. It was potently inhibited by phenobarbital. 6. We conclude that the dihydrodiol dehydrogenase activities of rabbit liver are associated with aldehyde and carbonyl reductase and with 3 alpha-hydroxysteroid and 17 beta-hydroxysteroid dehydrogenases.     

Characterisation of 11 beta-hydroxysteroid dehydrogenases in feline kidney and liver

11 Beta-hydroxysteroid dehydrogenases type 1 and 2 (11 beta-HSD1 and 11 beta-HSD2) are microsomal enzymes responsible for the interconversion of cortisol into the inactive form cortisone and vice versa. 11 beta-HSD1 is mainly present in the liver, and has predominantly reductase activity although its function has not yet been elucidated. 11 beta-HSD2, present in mineralocorticoid target tissues such as the kidney, converts cortisol into cortisone. Reduced activity due to inhibition or mutations of 11 beta-HSD2 leads to hypertension and hypokalemia resulting in the Apparent Mineralocorticoid Excess Syndrome (AMES). Like humans, cats are highly susceptible for hypertension. As large species differences exist with respect to the kinetic parameters (K(m) and V(max)) and amino acid sequences of both enzymes, we determined these characteristics in the cat. Both enzyme types were found in the kidneys. 11 beta-HSD1 in the feline kidney showed bidirectional activity with predominantly dehydrogenase activity (dehydrogenase: K(m) 1959+/-797 nM, V(max) 766+/-88 pmol/mg*min; reductase: K(m) 778+/-136 nM, V(max) 112+/-4 pmol/mg*min). 11 beta-HSD2 represents a unidirectional dehydrogenase with a higher substrate affinity (K(m) 184+/-24 nM, V(max) 74+/-3 pmol/mg*min). In the liver, only 11 beta-HSD1 is detected exerting reductase activity (K(m) 10462 nM, V(max) 840 pmol/mg*min). Sequence analysis of conserved parts of 11 beta-HSD1 and 11 beta-HSD2 revealed the highest homology of the feline enzymes with the correspondent enzymes found in man. This suggests that the cat may serve as a suitable model species for studies directed to the pathogenesis and treatment of human diseases like AMES and hypertension.     

Continuous-flow automated assay of steroids with nylon-tube-immobilized hydroxysteroid dehydrogenases

Several NAD(P)⁺-dependent hydroxysteroid dehydrogenases, namely 3α-hydroxysteroid dehydrogenase, β-hydroxysteroid dehydrogenase, 7α-hydroxysteroid dehydrogenase, and 12α-hydroxysteroid dehydrogenase were separately immobilized on nylon tubes for the continuous-flow automated assay of hydroxysteroids. 3α-Hydroxysteroid dehydrogenase was also immobilized on pore glass. Spectrophotometric monitoring in the visible region, where blank values were markedly reduced, was achieved through the Meldola blue catalyzed transfer of hydrogen from NAD(P)H to a tetrazolium salt. Nylon-tube-immobilized enzymes maintained 45–55% of the original activity after 1 month of intermittent use. The operational range, using the “end point” approach, was 1–25 nmol of steroid and the assay speed 10–15 samples/h. Reliable results were obtained in the determination of 3α-hydroxysteroids and 3β,17β-hydroxysteroids in urine and total bile acids in serum.     

The subcellular distribution and properties of aldehyde dehydrogenases in rat liver

1. Kinetic experiments suggested the possible existence of at least two different NAD(+)-dependent aldehyde dehydrogenases in rat liver. Distribution studies showed that one enzyme, designated enzyme I, was exclusively localized in the mitochondria and that another enzyme, designated enzyme II, was localized in both the mitochondria and the microsomal fraction. 2. A NADP(+)-dependent enzyme was also found in the mitochondria and the microsomal fraction and it is suggested that this enzyme is identical with enzyme II. 3. The K(m) for acetaldehyde was apparently less than 10mum for enzyme I and 0.9-1.7mm for enzyme II. The K(m) for NAD(+) was similar for both enzymes (20-30mum). The K(m) for NADP(+) was 2-3mm and for acetaldehyde 0.5-0.7mm for the NADP(+)-dependent activity. 4. The NAD(+)-dependent enzymes show pH optima between 9 and 10. The highest activity was found in pyrophosphate buffer for both enzymes. In phosphate buffer there was a striking difference in activity between the two enzymes. Compared with the activity in pyrophosphate buffer, the activity of enzyme II was uninfluenced, whereas the activity of enzyme I was very low. 5. The results are compared with those of earlier investigations on the distribution of aldehyde dehydrogenase and with the results from purified enzymes from different sources.     

Histochemical distribution of delta5-3beta- and 17beta-hydroxysteroid dehydrogenases in hamster trophoblast.

The histochemical distribution of delta5-3beta- and 17beta-hydroxysteroid dehydrogenases was demonstrated in hamster trophoblast between Days 8 and 15 of pregnancy. The delta5-3beta-hydroxysteroid dehydrogenase activity in the ectoplacental trophoblast of 8-day embryos was demonstrated by use of delta5-pregnenolone and dehydroepiandrosterone as substrates; between Days 11 and 15, activity was demonstrated in the trophoblastic giant cells of the placenta and in the intra-arterial trophoblast cells when delta5-pregnenolone was the substrate. Between Days 11 and 15, 17beta-hydroxysteroid activity was present in the spongiotrophoblast, labyrinth, placental giant cells and intra-arterial trophoblast cells, as shown by use of testosterone and oestradiol as substrates. Both enzymes were demonstrated in ectopic trophoblast cells, indicating that these activities are autonomous.     

Histochemical studies of dehydrogenases in the developing teeth

Summary Histochemical observations were made of oxidative and reducing enzymes in relation to several metabolic pathways in the developing tooth. The localization and activity of dehydrogenases and oxidases could be divided roughly into two types.Type one refers to enzymatic activities of lactic, succinic, malic and glucose-6-phosphate dehydrogenase and aconitase and cytochrome oxidase, which were low in undifferentiated dental epithelium and increased in proportion to cell differentiation. Comparatively high enzymatic activities were reflected in high cell function.Type two did not increase in the enzymatic activity during differentiation. Glyceraldehyde-3-phosphate, TPN isocitric, 6-phosphogluconate, glutamic, -glycerophosphate and -hydroxybutyric dehydrogenase and monoamine oxidase belong to this type.With 12 Figures in the TextPresented in Parts at the 3rd Annual Meeting in September, 1962 of the Japanese Histochemical Association.     

Human hydroxysteroid dehydrogenases and pre-receptor regulation: insights into inhibitor design and evaluation

Hydroxysteroid dehydrogenases (HSDs) represent a major class of NAD(P)(H) dependent steroid hormone oxidoreductases involved in the pre-receptor regulation of hormone action. This is achieved by HSDs working in pairs so that they can interconvert ketosteroids with hydroxysteroids resulting in a change in ligand potency for nuclear receptors. HSDs belong to two protein superfamilies the aldo-keto reductases and the short-chain dehydrogenase/reductases. In humans, many of the important enzymes have been thoroughly characterized including the elucidation of their three-dimensional structures. Because these enzymes play fundamental roles in steroid hormone action they can be considered to be drug targets for a variety of steroid driven diseases, e.g. metabolic syndrome and obesity, inflammation, and hormone dependent malignancies of the endometrium, prostate and breast. This article will review how fundamental knowledge of these enzymes can be exploited in the development of isoform specific HSD inhibitors from both protein superfamilies. Article from the Special issue on Targeted Inhibitors.     

Immobilized hydroxysteroid dehydrogenases for the transformation of steroids in water--organic solvent systems.

The hydroxysteroid dehydrogenases: beta-HSDH, 20 beta-HSDH, and 3 alpha-HSDH, were immobilized on CNBr-activated Sepharose. The effect of various immobilization conditions on the activity recovery and stability were examined. The presence of cofactor during the immobilization reaction increased the activity recovery (40--60% of the total) and also led to materials highly stable in the presence of organic solvents. For example, beta-HSDH maintained 60% of its original activity two months after continuous use in the water--ethyl-acetate system. Kinetic experiments showed that the increase of the apparent Km values is poor and demonstrated that the organic solvent behaves as a weak inhibitor (ki greater than 0.2M) for the substrate. The immobilized enzymes lyophilized in the presence of sucrose had full activity restored even after several months storage at room temperature. Immobilized hydroxysteroid dehydrogenases were shown to be suitable for preparative transformation of steroids in water--organic solvent systems.