Structural evidence for a programmed general base in the active site of a catalytic antibody.

The crystal structure of the complex of a catalytic antibody with its cationic hapten at 1.9-A resolution demonstrates that the hapten amidinium group is stabilized through an ionic pair interaction with the carboxylate of a combining-site residue. The location of this carboxylate allows it to act as a general base in an allylic rearrangement. When compared with structures of other antibody complexes in which the positive moiety of the hapten is stabilized mostly by cation-pi interactions, this structure shows that the amidinium moiety is a useful candidate to elicit a carboxylate in an antibody combining site at a predetermined location with respect to the hapten. More generally, this structure highlights the advantage of a bidentate hapten for the programmed positioning of a chemically reactive residue in an antibody through charge complementarity to the hapten.

"Spin-labelling" with the second hydrogen radioisotope: radicals formed from squalene by muonium addition.

By radio-labelling with muonium (the second hydrogen radioisotope), a free radical species has been clearly identified in squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene ) with a muon-electron hyperfine coupling of 240.7 MHz, as measured by the muon spin rotation (MuSR) technique. The radical is undetectable in pure squalene due to its high viscosity and the large molecular size which leads to extreme line broadening, but its signals may be resolved on reducing the viscosity of the medium by dilution with diethyl ether. The potential of the MuSR method is thus demonstrated as a means for spin-labelling radical species such as are formed from cellular antioxidants; ESR spectroscopy is unsuitable for studies of radicals formed from this molecule, due to extreme line-broadening and spectral complexity.

Mechanism of an antibody-catalysed allylic isomerization.

The catalytic antibody 4B2, which was generated against a substituted amidine 1, catalyses the allylic isomerization of beta, gamma-unsaturated ketones with an acceleration factor (k(cat)/k(uncat)) of 1.5x10(3). On the basis of the 'bait and switch' strategy, it was reasoned that the positively charged hapten could elicit, by charge complementarity, an acidic residue (Asp or Glu) in the antibody-binding site in the right position to catalyse this proton transfer reaction. The pH dependence curve of k(cat)/K(m) shows a bell-shaped feature with an optimum at approx. pH 4.5. By cloning and sequencing the light and heavy chains of the 4B2 antibody, we confirmed the presence of several Asp and Glu residues in the complementarity-determining region loops. The antibody catalyses the alpha-proton exchange on the same substrates, demonstrating the involvement of a dienol intermediate in the reaction mechanism. Kinetic studies with (2)H-NMR provide evidence that alpha-proton abstraction is stereospecific. Whether the process involves one or two acid/base residues in this simple proton transfer or whether it is a concerted mechanism is discussed.

Different in vitro metabolism of 7 alpha-methyl-19-nortestosterone by human and equine aromatases.

The ability of human and equine placental microsomes to aromatize 7 alpha-methyl-19-nortestosterone (MNT) was studied. Kinetic analysis indicates that MNT shares the androgen-binding site of human and equine placental microsomal aromatases. Human placental microsomal estrogen synthetase had about a 2.5-fold higher relative affinity for MNT than the equine placental enzyme (KiMNT/Km androstenedione of 32 versus 87). However, MNT was not metabolized by human placental microsomes, whereas it was very actively metabolized by equine placental microsomes. Further studies using purified equine cytochrome P-450arom indicated that the presence of a 7 alpha-methyl group and the absence of a C19 methyl group did not impair its conversion by the purified enzyme. The product of this reaction was separated and identified as 7 alpha-methylestradiol by gas chromatography coupled to mass spectrometry.

Estrogen synthetase in the horse. Comparison of equine placental and rat liver NADPH-cytrochrome c (P-450) reductase activities.

NADPH-cytochrome c (P-450) reductases from horse placenta and rat liver were purified and their biological activities compared using cytochrome c as substrate. Rat liver reductase was purified to electrophoretic homogeneity in one chromatographic step on 2',5'-ADP agarose, and had a relative mass of 85,000 Da as estimated by SDS-PAGE. Equine placental reductase was separated from cytochrome P-450 on aminohexyl-Sepharose 4B and further purified on 2',5-ADP agarose; this preparation exhibited two bands, one of 85,000 and one of 80,000 Da, on SDS-PAGE. The lower molecular weight form was assumed to be a proteolytic product of the higher molecular weight form. A high retention of activity was obtained in both preparations. Equine placenta and rat liver enzymes were found to exhibit very similar Vmax and Km, suggesting that they are not species specific.

Androgen and 19-norandrogen aromatization by equine and human placental microsomes.

The ability of equine and human placental microsomes to aromatize testosterone and 19-nortestosterone was studied. When 3 microM [1 beta,2 beta-3H]testosterone was used as substrate, the specific activity of equine placental microsomal aromatase was 2.5 times higher than that of the human microsomal enzyme. Although 19-nortestosterone was aromatized 67 times more rapidly by equine than by human aromatase, we found that equine aromatase exhibited a markedly weaker affinity for this substrate than did the human enzyme. Competitive inhibition of testosterone aromatization by 19-nortestosterone occurred with both equine and human aromatases. While having no effect on mare placental microsomes, Na+ and K+ (500 mM) stimulated testosterone aromatization by human placental microsomes by 73 and 52% respectively. If indeed a single enzyme is responsible for the aromatization of testosterone and 19-nortestosterone, which seems to be the case in both equine and human placental aromatase, our results show that differences in the structure of the active sites exist between equine and human aromatases.

Synthesis and aromatization of 19-norandrogens in the stallion testis.

The results of the measurement of 19-nortestosterone in the testiscular artery and vein of the stallion, the very low levels of this steroid in the peripheral blood of geldings and the similar patterns of increase in the peripheral levels of 19-nortestosterone and testosterone after hCG stimulation, show that 19-nortestosterone, like testosterone, is essentially synthesized in the testis. This testicular origin was confirmed by the ability of testicular tissue to synthesize 19-norandrogens from [4-14C]androgens in vitro. 19-Nortestosterone was 50% conjugated in the peripheral blood and almost entirely conjugated after biosynthesis in vitro. The sequence of appearance of steroids in the peripheral blood after a single injection of 10,000 IU hCG suggests that, in the equine testis, 19-norandrogens are produced by a specific C10-19 desmolase (estrene synthetase), stimulable by hCG. 19-Nortestosterone was aromatized into estradiol-17 beta by stallion testicular microsomes. The affinity of the aromatase for 19-nortestosterone was very low compared to that for testosterone. At low and presumably physiological levels, and at a high testosterone/19-nortestosterone ratio, testosterone did not inhibit 19-nortestosterone aromatization by more than 53%. Thus, 19-nortestosterone may be aromatized in vivo in the testis in spite of the endogenous concentrations of androgens. However, the low velocity of 19-nortestosterone aromatization by testicular microsomes at roughly physiological concentrations suggests that 19-norandrogen aromatization may only participate slightly in the testicular estrogen production. These results suggest that in the equine testis, two aromatizing enzyme systems may exist: one which aromatizes both androgens and 19-norandrogens, and a minority system more specific for 19-norandrogens.

Down-regulation of testicular aromatization in the horse.

A single i.m. injection of testosterone (750 mg of testosterone bexahydrobenzoate) or i.v. injection of human chorionic gonadotrophin (hCG) (10,000 IU) was given to geldings and stallions. Levels of unconjugated and conjugated (after solvolysis) androgens and estrogens were measured in blood and urine samples taken daily from the day of injection (D0) to the tenth day post-injection (D10). In the stallion, both treatments resulted in a sharp increase of plasma estrogens, which peaked one day before the androgen levels. Our results confirmed the testicular localization of a potent aromatase, which is able to aromatize androgens from endogenous as well as exogenous origin into conjugated estrogens. The very similar patterns of estrogen increase following testosterone or hCG administration suggest that the estrogen rise induced by hCG results at least partly from increased availability of testosterone. The abrupt drop in plasma estrogen levels cannot be explained by a lack of substrate, since two successive androgen injections did not succeed in maintaining the high estrogen levels. Since estrogens were unable to inhibit the aromatase activity in vitro, the drop in estrogen levels suggests a down-regulation of the aromatase synthesis.

Aromatization of testosterone and 19-nortestosterone by a single enzyme from equine testicular microsomes. Differences from human placental aromatase.

A single enzyme in the stallion testis was able to aromatize both testosterone and nortestosterone. This enzyme had a much lower affinity for nortestosterone than for testosterone. In contrast to human placental estrogen synthetase, this enzyme aromatized testosterone and 19-nortestosterone with similar efficiency. The differences observed (effects of monovalent cations, inhibition of androstenedione aromatization by testosterone and 19-nortestosterone and, above all, rate of norandrogen aromatization) suggest that the aromatase in the horse testis is not the same as that in the human placenta.