The enzymatic formation of novel bile acid primary amides

Bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the copper-, ascorbate-, and O(2)-dependent cleavage of C-terminal glycine-extended peptides and N-acylglycines to the corresponding amides and glyoxylate. The alpha-amidated peptides and the long-chain acylamides are hormones in humans and other mammals. Bile acid glycine conjugates are also substrates for PAM leading to the formation of bile acid amides. The (V(MAX)/K(m))(app) values for the bile acid glycine conjugates are comparable to other known PAM substrates. The highest (V(MAX)/K(m))(app) value, 3.1 +/- 0.12 x 10(5) M(-1) s(-1) for 3-sulfolithocholylglycine, is 6.7-fold higher than that for d-Tyr-Val-Gly, a representative peptide substrate. The time course for O(2) consumption and glyoxylate production indicates that bile acid glycine conjugate amidation is a two-step reaction. The bile acid glycine conjugate is first converted to an N-bile acyl-alpha-hydroxyglycine intermediate which is ultimately dealkylated to the bile acid amide and glyoxylate. The enzymatically produced bile acid amides and the carbinolamide intermediates were characterized by mass spectrometry and two-dimensional (1)H-(13)C heteronuclear multiple quantum coherence NMR.     

Interaction of steroid hormones with glutamate dehydrogenase]

The inhibitory action of sex hormones on the glutamate dehydrogenase activity is due to their binding to the enzyme protein. No binding of sex hormones to glutamate dehydrogenase is observed in the presence of L-amino acids, preventing the enzyme dissociation. Under those conditions the enzyme sensitivity to the inhibitory action of the hormones sharply decreases.     

Excitatory amino acid regulation of gonadotropin secretion: modulation by steroid hormones

Excitatory amino acids (EAAs) can potently modulate gonadotropin secretion in the male rat and monkey. In the present study we examined of EAAs on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the female rat under low estrogen (ovariectomized) and high estrogen (proestrus) backgrounds. In ovariectomized immature female rats (NMDA) inhibited LH but not FSH secretion at 30 min post-injection. In contrast, NMDA potently stimulated LH but not FSH secretion when administered on proestrus to adult female rats. Both glutamate and kainate were also found to stimulate LH but not FSH secretion in estrogen-treated ovariectomized immature rats. This study suggests that EAA neurotransmission may be an important component in the expression of gonadotropin surges and that EAA effects appear to be subject to gonadal steroid regulation.     

Time-resolved fluoroimmunoassay of steroid hormones

The use of europium chelates as labels in immunoassays and their sensitive quantitation based on time-resolved fluorescence is reviewed. The technique is applied on competitive solid-phase immunoassays for direct determination of progesterone and estradiol in serum samples. Both antigen- and antibody-labelled competitive assays are described. The nonisotopic label technology, which provides a very high specific activity, as well as the antibody-labelled competitive assays, present several advantages in the assay of haptens as e.g. steroids. As the optimal sensitivity of competitive methods is not limited by the specific activity of the label the steroid assays which employ europium chelates as labels do not show any marked increase in sensitivity as compared to that achieved by using 125I. The potential sensitivity provided by the high specific activity of the label is optimally utilized in noncompetitive immunometric assays.     

Nongenomic actions of steroid hormones

Steroid hormones modulate many physiological processes. The effects of steroids that are mediated by the modulation of gene expression are known to occur with a time lag of hours or even days. Research that has been carried out mainly in the past decade has identified other responses to steroids that are much more rapid and take place in seconds or minutes. These responses follow nongenomic pathways, and they are not rare.     

Sex chromatin in women treated with steroid hormones

3 groups of women, aged 15-71 years, were examined: a control group of 17 healthy women and 11 women with blood complications who had nver received steroid treatments, a 2nd group of patients with complications similar to the 1st groups' who were treated with the steroid preparation Enkorton-Polfa (for 5-10 days at 20-50 mg daily), and a 3rd group of 20 patients with similar prolonged complciations from 4 weeks to 5 years) who were treated with the steroid preparation Enkorton-Polfa in daily doses of 10-160 mg. Sex chromatin from these 3 groups was studied using the method of Sanderson and Stewart and the results compared. A lower percentage of sex chromatin bodies was found in those treated with steroids. Significant statistical differences were found in the comparison of the standard deviations of sex chromatin count: Group 1, + or -13%; Group 2, + or -10% before treatment and + or -5% during treatment; and Group 3, + or -8%.     

Horseradish peroxidase. Complex formation with anions and hydrocyanic acid

Equilibrium binding experiments have been performed with perchlorate, chloride, and acetate in the presence of horseradish peroxidase. The binding of perchlorate and acetate appears to be like that of nitrate, at a site other than the sixth coordination position of the heme iron. Competitive experiments using both nitrate and cyanide demonstrate that two different binding sites are present on the enzyme. Chloride appears to bind at the sixth coordination position as do both fluoride and cyanide. Temperature jump experiments indicate that it is likely the nitrate anion and not undissociated nitric acid which is the binding species. Competitive stopped flow experiments indicate that the bound nitrate slows both the association rate and dissociation rate of cyanide, indicating that nitrate binds close to the sixth coordination position.     

Interaction of lipoproteins in blood serum with steroid hormones

Interaction of human and serum lipoproteins with steroid hormones (corticosterone and cortisol) was studied. Methods of fluorescence quenching titration and equilibrium dialysis were used for quantitative evaluation of VLDL, LDL and HDL glucocorticoid-binding ability. Association constants were found to be 0.6-2.0 x 10(6) M-1 for corticosterone and 4.0-8.0 x 10(6) M-1 for cortisol. The number of binding sites varied from 3 to 300 for different classes of lipoproteins.