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An NAD(P)H-dependent H2O2 forming activity has been evidenced in thyroid tissue from patients with Grave's disease. Its biochemical properties were compared to those of the NADPH oxidase previously described in pig thyroid gland. Both were Ca2+-dependent and activated by inorganic phosphate anions in the same range of concentrations. Both are flavoproteins using FAD as cofactor, but the human enzyme was also able to utilize FMN. The apparent Km for NADPH of the human enzyme (100 microM) was 5-10 times higher than that of porcine enzyme. Vm was 3 to 10 times higher in pig (150 nmol x h(-1) x mg(-1)) than in man (14 to 45). Total content in human tissue was 7 to 9% of that in porcine tissue. An unidentified inhibitor has been detected in the 3000 g particulate fraction from most patients, which could account for this apparently low enzyme content. An NADH-dependent H2O2 production has also been observed in porcine and human thyroid tissues. This activity was only partly Ca2+-dependent (man, 50-70%; pig, 80-90%) and presented similar apparent Km values for NADH (man, 100 microM; pig, 200 microM). In pig thyrocytes, the expression of the Ca2+-dependent part of the NADH-oxidase activity was induced by TSH and down-regulated by TGFbeta, as was the NADPH oxidase activity. Furthermore, NADPH and NADH-dependent activities were not additive. We conclude that a single, inducible, NAD(P)H-oxidase can use NADPH or NADH as substrate to catalyse H2O2 formation, and that human and porcine NAD(P)H-oxidases are highly similar. Differences observed could be attributed to minor differences in enzyme structure and/or in membrane microenvironment. The NADH-dependent Ca2+-independent activity observed in human and porcine thyroid fractions could be attributed to a distinct and constitutive enzyme.

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Pig thyroid plasma membranes contain a Ca(2+)-dependent NADPH:O2 oxidoreductase, the thyroid NADPH-dependent H2O2 generator. This provided the H2O2 for the peroxidase-catalysed synthesis of thyroid hormones. The effect of the tervalent arsenical, phenylarsine oxide (PAO), on the NADPH oxidase was studied. PAO caused two directly related dose-dependent effects with similar half-effect concentrations of PAO (3 nmol of PAO/mg of protein): (i) partial inactivation of H2O2 formation by the Ca(2+)-stimulated enzyme, and (ii) desensitization of the enzyme activity to Ca2+. PAO had no effect on membranes that had been Ca(2+)-desensitized by alpha-chymotrypsin treatment. The NADPH oxidase in membranes treated with excess PAO had the same Vmax with and without Ca2+. This value was half the Vmax of the native enzyme. However, the K(m) for NADPH determined with Ca2+ (18 microM, identical with that of the native enzyme) was approx, one-third of the K(m) measured without Ca2+, showing the direct action of Ca2+ on the PAO-enzyme complex. PAO had the same effects, partial inactivation and Ca2+ desensitization, on the NADPH: ferricyanide oxidoreductase activity of the NADPH oxidase, suggesting that PAO acts on the flavodehydrogenase entity of the enzyme. Both partial inactivation and Ca2+ desensitization were completely and specifically reversed by 2.3-dimercaptopropanol, partly reversed by dithiothreitol and not reversed by 2-mercaptoethanol, indicating that PAO binds to vicinal thiol groups. These results suggest that thiol groups are involved in the control of thyroid NADPH oxidase by Ca2+; PAO bound to vicinal thiols might alter the structure of the enzyme so that electron transfer occurs without Ca2+ but more slowly.

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The thyroid plasma membrane contains a Ca(2+)-regulated NADPH-dependent H2O2-generating system which provides H2O2 for the thyroid-peroxidase-catalyzed biosynthesis of thyroid hormones. The molecular nature of the membrane-associated electron transport chain that generates H2O2 in the thyroid is unknown, but recent observations indicate that a flavoprotein containing a FAD prosthetic group is involved. Solubilization was reinvestigated using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), Triton X-100, and high salt concentrations. Chaps eliminated about 30% of the proteins, which included a ferricyanide reductase, without affecting the H2O2-generating system. Similarly, Triton X-100 alone did not extract the NADPH oxidase. An NADPH-oxidase activity, which was measured in the presence of the artificial electron acceptor potassium ferricyanide, was solubilized by increasing the ionic strength to 2 M KCl. This NADPH-ferricyanide reductase activity was shown to belong to the H2O2-generating system, although it did not produce H2O2. It was still Ca2+ dependent and H2O2 production was restored by decreasing the ionic strength by overnight dialysis. No H2O2 production activity was detected after sucrose density gradient centrifugation of the dialyzed solubilized enzyme, but a well-defined peak of NADPH oxidation activity with a sedimentation coefficient of 3.71 S was found in the presence of K3Fe(CN)6. These results suggest that some unknown component(s) (phospholipid or protein) is removed during sucrose density gradient centrifugation. Finally, thyrotropin, which induces NADPH oxidase and regulates H2O2 production in porcine thyrocytes in primary culture, also induced the NADPH-K3Fe(CN)6 reductase activity associated with the H2O2-generating system. Thus, this enzyme seems to be another marker of thyroid differentiation.

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A model tetradecapeptide, used for the purification of the RXVRG-endoprotease from Xenopus laevis skin exudate, has been studied by two-dimensional NMR, correlation (COSY) and NOE (NOESY) spectroscopy. This peptide has the 5-9 consensus sequence (RXVRG), along with an acidic moiety (1-4) and a hydrophobic domain (10-14). Variations with temperature of NH chemical shifts in a dimethyl sulfoxide solution (low thermal coefficients at residues 6, 7 and 8) and quantified NOE values from four spectra at different mixing times clearly showed a structural organization in the consensus domain with psi-angles around [-40, -10 degrees] for residues 7 and 8, and two NOE correlations of alpha HiNHi + 2 type (5-7 and 6-8). Moreover, a privileged rotamer in the side chain is established for three residues (Val2, Asp3 and Val7) and limited possibilities are discussed for seven others. Most of the folding trends were not observed in the [Ser7] derivative, underlying the relationship between the conformations and a full consensus sequence. In the model tetradecapeptide an equilibrium between two beta-turns of type I, fragments 4-7 and 5-8, seems the most probable. Comparison between this tetradecapeptide and its 4-14 fragment, also a substrate for RXVRG-endoprotease, shows that the 1-3 moiety (DVD) influences the consensus domain structure(s) and clearly stabilizes the folded one(s). Finally, two analytical methods are developed in order to determine: (1) the trifluoroacetic acid content of the peptide samples, on the basis of 19F NMR spectroscopy; (2) the mean phi- and psi-angles of each residue, from the whole set of NH/alpha H coupling constants (3JN alpha) and NOE data at a local level.

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Conformation of a tetradecapeptide with a RXVRG consensus sequence, Arg5-Asp-Val-Arg-Gly9, found in several precursors of antibacterian peptides, was investigated in dimethylsulfoxide solution by proton NMR spectroscopy. Complete resonance assignments and conformational parameters were obtained through correlated (COSY) and nuclear Overhauser (NOESY) techniques. The 3J(alpha H, beta H) coupling constants and the intramolecular NOE, NH...beta H, were used to analyse the conformers around the C alpha-C beta bond and, in four cases, to obtain stereospecific assignments. Use of restraints derived from NOE connectivities and 3J(NH, alpha H) coupling constants allows the determination of a range of phi and psi dihedral angles for all the residues in the sequence. The present NMR results provide favourable evidence for the formation of two bends in the consensus sequence of the tetradecapeptide. The first one has most of the features of a Glu4-Val7 beta-turn (low temperature coefficient of the Val7NH chemical shift, Arg5 alpha H...Val7NH and Asp6NH...Val7NH NOE correlations). The second one exhibits only the Asp6 alpha H...Arg7NH and Val7NH...Arg8NH NOE interactions. These consensus sequence organizations proposed were confirmed by molecular modeling based on low potential energy structure on the [4-9] fragment with high agreement of NOE data. Overall, the substitution of Ser12 by Ala12 shifts the conformation of the hydrophobic moiety [10-14] towards a quite random coil structure in this fragment and strongly destabilizes the folded structures of the consensus domain where only one NH (Val7) is solvent-shielded opposed to three (Asp6 to Arg8) in the [Ser12] tetradecapeptide. These conformational changes could be related to the processing enzyme activities on these model oligopeptides.

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Two synthetic fragments, corresponding to the 4-9 and 4-14 sequences of a tetradecapeptide used as a model to test the RXVRG-endoprotease activity from Xenopus laevis skin, have been studied by two-dimensional nmr spectroscopies, correlated spectroscopy, and nuclear Overhauser effect (NOE) spectroscopy. Both peptides wore the 5-9 consensus sequence found in several hormonal precursors. The nmr data for the 4-9 hexapeptide did not indicate any particular organization, either in water or in dimethylsulfoxide (DMSO), whereas, the 4-14 undecapeptide, a substrate for the RXVRG endoprotease, showed, in DMSO solution, significant trends of structural organization involving the amino acids pertaining to the consensus domain. From variations of integrated NOE peaks with temperature, the apparent interproton correlation times tau c were estimated and the maxima observed with Val7, the central residue in the consensus sequence. A defined tertiary structure in that domain was also supported by medium- and long-range NOEs between Asp6 and Arg8, Glu4 and Gly9, and by the likely involvement of Arg8 and Gly9 NHs in intramolecular hydrogen bonds. Most of these observations could be rationalized by an equilibrium between a 5-8 beta-turn and a 9 > 4 H-bonded loop. The predominance of one rotamer for the C alpha-C beta bond was established in four residues. Finally, the average phi and psi angles were derived from two models taking, or not, into account variations in the correlation times along the sequence. This allowed us to discuss the artefacts generated by using an average correlation time through the whole molecule.

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N-Arg dibasic convertase is a metalloendopeptidase from rat brain cortex and testis that cleaves peptide substrates on the N terminus of Arg residues in dibasic stretches. By using both an oligonucleotide and antibodies to screen a rat testis cDNA library, a full-length cDNA was isolated. The sequence contains an open reading frame of 1161 codons corresponding to a protein of 133 kDa that exhibits 35% and 48% similarity with Escherichia coli protease III (pitrilysin, EC 3.4.99.44) and rat or human insulinase (EC 3.4.99.45), respectively. Moreover, the presence of the HXXEH amino acid signature (XX = FL) clearly classifies N-Arg dibasic convertase as a member of the pitrilysin family of zinc-metalloendopeptidases. In addition, a Cys residue that may be responsible for the thiol sensitivity of the insulinase and N-Arg dibasic convertase was proposed. The protein sequence contains a distinctive additional feature consisting of a stretch of 71 acidic amino acids. We hypothesize that this metalloendopeptidase may be a member of a distinct class of processing enzymes.

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A novel metallo-endopeptidase from human neuroblastoma NB-OK-1 cells was partially purified and characterized. This enzyme activity was detected in the culture medium and could be detached from intact cells by gentle washing, suggesting a peripheral localization of the enzyme. This endopeptidase inactivated Atrial Natriuretic Peptide (ANP) by a unique and selective cleavage of the Ser123-Phe124 bond. It also produced hydrolysis at the Xaa-Phe, Xaa-Leu, or Xaa-Ile bonds of other peptide hormones such as bradykinin, somatostatin 14, litorin, substance P, neuromedin C and angiotensin II. The substrate selectivity and inhibition profile of the enzyme showed obvious similarities with the peptide hormone inactivating endopeptidase (PHIE) recently purified from Xenopus laevis skin secretions and indicated a thermolysin-like activity distinct from neutral endopeptidase (EC 3.4.24.11) and from angiotensin converting enzyme (EC 3.4.15.1).

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An endopeptidase was isolated from Xenopus laevis skin secretions. This enzyme, which has an apparent molecular mass of 100 kDa, performs a selective cleavage at the Xaa-Phe, Xaa-Leu, or Xaa-Ile bond (Xaa = Ser, Phe, Tyr, His, or Gly) of a number of peptide hormones, including atrial natriuretic factor, substance P, angiotensin II, bradykinin, somatostatin, neuromedins B and C, and litorin. The peptidase exhibited optimal activity at pH 7.5 and a Km in the micromolar range. No cleavage was produced in vasopressin, ocytocin, minigastrin I, and [Leu5]enkephalin, which include in their sequence an Xaa-Phe, Xaa-Leu, or Xaa-Ile motif. The endopeptidase activity was inhibited by divalent cation chelators and by phosphoramidon only at high concentrations (IC50 = 50 microM), whereas it was insensitive to classical inhibitors of chymotrypsin, angiotensin convertase, and serine and cysteine peptidases, as well as carboxypeptidases. It is hypothesized that this enzyme, which is distinct from neutral endopeptidase (EC 3.4.24.11), constitutes the prototype of a family of related metalloendopeptidases that inactivate peptide substrates by cleavage at the Xaa-Phe, Xaa-Leu, or Xaa-Ile bond.

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Comparison of the precursor sequence for several peptide hormones of Xenopus laevis skin revealed a consensus sequence around a single arginine cleavage site which is 100% conserved on four residues Arg-Xaa-Val-Arg-Gly (RXVRG). A tetradecapeptide substrate (Asp-Val-Asp-Glu-Arg-Asp-Val-Arg-Gly-Phe-Ala-Ser-Phe-Leu-NH2) was used as a probe to purify and characterize the putative processing endoprotease. A hydrophobic enzyme was purified at least 9000-fold from Xenopus skin exudate by a four-step procedure. This highly specific activity cleaves the Arg-Gly bond and has no effect on the Arg-Xaa bond. It was strongly inhibited by divalent ion chelators, moderately by phenylmethylsulfonyl fluoride, aprotinin, and 1-tosylamide-2-phenylethyl chloromethyl ketone, but was insensitive to soybean trypsin inhibitor. Tetradecapeptide derivatives selectively modified on each of the amino acids of the consensus sequence demonstrated the relevance of this conserved pattern to endoprotease action. This enzyme, which we refer to as RXVRG-endoprotease, is proposed to be involved in the post-translational processing of pro-caerulein, promagainin, pro-xenopsin, pro-glycyl-leucine amide, and pro-levitide of X. laevis skin secretory granules.

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We have previously reported that hypothyroidism is accompanied by modifications of the cytosolic rat liver glucocorticoid receptors. In the present study we have used molybdate, which stabilizes the glucocorticoid receptor from hypothyroid rats, allowing its characterization. We confirm a decrease in the number of glucocorticoid binding sites in the liver of hypothyroid rats as compared to that in normal rats (8000 per cell versus 23,000 per cell for normal rats). At 22 degrees C and in the absence of molybdate, thermal activation shows the same kinetics in both complexes from normal and hypothyroid rats, though activated hormone-receptor complexes from hypothyroid rats are progressively degraded (t 1/2 = 230 min). At the same temperature the dissociation rate of native complexes from hypothyroid rats is slower (k-1 = 8 X 10(-3) min-1) than normal (k-1 = 16 X 10(-3) min-1). Competitive dissociation studies using [3H]dexamethasone indicate that native complexes from hypothyroid rats show altered affinities for agonist and antagonist. Hypothyroidism decreases the number of rat liver glucocorticoid receptors and alters their properties, as evidenced by their greater instability and differences in steroid binding. These effects may be due to regulating factors and/or slight, probably post-transcriptional, modifications of the binding protein.

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