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M Pons

Publications and source records attributed to M Pons.

At least 145 records · Page 8Linked to original sources
PubMed1976

Affinity labelling of the estradiol-17 beta dehydrogenase from human placenta with substrate analogs.

Affinity labelling of the estradiol-17 beta dehydrogenase of human placenta has been performed using derivatives of estradiol-17 beta carrying alkylating groups in nine different positions on the steroid nucleus. The active-site-directed character of the inhibition is confirmed by the following observations: the affinity labels are substrates or competitive inhibitors, the enzyme is protected against inactivation and alkylation by the substrate and by the coenzyme, the stoichiometry of the alkylation is two moles of inhibitor per 68 000 g of enzyme (dimer). The alkylation of a histidine residue which is fast and extensive when the alkylation side chain is on the C-3 carbon atom, is dramatically decreased when alkylating side chain is shifted towards rings B and D. These results allow the location of this histidine in the vicinity of ring A and probably on the beta face of the steroid nucleus. The reactivity of a cysteine located on the active site was quite different, showing increasing alkylation when the alkylating substituent of the affinity labels was shifted from C-3 to C-16 of the steroid nucleus. The correlation of this result and that obtained using an alkylating analog of NAD (3-chloroacetyl-pyridine-adenine dinucleotide) indicates that this cysteine is located in the catalytic region of the active site, at the junction of the ring D of the steroid nucleus with the nicotinamide moiety of the coenzyme.

Acetamides↗
PubMed1976

Alkylation of estradiol 17beta-dehydrogenase from human placenta with 3-chloroacetylpyridine--adenine dinucleotide.

3-Chloroacetylpyridine--adenine dinucleotide, which is active as a hydride acceptor (Km = 0.6 mM), inactivates and alkylates estradiol 17beta-dehydrogenase. The kinetics of inactivation by 3-chloroacetylpyridine--adenine dinucleotide and the absence of inactivation by 3-chloroacetylpyridine ribose phosphate show that the alkylation follows the formation of a binary complex (Kd = 4.5 X 10(-4) M). Studies of the labelling by 3-chloro[2-14C]acetylpyridine--adenine dinucleotide and the rate of alkylation as a function of pH, give evidence to the alkylation of a cysteine, the stoichiometry being one mole per subunit. The 14C label is distributed between three chymotryptic peptides, one of which accounts for about 50% of the radioactive label.

Binding Sites↗
PubMedDate not supplied

3D structure of kaliotoxin: is residue 34 a key for channel selectivity?

Kaliotoxin (KTX) is a natural peptide blocker of voltage-dependent K+ channels. The 3D structure of a truncated analogue of KTX (Fernandez et al. (1994) Biochemistry 33, 14256-14263) was determined by NMR spectroscopy and showed significant differences from structures established for other related scorpion toxins. A recent publication with the structure of the complete toxin (Aiyar et al. (1995) Neuron 15, 1169-1181) did not confirm these differences. In this communication we report NMR data for KTX at pH 3.0, 5.5 and 7.2 and the 3D structure obtained from data at pH = 5.5. Complete KTX displays a folding similar to that of other toxins with an alpha-helix and a beta-sheet linked by two disulphide bonds. The pKa of His 34 is anomalously low (4.7-5.2 depending on the buffer) owing to its interaction with two Lys residues (including the essential Lys 27), the charged N-terminus and the side chain of Met 29. Charged residues are placed symmetrically with respect to an axis that approximately coincides with one of the principal components of the moment of inertia of the toxin. His 34, which occupies a well-defined position between two conserved Cys, is located on the centre of a layer of charged groups. Positively and negatively charged residues are found at the same position in related toxins. It is suggested that electrostatic effects modulate the distances between positive charges in flexible side chains, contributing to the fine tuning of the selectivity toward different channel subclasses and that the approximate coincidence between the moment of inertia and the charge axis facilitate the approach of the toxin to the channel. The very low pKa of His 34 implies that it will be completely unprotonated at physiological pH.

Hydrogen Bonding↗
PubMedDate not supplied

Stable luciferase transfected cells for studying steroid receptor biological activity.

In the course of steroid hormone research, firefly luciferase was used as a reporter gene to construct chimeric cellular models in which the firefly luciferase expression mimics natural hormonal response. Cells containing the endogenous receptor of interest were stably transfected with a reporter gene whose expression is controlled by this endogenous receptor. Based on the detection of luciferase activity in intact cells using a photon-counting camera, various stable transfected cell lines were established. We present potential experimental uses of these cellular models such as for screening new (anti)hormonal molecules. We also show that the hormonal responses can be modulated at any step, suggesting that these stable cell lines may be helpful in studying hormonal interactions. For example, we have detected the antiestrogen activity of molecules able to mediate their effect via a pathway other than the estrogen receptor. Lastly, we show that the detection of luciferase activity in intact living cells is particularly helpful in investigating the variation of the hormonal responses with time. Since chimeric response faithfully reflects hormone (or effector) actions in the cell, we conclude that stable transfected cells can be used in both pharmacological and fundamental studies to investigate different aspects of the endocrine research.

Animals↗