INVESTIGADORES
GALIGNIANA Mario Daniel
artículos
Título:
Comparative inhibition by hard and soft metal ions of steroid-binding capacity of renal mineralocorticoid receptor cross-linked to the 90-kDa heat-shock protein heterocomplex
Autor/es:
GALIGNIANA MD, PIWIEN-PILIPUK G
Revista:
BIOCHEMICAL JOURNAL
Editorial:
PORTLAND PRESS LTD
Referencias:
Año: 1999 vol. 341 p. 585 - 592
ISSN:
0264-6021
Resumen:
We analysed the inhibitory effects in vitro and in vivo of several metal
ions on aldosterone binding to the rat kidney mineralocorticoid
receptor with the purpose of assessing possible toxic effects of those
ions on sodium retention, as well as to obtain information on receptor
structural requirements for ligand binding. For the assays in vitro, the
inhibitory effects of 20 metal ions were analysed on steroid-binding
capacity for renal receptor cross-linked to 90-kDa heat-shock protein
(hsp90) by pretreatment with dimethyl pimelimidate. Cross-linking
prevented the artifactual dissociation of hsp90 (and, consequently, the
loss of steroid binding) from the mineralocorticoid receptor due to the
presence of high concentrations of salt in the incubation medium.
Cross-linked heterocomplex showed no difference in ligand specificity
and affinity with respect to native receptor, but increased stability
upon thermal- or ionic-strength-induced destabilization was observed.
Treatments in vitro with metal ions in the range 10(-8)-10(-1) M
resulted in a differential inhibitory effect for each particular ion on
aldosterone binding. Using the negative logarithm of metal concentration
for 50% inhibition, the ions could be correlated with their Klopman
hardness constants. The analysis of this relationship led us to
postulate three types of reaction: with thiol, imidazole and carboxyl
groups. The essential role played by these residues in steroid binding
was confirmed by chemical modification of cysteines with
dithionitrobenzoic acid, histidines with diethyl pyrocarbonate and
acidic amino acids with Woodward's reagent
(N-ethyl-5-phenylisoxazolium-3'-sulphonate). Importantly, the toxic
effects of some metal ions were also observed by treatments in vivo of
adrenalectomized rats on both steroid-binding capacity and
aldosterone-dependent sodium-retaining properties. We suggest that those
amino acid residues are involved in the activation process of the
mineralocorticoid receptor upon steroid binding. Thus toxic effects
observed with these metal ions may be a consequence of modifications of
those essential groups. Our results support the notion that toxicity of
metals on renal mineralocorticoid function may be predicted according to
their chemical hardness.