title: Characterization of cytosolic glucocorticoid receptor of fetal rat epiphyseal chondrocytes.
abstract: The cytosolic glucocorticoid receptor of 21st gestational day rat epiphyseal chondrocytes has been evaluated. The receptor, a single class of glucocorticoid binding component approached saturation, utilizing [3H]triamcinolone acetonide ([3H]TA) as the radiolabeled ligand, at approximately 1.8-2.0 x 10(-8) M. The dissociation constant (Kd) reflected high-affinity binding, equaling 4.0 +/- 1.43 x 10(-9) M (n = 7) for [3H]TA. The concentration of receptor estimated from Scatchard analysis was approximately 250 fmol/mg cytosolic protein and when calculated on a sites/cell basis equalled 5800 sites/cell. The relative binding affinities of steroid for receptor were found to be triamcinolone acetonide greater than corticosterone greater than hydrocortisone greater than progesterone greater than medroxyprogesterone acetate much greater than 17 alpha-hydroxyprogesterone much greater than testosterone greater than 17 beta-estradiol. Cytosolic preparations activated in vitro by warming (25 degrees C for 20 min) were shown to exhibit an increased affinity for DNA-cellulose. 46% of the total specifically bound activated ligand-receptor complex was bound to DNA-cellulose. Cytosol maintained at 0-4 degrees C in the presence of 10 mM molybdate or activated in vitro in the presence of molybdate, bound to DNA-cellulose at 8 and 10% respectively. DEAE-Sephadex elution profiles of the nonactivated receptor were indicative of a single binding moiety which eluted from the columns at 0.4 M KCl. Elution profiles of activated receptor were suggestive of an activation induced receptor lability. The 0.4 M KCl peak was diminished, while a concomitant increase in the 0.2 M KCl peak was only modestly discernible. Evaluation of endogenous proteolytic activity in chondrocyte cytosol using [methyl-14C]casein as substrate show a temperature-dependent proteolytic activity with a pH optimum of 5.9-6.65. The proteolytic activity was susceptible to heat inactivation and was inhibitable, by 20 mM EDTA. The sedimentation coefficient of the nonactivated receptor was 9.3s (n = 6) on sucrose density gradients and exhibited steroid specificity and a resistance to activation induced molecular alterations when incubated in the presence of 10 mM molybdate. Receptor activation in vitro, in the absence of molybdate induced an increased receptor susceptibility to proteolytic attack and/or enhanced ligand receptor dissociation as evidenced by a diminution of the 9.3s binding form without a concomitant increase in 5s or 3s receptor fragments.
authors.affiliation: Stein Research Center, Thomas Jefferson University, Phildelphia, PA.
authors.forename: J P
authors.initials: JP
authors.lastname: Carbone
authors.forename: R C
authors.initials: RC
authors.lastname: Baldridge
authors.forename: T R
authors.initials: TR
authors.lastname: Koszalka
authors.forename: A M
authors.initials: AM
authors.lastname: Bongiovanni
authors.forename: R L
authors.initials: RL
authors.lastname: Brent
mesh_terms: D000818:Animals; D002154:Calpain / Q000032:analysis / Q000502:physiology; D002356:Cartilage / Q000032:analysis* / Q000166:cytology; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000032:analysis; D004492:Edetic Acid / Q000494:pharmacology; D004838:Epiphyses / Q000032:analysis; D005260:Female; D005333:Fetus / Q000378:metabolism; D007700:Kinetics; D008666:Metalloendopeptidases; D010447:Peptide Hydrolases / Q000032:analysis; D011247:Pregnancy; D051381:Rats; D011919:Rats, Inbred Strains; D011965:Receptors, Glucocorticoid / Q000032:analysis*; D014222:Triamcinolone Acetonide / Q000378:metabolism
chemical_list: D011965:Receptors, Glucocorticoid; D004492:Edetic Acid; D010447:Peptide Hydrolases; D002154:Calpain; D008666:Metalloendopeptidases; C030895:caseinase; D014222:Triamcinolone Acetonide