title: Purification of putative thyroid hormone receptor from the ovarian nuclei of fresh water perch, Anabas testudineus.
abstract: Perch ovarian putative T3 (3,5,3'-triiodo-L-thyroxine) receptor was purified to 580-fold by extracting the ovarian nuclear preparation with 0.4 M KCl, gel filtration on Sephadex G-25, DEAE-Sephacel chromatography and FPLC Superose 6 chromatography. To monitor the T3 binding protein at each purification step, aliquots from each peak protein fractions were incubated with (125)I-T3 (0.16 pmol to 3.2 nmol) in the absence or presence of 500-fold excess of unlabelled T3. Maximum binding capacity (Bmax) obtained from the Scatchard plot analysis was estimated to determine the extent of purification at each step. Purified putative T3 receptors showed a single band in polyacrylamide gel electrophoresis (PAGE) indicating homogeneity of the putative receptor protein. The molecular weight of the putative T3 receptor protein, as determined on a FPLC Superose 6 column, was 50 kD. Treatment of putative T3 receptor protein with β-mercaptoethanol followed by SDS-PAGE resulted in two subunits of 26 and 31 kD. Purification increased the specific activity of the receptor, but did not alter its affinity. Analogue specificity of the purified receptor corresponded to that of the crude nuclear preparation. Triiodothyroacetic acid (Triac) and T3 equally competed in inhibiting radiolabelled T3 binding while thyroxine (T4) was a poor competitor. T3 receptor antiserum crossreacted with the receptor protein. (125)I-labelled receptor protein binding with its antiserum was inhibited by increasing logarithmic concentrations of unlabelled receptor. In contrast to earlier reports on hepatic T3 receptor, which is a monomer, present investigation demonstrated T3 binding protein in the perch ovary to be a heterodimer held together by disulphide bond.
authors.affiliation: Department of Zoology, Visva-Bharati University, Santiniketan-731 235, W. Bengal, India.
authors.forename: A
authors.initials: A
authors.lastname: Bandyopadhyay
authors.forename: S
authors.initials: S
authors.lastname: Bhattacharya