Purification and properties of an Indian major carp (Cirrhinus mrigala, Ham.) pituitary thyrotropin

Abstract

Thyroid stimulating hormone (TSH) was isolated and purified from the pituitaries of Indian major carp, Cirrhinus mrigala, with the help of a sensitive in vitro bioassay system, based on incubating isolated thyroid follicles from murrel, Channa gachua. In this assay, addition of test material containing TSH increased thyroxine (T₄) release into the medium which was then measured by radioimmunoassay (RIA). TSH activity was eluted as one major peak on Sephadex G-100 gel filtration (peak 1), which also contained gonadotropin (GtH) and extraneous proteins as contaminants. GtH was monitored by RIA. Chromatography of peak I of gel filtration on concanavalin A-Sepharose was useful in harvesting glycoprotein hormones; adsorbed (Con A-II) material showed strong TSH activity and low GtH content. To separate TSH from GtH completely, immunoaffinity chromatography was used; more than a 200-fold purification was achieved. Polyacrylamide gel electrophoresis of carp TSH (cTSH) showed a single band, indicating it to be a homogenous protein. The molecular weight (MW) of cTSH was estimated by Sephadex G-100 gel filtration to be 42.000 Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cTSH revealed two dissimilar subunits, α and β, and MW of α was 21,000 Da while that of β was 24,000 Da. When tested in similar doses, cTSH released more T₄ from murrel thyroid follicle than bovine TSH (bTSH), cTSH stimulated the T₄ release from rat and goat thyroid but its activity was less than that of bTSH. Radiolabeled cTSH (¹²⁵]-cTSH) bound specifically to a murrel thyroid follicular plasma membrane preparation, and Scatchard analysis showed the K_d to be 0.17 × 10^-10M and the maximum binding (B_max) to be 15.2 fmol/mg protein. ¹²⁵ I-cTSH also binds to the goat thyroid plasma membrane preparation with a K_d = 0.23 × 10^-10M and a B_max 9.8 fmol/mg protein. Findings indicate that carp pituitary thyrotropin is approximately similar in size to that of vertebrates, its biological activity is not restricted only to fish but is also observed in rat and goat, and it has a receptor in teleost and goat thyroid.