Fish Gonadotropin(s). II. Isolation of Gonadotropin(s) from Chum Salmon Pituitary Glands Using Affinity Chromatography

Abstract

A highly purified gonadotropic hormone preparation has been obtained from chum salmon (Oncorhynchus keta) pituitaries by extraction with ethanolic or aqueous buffer, affinity chromatography on Con A Sepharose and gel filtration on Sephadex G-75 superfine. A purified fraction from Sephadex G-75 averaged 448 μg NIH-LH-S18/mg glycoprotein as measured by the uptake of radiophosphate into chick testes. A total of 1.1 g of salmon gonadotropin (s) (SG)/kg fresh tissue was recovered when the isolation began with an aqueous extraction.

Analytical polyacrylamide gel electrophoresis (P.A.G.E.) of the purified fraction from Sephadex G-75 displayed a single broad zone in non-dissociating conditions and two bands in 8 M urea. Polyacrylamide gel electrofocusing yielded six sharp bands with an isoelectric point range of 4.38 to 5.05, and four bands with an isoelectric point range of 4.31 to 4.95 in 8 M urea. A molecular weight of 41,000 was determined by gel. filtration. A subunit molecular weight of 17,800 ± 10% was found by P.A.G.E. in 0.1% sodium dodecyl sulphate (SDS), suggesting that native SG consists of two subunits. Purified preparations were highly stable in Tris-Cl buffers and retained their activity for several months when stored at-73°C