Use of immobilized metal ion affinity chromatography and dye-ligand chromatography for the separation and purification of rainbow trout pituitary gonadotropins, GTH I and GTH II.
A new procedure is described for the purification of gonadotropic hormones (GTHs) from the pituitary glands of vitellogenic rainbow trout. The procedure utilizes immobilized metal ion affinity chromatography (IMAC) on a column containing immobilized iminodiacetic acid (Toyopearl AF Chelate) charged with Cu2+ ions as a critical step for the efficient separation of GTH I and GTH II. Further purification of both GTH fractions on Cibacron Blue F3GA immobilized on Toyopearl was followed by HPLC size-exclusion for GTH II. The resulting electrophoretically homogeneous preparations possessed a characteristic range of biological activity in the stimulation of steroidogenesis in vitro. N-Terminal sequences of the GTH I and GTH II subunits purified using reversed-phase HPLC revealed a high level of homology with those of GTH subunits found in three other salmonid fish species. In contrast to salmon GTH II, two different alpha-subunits were observed in trout GTH II. The cross-reactivity between GTH I and GTH II was studied in radioimmunoassay using antibodies against Chinook salmon GTH II beta-subunit and rainbow trout GTH I dimer.
Govoroun
M S
MS
Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, Rennes, France.
Huet
J C
JC
Pernollet
J C
JC
Breton
B
B
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004396:Coloring Agents; D004591:Electrophoresis, Polyacrylamide Gel; D006065:Gonadotropins, Pituitary / Q000737:chemistry / Q000302:isolation & purification*; D008670:Metals; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss
D004396:Coloring Agents; D006065:Gonadotropins, Pituitary; D008670:Metals