GONADOTROPINS OF THE SWINE PITUITARY II. PREPARATION, AND BIOLOGICAL AND PHYSICO-CHEMICAL CHARACTERIZATION OF A PROTEIN APPARENTLY IDENTICAL WITH METAKENTRIN (ICSH)

Abstract

II. A simplified procedure to isolate the protein metakentrin by isoelectric precipitation at pH = 7.4 in 1/3 saturated (NH4)sSO4 soln. is described. Attempts were made to separate a highly active, hypothetical substance from the homogeneous protein by fractionation with salts, by ultracentrifugation and by electrophoresis. Biological assays of the fractions obtained by any one of the above-mentioned methods not only failed to furnish any evidence of the presence of an impurity more active than the homogeneous protein, but also showed that all these fractions have constant biological activity per given wt. of the protein. Therefore it is believed that the protein is the hormone. A comparison of the electrophoretic behavior between pure swine metakentrin and a highly purified sheep metakentrin showed that the former has an isoelectric point of pH = 7.45 whereas that of the latter is about pH = 4.8 to 5.0[long dash]III. Metakentrin (ICSH) isolated from the hog pituitary gland in a pure state, as shown by electrophoretic or ultracentrifugal analysis or by solubility measurement, stimulated the production of antibodies in rabbit sera. The presence of antibodies was demonstrable by both the precipitin and complement-fixation reactions. Hog metakentrin anti-serum of rabbits reacted only with crude or pure metakentrin from hog pituitary glands but not with metakentrin extracts prepared from pituitary glands of sheep or oxen. The anti-serum of hog metakentrin did not react with other hormones from different or even the same lobes of the pituitary gland of the same sp. of animals. Hog metakentrin is therefore "species-," "lobe-" and "hormone"-specific.