Purification of luteinizing hormone from sheep pituitary glands and evidence for several physicochemically distinguishable active components

Abstract

Listen

Translate article

1. 1. A highly purified preparation of luteinizing hormone has been obtained from sheep pituitary glands by extraction with ethanol, metaphosphoric acid and (NH 4) 2SO 4 fractionations, and ion-exchange chromatography on CM-Sephadex. This luteinizing hormone preparation designated CM3 contained 1.6 and 1.9 units of NIH-LH-S1 per mg dry wt. as measured by ovarian ascorbic acid depletion bioassays and the ventral prostate bioassay, respectively. The level of follicle-stimulating hormone contamination was approx. 0.025% as judged by augmentation bioassay. 2. 2. Three major luteinizing hormone fractions (Fr 1, Fr 2, Fr 3) were obtained when the purified luteinizing hormone preparation CM3 was chromatographed on hydroxylapatite. Ovarian ascorbic acid depletion bioassays and the ventral prostate bioassay indicated that two of the luteinizing hormone fractions (Fr 2, Fr 3) contained 2.0–2.6 units of NIH-LH-S1 per mg dry wt. Augmentation bioassays and hypophysectomized female rat bioassays indicated the low level of follicle-stimulating hormone contamination in the preparation CM3 was concentrated in Fr 1 after hydroxylapatite chromatography. Therefore, the highly active luteinizing hormone fractions (Fr 2, Fr 3) were essentially free of follicle-stimulating hormone contamination. 3. 3. Physicochemical analyses of the three luteinizing hormone fractions, as well as the purified luteinizing hormone preparation CM3, were conducted employing gel filtration, ultracentrifugation, analytical acrylamide disc gel electrophoresis, preparative acrylamide disc gel electrophoresis, electrofocusing, amino acid analysis, carbohydrate analyses, and immunodiffusion in agar gels. Perceivable differences among the three luteinizing hormone fractions could be detected with analytical acrylamide disc gel electrophoresis, preparative acrylamide disc gel electrophoresis, and electrofocusing. Moreover, amino acid and carbohydrate analyses suggested there might be differences in the amino acid and carbohydrate compositions among the luteinizing hormone fractions. Dissimilarity in molecular size and immunological specificity was not detected. In addition to the differences found among the luteinizing hormone fractions, data are presented which show that electrophoretically distinguishable luteinizing hormone components were contained within two fractions (Fr 1, Fr 2), as well as preparation CM3. 4. 4. These results suggest a broad spectrum of physicochemically similar luteinizing hormone components which differ from one another to the degree necessary for chromatographic and electrophoretic resolution.