Abstract
Abstract Three species of luteinizing hormone (LH) designated as LH-A, -B, and -C, and containing 9, 8, and 4 NIH-LH-S1 units per mg were isolated from human pituitary glands by isoelectric focusing. The LH-A, -B, and -C each sedimented as a single boundary in the ultracentrifuge with s20,w of 2.34, 2.24, and 2.70 S, respectively. Amino acid analyses of LH-A, -B, and -C were very similar, although minor differences, whose significance remains to be determined, were observed. LH-A, -B, and -C contained 13.3, 13.2, and 6.9% of neutral sugars and 0.5, 1.3, and 1.8% of sialic acid, respectively. Minor differences in the physicochemical properties of the three forms of LH suggested microheterogeneity of the LH molecule caused by the modifications in the glycosidic linkages involving labile carbohydrate and sialic acid residues. Incubation of LH in 8 m urea followed by ion exchange chromatography on diethyl amino ethyl Sephadex A-50 resulted in the separation of α and β subunits. The α and β subunits were further purified by gel filtration on Sephadex G-100 and contained 0.8 and 1.1 units of LH activity per mg, respectively. Incubation of the α and β subunits in equal quantities (w/w) showed a 4-fold augmentation of the biological activity thus suggesting the recombination of the subunits. The gel filtration on Sephadex G-100, disc electrophoreses, amino acid analyses, and bioassays indicated that the α and β subunits of LH represent two nonidentical and noncovalently linked components of LH. The α subunits of human LH, follicle-stimulating hormone, and human chorionic gonadotropin could be substituted for each other in combination with the β subunit of each hormone suggesting that the β subunits are hormone specific and the α subunits of LH, follicle-stimulating hormone, and human chorionic gonadotropin are homologous.
Highlights
Isolation of LH-Acetone-preserved human pituitary glands were extracted and purified to obtain a fraction rich in LH and TSH activity [9], which was fractionated by isoelectric focusing in sucrose gradient containing carrier ampholytes of pH range 3 to 10 [2]
LH-A, -B, and -C each showed a single protein band of different electrophoretic mobility, showing the effectiveness of isoelectric focusing in the isolation of proteins with narrow differenees in their isoelectric points
LH-A, -B, and -C each sedimented as a single boundary in the analytical ultracentrifuge with s20,Wof 2.35 f 0.03,2.24 i 0.02, and 2.70 =I=0.01 S which are not widely divergent and are comparable to the ~20,~of LH preparations reported earlier [2, 29]
Summary
Each protein fraction containing LH activity was purified by a second isoelectric focusing (Fig. 1). In present studies, the LH content of the preparations used for bioassay was determined from the human LH standard and the potencies were calculated on weight basis.
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