Isolation and Properties of Subunits of Bovine Pituitary Luteinizing Hormone

• Published in: The Journal of biological chemistry 1969-10, Vol.244 (19), p.5110-5117
• Main Authors: Reichert, Jr, L E, Rasco, M A, Ward, D N, Niswender, G D, Midgley, Jr, A R
• Format: Article
• Language: English
• Subjects: Amino Acids - analysis; Animals; Antigens; Arginine - analysis; Biological Assay; Carbohydrates - analysis; Carboxypeptidases; Cattle; Chromatography, Gel; Countercurrent Distribution; Dextrans; Diffusion; Electrophoresis, Disc; Immune Sera; Leucine - analysis; Luteinizing Hormone - analysis; Luteinizing Hormone - isolation & purification; Lysine - analysis; Methods; Molecular Weight; Pituitary Gland - analysis; Proline - analysis; Radioimmunoassay
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)63634-8

Subunits of bovine pituitary luteinizing hormone (LH) have been isolated by countercurrent distribution, using a modification of the system employed by Papkoff and Samy ( Biochim. Biophys. Acta , 147, 175 (1967)) in studies on ovine LH. The subunit having a low partition coefficient was designated C-1, and that with a high partition coefficient, C-2. Gel filtration data indicated intact LH to have a Stokes radius of 2.76 mµ and a molecular weight of 29,258. Each subunit had a Stokes radius of 2.28 mµ and a molecular weight of 14,609. Diffusion coefficients and frictional ratios were also calculated. There were significant differences in chemical composition between the subunits. Subunit C-2 had 2.64 times as much proline and 3.19 times as much leucine as did C-1. The lysine to arginine ratio for C-1 was 3:1, while for C-2 it was 1:3. In addition, the ratio of hydrophobic to hydrophilic amino acids for subunit C-2 was practically double that found in C-1. No NH 2 -terminal amino acids could be found for either subunit, but the COOH-terminal amino acids appeared to be serine for C-1 and leucine for C-2, on the basis of carboxypeptidase A digestion and hydrazinolysis studies. Each subunit possessed definite biological and immunological activity. The biological activities of C-1 and C-2 were 30% and 3.9% of intact LH. The immunological activities of C-1 and C-2 were 24% and 2% of intact LH. It was possible to achieve a reassociation of the subunits by incubation in 0.01 m phosphate buffer, pH 7.0, for 16 hours at 40°. This reassociation could be demonstrated by gel filtration and biological measurements. The physical properties of the recombined C-1 plus C-2 subunit molecule were essentially identical with those of intact LH. When combined in a 1:1 ratio by weight, the biological potency of the recombined molecule was augmented 3.35 times over that predicted on the basis of individual subunit potencies. Immunological potencies of the recombined molecule were augmented 3.42 times over predicted values. When compared to intact LH, recovery of biological activity was 57% and immunological activity, 44%.