Growth Hormone Receptors in Testis and Liver: MATERIALS AND METHODS(3)

20 Nov

Five micrograms rtGH was radiolabeled with 0.5 mCi 125I-Na (IMS 30; Amersham, Les Ulis, France) by the chlo-ramine T method , with the modification introduced by Martal . The specific activity of 125I-rtGH, calculated by self-displacement with hepatic membrane preparation (5 mg of pellet per tube), was 20 (iCi/mg for studies on testis and 46 |xCi/mg for studies on liver. Maximum binding activity (MBA) of 125I-rtGH, estimated with an excess amount of liver membrane preparation, ranged from 60% to 65% of total added hormone. The labeled hormone was stable for about 3 wk when stored in glycerol (1:1, v:v) at -20°C. cialis professional

Tissue Preparations

Crude testicular fractions were obtained according to the method of crude hepatic preparation of Yao et al. with some modifications. The entire procedure involving tissue preparation was conducted at 0-4°C using chilled buffers. Testes were minced finely by hand and homogenized (1:5, w:v) with a Polytron (Kinematica AG, Luzern, Switzerland) homogenizer (2 X 15 sec, 8000 rpm) in ice-cold homoge-nization buffer (20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 5 mM CaCl2 5, 0.1% NaN3) containing paraaminobenzami-dine (0.25 mg/ml), 4-(2-aminoethyl)-benzenesulfonyl fluoride (0.1 mg/ml), and soybean trypsin inhibitors (0.05 mg/ml). The preparation was further homogenized by being passed through a Dounce homogenizer and centrifuged at 3200 X g for 30 min. The pellet was resuspended in 5 vol of the homogenization buffer and centrifuged at 3200 X g for 30 min. The supernatant was removed, and the Апя! pellet containing crude membrane fraction was weighed and resuspended in ice-cold incubation buffer (homogenization buffer containing soybean trypsin inhibitors [0.05 mg/ml], 0.5% BSA).