Binding of hormone to tissue: the first step in polypeptide hormone action.

Polypeptide hormones produce a wide range of metabolic and morphologic responses in specific target tissues.' Clearly the initial interaction of hormone with sensitive tissue must be binding of the hormone to some cellular constituent. To delineate this interaction we have studied the effects of thyroid-stimulating hormone (TSH) on thyroid slices and of insulin on skeletal muscle. Tissues that were exposed to hormone and washed thoroughly in hormone-free medium showed a persistent hormone effect. However, when the washed tissues were subsequently exposed to antibody or to trypsin, this effect was obliterated. Thus, the persistent hormone effect was due to the continued presence of relatively intact hormone at a readily accessible tissue site. Experimental Procedure.-Bovine TSH at 4 IU/mg (#1493A) and at 20 IU/mg, and human TSH at 5 IU/mg were gifts of Dr. Peter Condliffe, NIH. During this study TSH 1493A lost some activity which was not corrected for in the presentation of the data. Rabbit gamma globulin (RGG), purified on DEAE-cellulose, was a gift of Dr. Henry Metzger, NIH. Crystalline trypsin was purchased from Worthington, and soybean trypsin inhibitor from Sigma. Bovine serum albumin (BSA, Armour) and RGG were labeled with Il25 at specific activities of 0.2 mc/mg and bovine TSH (20 IU/mg) with I'll at 20 mc/mg.2 Radioactive contaminants were removed from BSA-1125 and RGG-1125 by batch adsorption to Dowex 1X10, and from TSH-I131 by filtration on Sephadex G-100.3 On chromatoelectrophoresis in 0.05 M phosphate, pH 7.4, at 500 v at 40 on Whatman 3MM chromatography paper,4 over 95% of the radioactivity of each of the labeled proteins migrated appropriately as a single peak. Substantially less than 1% of the radioactivity was iodide. At least 80% of the TSH-Il3l was bound by TSH antibody. Labeled proteins were stored at -20? aind used within 72 hr of preparation. Antibodies were produced in a burro by four intramuscular injections of 6 mg of bovine TSH (4 IU/mg) in complete Freund's adjuvant (Difco). Antibody was prepared from citrated plasma by defibrination with calcium and thrombin and precipitation at 230 with ammonium sulfate at 40% saturation. The guinea pig anti-insulin serum (Suburban Laboratories) was similarly purified. Hereafter antiserum refers to the purified gamma globulin. The anti-TSH serum at a final dilution of 1: 5 neutralized bovine TSH at 50 mU/ml but had little effect on human TSH. The anti-insulin serum at a 1: 20 dilution neutralized insulin at 10 mU/ml. Neutralization indicates that a solution of hormone and antibody, allowed to mix for 30 min, failed to stimulate glucose1-C14 oxidation in thyroid or glucose-1-C14 incorporation into rat diaphragms. An aliquot of burro anti-TSH serum was treated with papain (Worthington) at pH 6 for 5 hr.5 Papain was inactivated by iodoacetamide, and Porter fraction III was precipitated by dialysis in water and removed by centrifugation. Simultaneous with the preparation of each antiserum, serum from an unimmunized animal was treated identically and served as control throughout. All sera were dialyzed against Krebs-Ringer bicarbonate before use. The buffer for in vitro experiments was Krebs-Ringer bicarbonate pH 7.4 containing bovine fraction V (fr. V, Armour) at 5 mg/ml. Routinely, dog thyroid slices weighing 15-30 mg were preincubated with bovine TSH (4 IU/mg) in 2-5 ml of buffer anid washed three times by agitation for 1 m inn 10 ml of hormone-free buffer at 1?. The slices were then blotted, weighed, and incubated at 370 in 1 ml of buffer containing 1 mg of glucose and 0.5 ,ic of glucose-i-C14. The C1402 was collected in hyamine.6 Data are expressed as cpm/mg of thyroid slice per hr of incubation. The thyroid of a single normal dog was used for each experiment. The effect of insulin on glucose utilization in the rat diaphragm was studied by a modification