Active-site-directed photoaffinity radioiodination of androgen-binding proteins.
Androgen-binding protein (ABP) in rat epididymal cytosol and sex hormone-binding globulin (SHBG) in rabbit serum and SHBG purified from human serum were active-site-directed photoaffinity radiolabeled with 17 alpha-[(E)-2-[125I]iodoethenyl]androstan-4,6-dien-17 beta-ol-3-one ([125I]1). The interaction of this compound with binding components in epididymal cytosol was dependent on exposure of the mixture to ultraviolet light and on the duration of exposure. Photolysis in the presence of [125I]1 and 5 alpha-dihydrotestosterone (5 alpha-DHT) resulted in a 40% inhibition of binding of [125I]1 to cytosolic components. These result indicate that, while [125I]1 interacted with 5 alpha-DHT binding sites, it also formed adducts with other sites. To characterize the labeled species, the photolysis mixture was subjected to electrophoresis under denaturing and reducing conditions. Autoradiography of the gel revealed that ABP and SHBG were labeled with [125I]1, but in cytosol and serum, higher and lower molecular weight components were also labeled. Purified SHBG was labeled, but no labeled contaminating protein was detected. The presence of 5 alpha-DHT completely inhibited [125I]1 photolabeling of human and rabbit SHBG and of ABP. However, in cytosol, the presence of 5 alpha-DHT also eliminated photolabeling to a component that may be albumin, but 5 alpha-DHT did not affect [125I]1 photolabeling of other contaminating proteins in cytosol. Thus, while [125I]1 is an effective photoaffinity radiolabel for ABP and SHBG, the observation that it also photolabels other proteins limits its practical use to the radiolabeling of purified ABP and SHBG preparations.
Diaz Cruz
P J
PJ
Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, USA.
Danzo
B J
BJ
Smith
H E
HE
D000345:Affinity Labels; D000727:Androgen-Binding Protein / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D013196:Dihydrotestosterone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis / Q000737:chemistry; D006801:Humans; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D008297:Male; D010782:Photolysis; D011817:Rabbits; D051381:Rats; D012738:Sex Hormone-Binding Globulin / Q000737:chemistry / Q000378:metabolism*
D000345:Affinity Labels; D000727:Androgen-Binding Protein; D007457:Iodine Radioisotopes; D012738:Sex Hormone-Binding Globulin; D013196:Dihydrotestosterone; C078296:17-(2-iodoethenyl)androsta-4,6-dien-17-ol-3-one