The Microheterogeneity of Rat Androgen-Binding Protein from the Testis, Rete Testis Fluid, and Epididymis, as Demonstrated by Immunoelectrophoresis and Photoaffinity Labeling*

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Abstract
Studies on the microheterogeneity of rat androgen-binding protein (rABP) were conducted using crossed immunoelectrophoresis (CIE) and photoaffinity labeling. rABP exhibits two molecular weight components of 45,000 (45K) and 41,000 (41K) which are designated heavy (H) and light (L) according to their mobilities during electrophoresis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two-dimensional electrophoresis of epididymal rABP revealed that the charge heterogeneity of the H differed from that of the L component. On the other hand, when highly purified rABP from rat epididymis was fractionated by CIE using isoelectric focusing, followed by electrophoresis into antibody-containing agarose gel, three species with identical immunological determinants were demonstrated. CIE was then used as a convenient technique to study partially purified samples from testis, rete testis fluid, and different portions of the epididymis. The results indicated that there is a loss of more acidic and an appearance of more basic species of rABP as this protein moves from testis to cauda epididymis. Photolysis of purified rABP from testis and epididymis with [3H]Δ6-testosterone ([l,2-3H]17β-hydroxy-4,6-androstadien-3- one) resulted in the specific labeling of its binding site. Analysis of photolabeled products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two radioactive specific products with electrophoretical mobilities identical to those of the H and L components of native rABP. These labeling patterns were invariably present in the different portions of the reproductive tract. Experiments using isoelectric focusing of photolabeled rABP obtained from the testis and different portions of the epididymis confirmed the observation obtained by CIE. We conclude from these studies that: 1) the results support previous postulates that rABP exists as a dimeric hybrid composed of combinations of the H and L components; 2) both the H and L components exhibit chrge as well as size heterogeneity; 3) native rABP has a variable charge heterogeneity which is dependent upon its localization in the male reproductive tract; and 4) the isoelectric species of rABP have common antigenic determinants.