Multiple Forms of Rat Testicular Androgen Binding Protein: Physicochemical Properties and the Effect of Ca++

Abstract

The presence of two forms of androgen binding protein (ABP) was confirmed in rat testicular cytosol. Each was distinctly separated from the other by DEAE‐cellulose chromatography and showed a single peak with a slightly different Rf on polyacrylamide gel electrophoresis (PAGE) (0.49 ± 0.03 (mean ± SD) for one (ABP I) and 0.55 ± 0.04 for the other (ABP II)). ABP I retained 90% of its binding capacity for dihydrotestosterone for 2 hours at 60 C, whereas ABP II lost more than 90% of its binding capacity within 10 minutes at 60 C. Other physicochemical properties of the two were very similar: identical values of Einstein's stokes radius of 47 A; sedimentation coefficient of 4.6–4.7 S; 5–8 minutes as half dissociation time of [3H]‐dihydrotestosterone‐ABP complex; identical elution positions from Sephadex G‐200 chromatography; and association constants for dihydrotestosterone of 2.1 × 108 M −1 for ABP I and 4.0 × 108 M 1 for ABP II. The order of binding affinity of the two forms was dihydrotestosterone > testosterone > estradiol‐17β >progesterone. The presence of Ca++ in the elution buffer caused the two forms to elute at lower ionic concentration off DEAE‐cellulose. This was reversed by the removal of Ca++ with the addition of (ethylenebis (oxyethylene nitrilo)) tetraacetic acid.