Purification of the intact monomeric 110 kDa form of the androgen receptor from calf uterus to near homogeneity.
In the present study, calf uterine tissue has been used for isolation of androgen receptors. This tissue appeared to be a favourable source for large-scale purification of androgen receptors, because of the relatively high level of androgen receptors and the low concentration of proteolytic enzymes. The purification involved differential phosphocellulose and DNA affinity chromatography as first steps. The non-transformed receptor was passed through these matrices in order to remove contaminating DNA-binding proteins. After a transformation step to the DNA-binding state, the receptor was bound to DNA cellulose and subsequently eluted with MgCl2. A 0.5% pure androgen receptor preparation was obtained. Photoaffinity labelling with [3H]R1881 (methyltrienolone) was used to determine the size of the receptor at this stage of purification and during the following steps. Subsequently, isoelectric focussing of the partially purified androgen receptor preparation in an aqueous glycerol gradient was performed. In this step, the progesterone receptor, which is copurified with the androgen receptor protein during the first part of the purification procedure, focussed at pH 5.5 while the androgen receptor could be isolated at pH 5.8. The isoelectric focussing procedure could be applied in a preparative way for further purification of androgen receptors. After this step an approx. 8% pure preparation was obtained. Polyacrylamide gel electrophoresis of S-carboxymethylated androgen receptor was used as the final purification step. The [3H]methyltrienolone labelled androgen receptor from calf uterus was purified to homogeneity and consisted of one polypeptide with a molecular mass of 110 kDa.
van Loon
D
D
Department of Biochemistry (Division of Biochemical Endocrinology), Erasmus University Rotterdam, The Netherlands.
Voorhorst
M M
MM
Brinkmann
A O
AO
Mulder
E
E
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003600:Cytosol / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010777:Photochemistry; D011944:Receptors, Androgen / Q000302:isolation & purification*; D014599:Uterus / Q000032:analysis*
D000345:Affinity Labels; D011944:Receptors, Androgen