Enzymic synthesis of steroid sulphates. XV. Structural domains of oestrogen sulphotransferase.

Pure preparations of oestrogen sulphotransferase (3'-phosphoadenylylsulphate:oestrone sulphotransferase, EC 2.8.2.4), exhibiting the normal four-isoenzyme pattern on gel electrophoresis, revealed limited proteolytic splits in the protein chain when examined by SDS-polyacrylamide gel electrophoresis. In addition to the normal 74000 molecular weight (74 kDa) protein band, an additional major band was seen at 36 kDa, often accompanied by band at 24kDa and 12kDa. Such preparations, either alone, or after reduction and S-carboxymethylation, showed an extremely strong resistance to dissociation, a concentration of 2% SDS being required for dissociation on Sephadex G-100 column chromatography. These lower molecular weight fragments, isolated by several techniques employing dissociative conditions, all showed a remarkable ability to reassociate to a species of approx. Mr 70000 when examined by SDS-polyacrylamide gel electrophoresis. In addition, the 12kDa fragment yielded dimeric, trimeric, tetrameric, pentameric and hexameric forms. Results of amino acid analyses and tryptic digestion fingerprints of the 36kDa, 24kDa and 12kDa fragments, in conjunction with N-terminal amino acid determinations, suggested in initial protease cleavage at a susceptible region midway in the chain. One, or both, of the resultant 36kDa lobes was then further attacked to yield the 12kDa and 24kDa species - the latter again being capable of cleavage to two 12kDa species. Tryptic maps indicated that the 12kDa, 36kDa and 72kDa species were discrete polypeptide chains and not composed of subunits of the 12kDa species. These data suggest that the enzyme contains a number of domains and that strong interaction occurs between them. If these domains possess oestrogen-binding properties this would explain the most unusual wave-like kinetics exhibited by the enzyme consistent with a rate equation of degree greater than 4. Such properties also provide evidence further to that previously reported which suggests that the enzyme may be genetically related to serum albumin.