Characterization of two uterine proteases and their actions on the estrogen receptor

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Abstract
Two previously undetected proteases from the calf uterine cytosol were characterized and their actions on the estrogen receptor were measured. One is an exopeptidase, purified 60-fold, that hydrolyzed amino acid (lysine-, and alanine-, or leucine-) p-nitroanilide substrates and leucyl-glycylglycine, did not hydrolyze [14C]methemoglobin, was completely inhibited by 1 mM bestatin or puromycin (specific inhibitors of leucine aminopeptidase-like enzymes), and was unable to influence the sedimentation of the 8S form of the estrogen receptor in sucrose gradients containing dilute Tris buffer. A commercial porcine leucine aminopeptidase, like the calf uterine aminopeptidase, did not convert the 8S estrogen receptor to a 4S form. Evidently, removal of the N-terminal amino acid(s) from the estrogen receptor by exopeptidase action cannot alter the sedimentation of the 8S form of the receptor, or the N-terminal amino acid(s) of the receptor is (are) unaccessible or resistant of exopeptidase activity. The second, a receptor-active protease, is an endopeptidase that did not hydrolyze any of the synthetic amide or peptide substrates tested but did possess [14C]methemoglobin-degrading activity and the ability to convert the 8S estrogen receptor to a modified 4S form in sucrose gradients containing dilute Tris buffer. The modified 4S receptor was separable from the native receptor by DEAE-cellulose chromatography. The endopeptidase did not require Ca2+ for activity, and its chromatographic properties were distinctly different from a previously isolated Ca2+-activated protease. It was inhibited by leupeptin or dipyridyl disulfide, suggesting the presence of a thiol group that is essential for its activity. A decrease in the sedimentation rate of the estrogen receptor in sucrose gradients with low salt or a change in the receptor''s elution on DEAE-cellulose chromatography is not related to receptor activation but is produced by the receptor-active protease or other proteases.

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