Purification and Characterization of the Thyrotropin‐releasing‐hormone‐degrading Ectoenzyme

The membrane‐bound enzyme which catalyzes the degradation of thyrotropin‐releasing hormone (TRH; Glp‐His‐Pro‐NH2) could be released from membranes of rat and pig brain by treatment with trypsin under very mild incubation conditions. The solubilized enzyme was purified 200000‐fold, with an overall yield of 20%, by conventional chromatographic methods.The enzyme preparation appeared to be electrophoretically homogenous since SDS/PAGE analysis revealed a single band with a molecular mass of 116000 Da. By gel‐filtration chromatography, a molecular mass of 230000 Da was estimated, suggesting that the enzyme consists of two identical subunits.The enzyme could be identified as a glycoprotein by lectin‐binding analysis and by the reduction of the molecular mass to 97000 Da upon treatment of the denatured enzyme with endoglycosidase‐F/N‐glycosidase F. In its native form, however, the enzyme was only partially deglycosylated and retained full enzymic activity.In addition to TRH, the enzyme also hydrolyzed l‐5‐oxoprolyl‐β‐naphthylamide, and thus a convenient fluorimetric assay could be established to determine high enzyme activities. The hydrolysis of both substrates was found to obey Michael