Purification and characterization of the thyrotropin-releasing hormone (TRH)-degrading serum enzyme and its identification as a product of liver origin.
Previous biochemical studies have indicated that the membrane-bound thyrotropin-releasing hormone (TRH)-degrading enzyme (TRH-DE) from brain and liver and the serum TRH-DE are derived from the same gene. These studies also suggested that the serum enzyme is of liver origin. The present study was undertaken to verify these hypotheses. In different species, a close relationship between the activities of the serum enzyme and the particulate liver enzyme was noticed. The activity of the serum enzyme decreased when rats were treated with thioacetamide, a known hepatotoxin. With hepatocytes cultured in a sandwich configuration, release of the TRH-DE into the culture medium could also be demonstrated. The trypsin-solubilized particulate liver TRH-DE and the serum TRH-DE were purified to electrophoretic homogeneity. Both enzymes and the brain TRH-DE were recognized by a monoclonal antibody generated with the purified brain enzyme as antigen. Lectin blot analysis indicated that the serum enzyme and the liver enzyme are glycoproteins containing a sugar structure of the complex type, whereas the brain enzyme exhibits an oligomannose/hybrid glycostructure. A molecular mass of 97 000 Da could be estimated for all three enzymes after deglycosylation and SDS/PAGE followed by Western blotting. Fragment analysis of the serum TRH-DE revealed that the peptide sequences correspond to the cDNA deduced amino-acid sequences of the membrane-bound brain TRH-DE, whereby two peptides were identified that are encoded by exon 1. These data strongly support the hypothesis that the TRH-DEs are all derived from the same gene, whereby the serum enzyme is generated by proteolytic cleavage of the particulate liver enzyme.[1]References
- Purification and characterization of the thyrotropin-releasing hormone (TRH)-degrading serum enzyme and its identification as a product of liver origin. Schmitmeier, S., Thole, H., Bader, A., Bauer, K. Eur. J. Biochem. (2002) [Pubmed]
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