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.
Schmitmeier
Stephanie
S
Max-Planck-Institut für experimentelle Endokrinologie, Hannover, Germany.
Thole
Hubert
H
Bader
Augustinus
A
Bauer
Karl
K
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001798:Blood Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D051381:Rats; D020539:Sequence Analysis, Protein; D013552:Swine; D013853:Thioacetamide / Q000494:pharmacology; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*; D014018:Tissue Distribution
D001798:Blood Proteins; D006023:Glycoproteins; D010446:Peptide Fragments; D013853:Thioacetamide; D013973:Thyrotropin-Releasing Hormone; D000626:Aminopeptidases; C023166:pyroglutamyl-peptidase II; D011761:Pyrrolidonecarboxylic Acid