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 200,000-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 116,000 Da. By gel-filtration chromatography, a molecular mass of 230,000 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 97,000 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 enzymatic activity. In addition to TRH, the enzyme also hydrolyzed L-5-oxoprolyl-beta-naphthylamide, and thus a convenient fluorimetric assay could be established to determine high enzyme activities. The hydrolysis of both substrates was found to obey Michaelis-Menten kinetics, but considerable differences in the respective Km and Vmax values were noticed.
Bauer
K
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Max-Planck-Institut für experimentelle Endokrinologie, Hannover, Germany.
D000626:Aminopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D010450:Endopeptidases / Q000378:metabolism; D007700:Kinetics; D037102:Lectins; D046911:Macromolecular Substances; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D051381:Rats; D013379:Substrate Specificity; D013552:Swine
D037102:Lectins; D046911:Macromolecular Substances; D011480:Protease Inhibitors; D010450:Endopeptidases; D000626:Aminopeptidases; C023166:pyroglutamyl-peptidase II; D011761:Pyrrolidonecarboxylic Acid