Purification and characterization of a thyrotropin-releasing hormone deamidase from rat brain

Publisher Website
Google Scholar
Abstract
The purification of a rat brain thyrotropin-releasing hormone (TRH) deamidating enzyme to apparent homogeneity was described. Criteria for purity included sodium dodecyl sulfate [SDS] and disc gel electrophoresis, as well as isoelectric focusing (pI [isoelectric point] = 4.5). Enzyme purification was facilitated by development of a rapid and sensitive continuous assay using the substrate L-pyroglutamyl-Nim-benzylhistidyl-L-prolyl-.beta.-naphthylamide, which, upon hydrolysis of the naphthylamide, resulted in the appearance of the fluorescent product, .beta.-naphthylamine (.beta.NA). With this substrate the homogeneous enzyme had a specific activity of 14.5 .mu.mol of .beta.NA min-1 mg-1. The only peptide product formed was L-pyroglutamyl-Nim-benzylhistidyl-L-proline. Hydrolysis of [L-prolyl-2,3-3H]TRH yielded L-pyro-glutamyl-L-histdyl-L-proline as the only radiolabeled product. Characterization of the brain deamidase by gel filtration chromatography and SDS gel electrophoresis indicated that the enzyme consisted of a single polypeptide chain having MW of 70,000 and 73,500, respectively. Rat brain TRH deamidase had an apparent Km of 34 .mu.M, and a pH optimum between 7 and 8 using L-pyroglutamyl-Nim-benzylhistidyl-L-prolyl-.beta.-naphthylamide as a substrate. With this substrate, TRH was a competitive inhibitor with an apparent Ki [inhibition constant] of 120 .+-. 20 .mu.M.