A study of a synaptosomal thyrotropin releasing hormone-inactivating pyroglutamate aminopeptidase from bovine brain.
Pyroglutamate aminopeptidase type II is a highly specific membrane-bound neuropeptidase that has the ability to remove N-terminal pyroglutamate (Glp) from Thyrotropin Releasing Hormone (Glp-His-Pro-NH2) or very closely related tripeptides or tripeptide amides. In this paper we report on the purification and characterisation of a pyroglutamate aminopeptidase activity from the synaptosomal membranes of bovine brain. The Triton X-100 solubilised enzyme was purified nearly 600-fold by a combination of conventional column chromatography steps with a recovery/yield of 17.0%. Phase-partitioning experiments with Triton X-114 showed the activity to be an integral membrane protein. This detergent-solubilised pyroglutamate aminopeptidase activity was found to have a relative molecular mass of 240 kDa on a calibrated S-200 column. HPLC analysis on a C18 reverse-phase column showed that the purified activity displayed a very narrow substrate specificity cleaving only Thyrotropin Releasing Hormone (TRH) or the very closely related acid-TRH, LHRH (1-3) and the TRH-analogue (methyl-His)-TRH and had a Km of 100 microM for the fluorimetric substrate Glp-His-Pro-methyl-coumarin. The enzyme was inactivated by the metalchelator 1,10-ortho-phenanthroline but showed less sensitivity to EDTA. It also showed some inhibition by thiol protease inhibitors such as iodoacetate and n-ethyl-maleimide. In summary, we have purified a pyroglutamate aminopeptidase from the synaptosomal membrane of bovine brain. This enzyme displays characteristics consistent with it being classified as a PAP type II neuropeptidase with only minor differences from other proteases in this group.
O'Leary
R M
RM
School of Biological Sciences, Dublin City University, Ireland.
O'Connor
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology / Q000378:metabolism*; D002417:Cattle; D008969:Molecular Sequence Data; D011751:Pyroglutamyl-Peptidase I / Q000037:antagonists & inhibitors / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism*; D013570:Synaptic Membranes / Q000201:enzymology; D013574:Synaptosomes / Q000201:enzymology*; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*
D013973:Thyrotropin-Releasing Hormone; D011751:Pyroglutamyl-Peptidase I