Localization of a narrow-specificity thyroliberin hydrolyzing pyroglutamate aminopeptidase in synaptosomal membranes of guinea-pig brain.
ABSTRACT In this paper we report the presence of a particulate pyroglutamate aminopeptidase in guinea-pig brain tissue. This activity appears to reside in the synaptosomal membrane and could be released from the membrane by treatment with papain or Triton X-100. By contrast with a previously described broad-specificity soluble pyroglutamate aminopeptidase from guinea-pig brain tissue, the enzyme released from the synaptosomal membrane preparation removed pyroglutamic acid from thyroliberin, acid thyroliberin and less than Glu-His-Gly alone of the peptides tested. Unlike the soluble tissue enzyme the present enzyme was inhibited by the presence of EDTA and the activity released from synaptosomal membranes by papain was found to have a relative molecular mass of 230 000, almost one order of magnitude greater than that reported for the soluble enzyme.
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ABSTRACT: 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 200000-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 116000 Da. By gel-filtration chromatography, a molecular mass of 230000 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 97000 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 enzymic activity.In addition to TRH, the enzyme also hydrolyzed l-5-oxoprolyl-β-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.08/1994; 224(2):387 - 396. DOI:10.1111/j.1432-1033.1994.00387.x
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ABSTRACT: Pyroglutamyl peptidase II (PPII; E.C. 3.4.19.-) is a highly specific membrane-bound ectoenzyme degrading thyrotropin releasing hormone (TRH). The ontogenesis of this enzyme was measured in rat brain regions, adenohypophysis and pancreas. In hypothalamus PPII activity was maximal at day 8 postnatal, decreasing to adult values at day 45. The postnatal ontogenic patterns in posterior cerebral cortex and hypothalamus were similar. In olfactory bulb, two peaks of activity were observed (3th and 22nd day) while in adenohypophysis it appeared only at day 8, increased to day 30, decreasing thereafter to adult values.Developmental Brain Research 05/1992; DOI:10.1016/0165-3806(92)90087-D · 1.78 Impact Factor
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ABSTRACT: Pyroglutamyl aminopeptidase type-1 (PAP-I) is reported to be a soluble, broad specificity aminopeptidase, capable of removing the pyroglutamic acid (pGlu) residue from the amino terminus of pGlu-peptides (e.g. TRH, LHRH, neurotensin and bombesin). The central aim of this study was to undertake, for the first time, the complete purification and characterisation of a PAP activity observed within the cytosolic fraction of bovine whole brain and to compare the properties of the enzyme with previous findings. A series of chromatographic steps (DEAE-Sepharose, Sephacryl S-200 and Activated Thiol Sepharose 4B) generated a soluble PAP activity purified to near homogeneity with a total active yield of 6.6%. The enzyme displayed a native molecular mass of approximately 23,700 Da, which compares well with that value obtained under denaturing conditions via SDS-PAGE (24,000 Da), suggesting that the enzyme exists as a monomer. The expression of PAP activity displayed an absolute requirement for the presence of a disulphide bond-reducing agent such as DTT, whilst optimum activity was observed at pH 8.5. Strong inhibition of PAP activity was observed with a number of different agents, including transition metal ions, sulphydryl-blocking agents and 2-pyrrolidone (a pGlu analog). A broad pyroglutamyl substrate specificity, which excludes substrates commencing with the pGlu-Pro bond, was also demonstrated for the bovine brain enzyme. Based on a comparison of these findings with those reported for PAP-I in other mammalian tissues, the soluble PAP activity observed in bovine whole brain can tentatively be classified as a pyroglutamyl aminopeptidase type-1 (EC 126.96.36.199).The International Journal of Biochemistry & Cell Biology 09/1996; DOI:10.1016/1357-2725(96)00034-9 · 4.24 Impact Factor