Characterization of a neutral endopeptidase localized in the mitochondrial matrix of rat anterior pituitary tissue with GnRH as a substrate.
We have determined the subcellular localization of an endopeptidase activity able to degrade gonadotropin releasing hormone (GnRH) and present in the rat adenohypophysis. After fractionation of tissue homogenates in 0.25 M sucrose by differential centrifugation, about 25% of the total cellular GnRH degrading activity was found to be sedimentable and recovered from heavy (M) and light (L) mitochondrial fractions with a distribution pattern similar to that of the mitochondrial and lysosomal reference enzymes cytochrome oxidase and beta-galactosidase. Upon further fractionation on sucrose density gradients, the activity comigrated with mitochondria. The peptidase appears endowed with a structure-linked latency; the activity is low in a freshly prepared mitochondrial fraction and increases upon treatment with membrane disrupting agents in a manner similar to that of malate dehydrogenase, a component of the mitochondrial matrix. Determination of GnRH cleavage sites was performed by amino acid analysis of the fragments obtained after incubation of the peptidase with (3H)-GnRH labelled on the pyroglutamic acid residue, in presence of carboxypeptidase and peptidyldipeptidase inhibitors. The fragments were separated by ion-exchange chromatography on an Aminex Q-15S column and purified by chromatography on silica gel plates. Fragments 1-2, 1-3, 1-4, 1-5 and 1-6 were all present as early as 1 min after the beginning of incubation. Formation of each of them was inhibited to the same extent by EDTA, mersalyl acid, dithioerythritol and Na deoxycholate. The same fragmentation pattern was observed after partial purification of the enzyme by gel filtration. These data indicate that cleavage of several peptide bonds may result from a possibly single endopeptidase located in the mitochondrial matrix space.
D000818:Animals; D002498:Centrifugation; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002621:Chemistry; D010450:Endopeptidases / Q000032:analysis / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D010903:Pituitary Gland, Anterior / Q000201:enzymology*; D010906:Pituitary Hormone-Releasing Hormones / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D010906:Pituitary Hormone-Releasing Hormones; D010450:Endopeptidases