Localization of Endo‐Oligopeptidase (EC 3.4.22.19) in the Rat Nervous Tissue

Abstract

The subcellular and regional distribution of endo-oligopeptidase (EC 3.4.22.19), an enzyme capable of generating enkephalin by single cleavage from enkephalin-containing peptides, was determined by an enzymatic assay using metorphamide and by immunochemical techniques in the CNS of the rat. The rat CNS contains a membrane-associated form of endo-oligopeptidase, an enzyme predominantly associated with the soluble fraction of brain homogenates. Subcellular fractionation showed that approximately 17% of the total activity of the enzyme is associated with membrane fractions including synaptosomes. Synaptosomal membranes were prepared from neocortex, striatum, hypothalamus, medulla, spinal cord, and cerebellum. The amount of EC 3.4.22.19 activity solubilized by 3-[( 3-cholamidopropyl]dimethylammonio)-1-propanesulfonate from synaptosomal membranes was similar in neocortex, striatum, and hypothalamus, being three- to 10-fold greater than in spinal cord, cerebellum, and medulla. A polyclonal antibody exhibiting high affinity for endo-oligopeptidase was raised in rabbits against the purified rat brain enzyme and used to localize endo-oligopeptidase by Western blotting and by immunoperoxidase techniques. A strong band corresponding to the Mr of EC 3.4.22.19 was found in solubilized proteins obtained from synaptosomal membranes prepared from hypothalamus, neocortex, and striatum when subjected to Western blotting. The immunohistochemical localization of endo-oligopeptidase indicated that the immunoreactivity was confined to gray matter in regions known to be rich in peptide-containing neurons such as the striatum. In the cerebellum, a region poor in peptides, no staining could be detected. The nonuniform distribution of endo-oligopeptidase in rat brain suggests a role in neurotransmitter processing in the CNS.

Comparative fine structural distribution of endopeptidase 24.15 (EC3.4.24.15) and 24.16 (EC3.4.24.16) in rat brain

“…As previously reported (Oliveira et al, 1990;Healy and Orlowski, 1992;Woulfe et al, 1992;Massarelli et al, 1999), EP24.15 and EP24.16 antibodies both gave rise to widespread immunostaining throughout the rat CNS. The present study focuses on three regions that showed exten-sive and dense immunolabeling for both antibodies: the cerebral cortex, the cerebellar cortex, and the NTS.…”

“…Both are highly concentrated in the brain and are reportedly enriched in areas containing putative peptide substrates (Heally and Orlowski, 1992;Checler et al, 1995). Thus, EP24.15 was found to colocalize with Leu-enkephalin in neurons of the pigeon retina (Ferro et al, 1991) and to predominate in brain areas rich in opioid peptides, such as the striatum and the hypothalamus (Oliveira et al, 1990;Healy and Orlowski, 1992). Surprisingly, however, the highest levels of EP24.15 activity were detected in the cerebellar cortex, which has a notoriously low peptide content (Chu and Orlowski, 1985).…”

“…Light and electron microscopic immunohistochemical studies have indicated that within the brain, EP24.15 and EP24.16 were present in both neurons and glial cells (Oliveira et al, 1990;Healy and Orlowski, 1992;Woulfe et al, 1992;Vincent et al, 1996;Massarelli et al, 1999). EP24.15 and EP24.16 activity was also demonstrated in synaptosomal preparations from dog ileum (Acker et al, 1987;Barelli et al, 1989), suggesting that both enzymes were present in axon terminals.…”

Rat kidney endopeptidase 24.16

“…Subcellular fractionation demonstrated that 20-25% of bovine endopeptidase 24.15 was associated with synaptosomal membrane fkction (Acker et al, 1987). By the same approach Oliveira et al (1990) established a similar dual localization for endooligopeptidase A. Pyroglutamyl-aminopeptidase 11, which could be recovered as a soluble activity in body fluids and serum (Taylor and Dixon, 1978), was also shown to display a neuronal localization in primary cultures of fetal mouse brain (Cruz et al, 1991).…”

Secretion of Metalloendopeptidase 24.15 (EC 3.4.24.15)

“…Thus, this mainly cytosolic enzyme must access the outside of the cell to elicit its well-characteriz ed neuropeptide degrading/ processing capabilities. Cell fractionation studies show the EP24.15 enzyme is present in a minor form in the particulate subcellular fractions (Acker et al, 1987;Oliveira et al, 1990), but the reported amino acid sequences derived from cDNA clones isolated from sev-eral species do not contain a classical secretory signal for a secreted protein. Previous studies indicate calcium-independ ent secretion of EP24.15 from C6 glioma cells (Ferro et al, 1993).…”