Carboxypeptidase E (enkephalin convertase): mRNA distribution in rat brain by in situ hybridization.
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Carboxypeptidase E (CPE), also referred to as enkephalin convertase or carboxypeptidase H (EC 3.4.17.10), is present in neurotransmitter secretory granules and can remove C-terminal basic residues following endopeptidase cleavage during peptide processing. Using in situ hybridization with 35S-labeled oligonucleotide probes, we have mapped the localization of CPE mRNA in the rat brain. Specificity for CPE was confirmed by control experiments, which included production of identical patterns hybridization with 3 different antisense oligonucleotide probes, loss of label with RNase pretreatment of sections or co-incubation with excess unlabeled probe, and lack of labeling with sense orientation probes. In addition, the regional distribution of CPE mRNA by Northern blot analysis corresponded with distribution of labeling by in situ hybridization. The highest levels of CPE mRNA were found to be present in the pyramidal cells of the hippocampus, the pituitary anterior and intermediate lobes, the ependymal cells of the lateral ventricle, the endopiriform nucleus, the basolateral amygdala, the supraoptic nucleus, and the paraventricular nucleus. Intermediate levels were present in the thalamus, medial geniculate nucleus, lateral septal nucleus, piriform and entorhinal cortex, nucleus of the tractus solitarius, cerebellar cortex, pontine nuclei, and inferior olive. The lowest levels were found in the hippocampal granule cell layer, lateral hypothalamus, globus pallidus, and brain stem reticular formation. Ibotenic acid lesions of the hippocampus eliminated the majority of the label, which had been present over pyramidal cells, though labeling was increased over areas of reactive gliosis, suggesting that activated astrocytes can also synthesize CPE mRNA. In general, the localization of CPE mRNA in the rat brain corresponded to the distribution of enkephalin and other peptide neurotransmitter-synthesizing neurons, though CPE mRNA was also present in neurons that do not secrete known peptides and in reactive glia. The widespread yet specific localization of CPE mRNA in the rat brain suggests it may be an excellent marker for peptide synthesizing cells in the CNS. (+info)
Carboxypeptidase E/NFalpha1: a new neurotrophic factor against oxidative stress-induced apoptotic cell death mediated by ERK and PI3-K/AKT pathways.
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Identification of zymogen and mature forms of human carboxypeptidase H. A processing enzyme for the synthesis of peptide hormones.
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Carboxypeptidase H (CPH) is one of several processing enzymes required for the conversion of peptide hormone precursors into their smaller active forms. In this study, high levels of CPH activity was found in a liver metastasis of a human ileal carcinoid which expresses beta-preprotachykinin mRNA and the tachykinin neuropeptides, substance P and substance K. This human CPH showed properties of a zinc-metallopeptidase that is structurally similar to bovine and rat CPH. Immunoblots of the human ileal carcinoma with anti-bovine CPH showed that CPH activity is represented by two proteins of apparent molecular masses 57 and 55 kDa. Cell-free translation of poly(A)+ RNA followed by immunoprecipitation with anti-bovine CPH showed that human CPH mRNA encodes a precursor protein of apparent molecular mass 75 kDa. These data demonstrate that human CPH is synthesized as a zymogen, prepro-CPH, which must be cleaved to form catalytically active CPH. (+info)
Co-secretion of carboxypeptidase H and insulin from isolated rat islets of Langerhans.
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The release of carboxypeptidase H activity from isolated rat islets was determined and compared to the secretion of immunoreactive insulin. Analysis of pancreatic islet cells sorted into beta and non-beta types indicated that approx. 80% of islet carboxypeptidase H activity is present in the beta cell. The release of both insulin and carboxypeptidase H was stimulated markedly by increasing the glucose concentration in the medium from 2.8 to 28 mM. The fractional release was in accordance with the observed cellular distribution of both proteins. The secretory response was biphasic with time, with an initial rapid transient phase of release within 5 min, followed by a more sustained response. The concentration-dependencies of glucose stimulation of release of insulin and carboxypeptidase H were similar, with a threshold for stimulation around 5.6 mM-glucose and maximal stimulatory response at 16.7-28 mM-glucose. The release of both proteins was inhibited by 20 mM-mannoheptulose, removal of Ca2+ from the medium and addition of 1 microM-noradrenaline. The combination of 10 mM-4-methyl-2-oxopentanoate and 10 mM-glutamine stimulated the release of carboxypeptidase H and insulin, as did 3-isobutyl-1-methylxanthine and 350 microM-tolbutamide in the presence of glucose. It is evident that carboxypeptidase H is released from the pancreatic beta-cell by an exocytotic process from the same intracellular compartment as insulin. The release of carboxypeptidase H by a constitutive process was at best equivalent to 0.4%/h, or less than 2% of the maximal rate of release via the regulated pathway. It is concluded that carboxypeptidase H can be used as a sensitive index of beta-cell secretion and an alternative marker to the insulin-related peptides. (+info)
Carboxypeptidase H. A regulatory peptide-processing enzyme produced by human hepatoma Hep G2 cells.
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Human hepatoma (Hep G2) cells have been shown to secrete nanogram quantities of carboxypeptidase N (Grimwood, B. G., Plummer, T. H., Jr., and Tarentino, A. (1988) J. Biol. Chem. 263, 14397-14401). A second carboxypeptidase with an acidic pH optimum (pH 5.5) is also secreted at levels 2-3-fold greater than carboxypeptidase N. This enzyme was partially purified from the conditioned medium and compared with pure bovine pituitary carboxypeptidase H. The two enzymes behaved in a similar fashion in DE52 ion-exchange chromatography and on gel filtration, with the Hep G2 enzyme being slightly larger than the bovine pituitary enzyme (52-54 versus 50-52 kDa). Both enzymes hydrolyzed COOH-terminal basic amino acids from typical synthetic substrates as well as from natural leuenkephalin peptides and were identical based on pH activity profiles, inhibition by EDTA or guanidinoethyl mercaptosuccinic acid, and stimulation by Co2+ ions. Inhibition of enzyme secretion from Hep G2 cells by tunicamycin indicated that the Hep G2 enzyme was glycosylated. This finding was confirmed by a parallel deglycosylation of the Hep G2 and bovine pituitary carboxypeptidase H enzymes with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Immunoblots using mouse antiserum to bovine pituitary carboxypeptidase H revealed that the Hep G2 enzyme was immunocross-reactive with the bovine enzyme but was slightly larger in size (54 versus 52 kDa). Continuous [35S]methionine labeling and purification to near homogeneity using an affinity matrix corroborated the observations that the secreted Hep G2 carboxypeptidase H was slightly larger than bovine pituitary carboxypeptidase H. The Hep G2-secreted enzyme in pulse-chase experiments was initially detected intracellularly after a 15-min pulse as a single protein of about 54 kDa and was present in the 30-min chase medium with no evidence for pre- or postsecretion proteolytic processing. The human adrenergic cell line IMR-32 continuously labeled with [35S]methionine also secreted carboxypeptidase H of the same size as the Hep G2 enzyme. (+info)
Adrenal chromaffin granules and secretory granules from thyroid parafollicular cells have several common antigens.
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The presence of various antigens in two types of isolated endocrine vesicles (chromaffin granules and secretory vesicles of thyroid parafollicular cells) was investigated by immunoblotting. The two types of vesicles have three common secretory proteins: chromogranin A, chromogranin B and secretogranin II. Furthermore, six common membrane antigens were found: cytochrome b-561, carboxypeptidase H, glycoprotein II, glycoprotein III, synaptin/synaptophysin and SV 2. These results demonstrate that vesicles obtained from neural crest-derived endocrine cells not only share several common secretory peptides and proteins, but also have common properties as far as their membrane antigens are concerned. (+info)