Posttranslational Processing of Carboxypeptidase E, a Neuropeptide-Processing Enzyme, in AtT-20 Cells and Bovine Pituitary Secretory Granules

Abstract: Carboxypeptidase E (CPE) functions in the posttranslational processing of peptide hormones and neurotransmitters. Like other peptide processing enzymes, CPE is present in secretory granules in soluble and membrane-associated forms that arise from posttranslational processing of a single precursor, “proCPE.” To identify the intracellular site of proCPE processing, the biosynthesis and posttranslational processing were investigated in the mouse anterior pituitary-derived cell line, AtT-20. Following a 15-min pulse with [³⁵S]Met, both soluble and membrane-bound forms of CPE were identified, indicating that the posttranslational processing event that generates these forms of CPE occurs in the endoplasmic reticulum or early Golgi apparatus. The relative proportion of soluble and membrane-bound forms of CPE changed when cells were chased for 2 h at 37°C but was unaffected when cells were chased at either 20 or 15°C, suggesting that further processing of membrane forms to the soluble form occurs in a post-Golgi compartment. Treatment of the cells with chloroquine did not alter the relative distribution of soluble and membrane forms, suggesting that an acidic compartment is not required for this processing event. Overexpression of CPE did not influence the distribution of soluble and membrane forms of CPE, indicating that the CPE-processing enzymes are not rate-limiting. To examine directly CPE-processing enzymes, bovine anterior pituitary secretory vesicles were isolated. An enzyme activity that releases the membrane-bound form of CPE was detected in the purified secretory vesicle membranes. This enzyme, which removes the C-terminal region of CPE, is partially inhibited by EDTA and phenylmethylsulfonyl fluoride and is activated by CaCI₂. Together, the data indicate that posttranslational processing of CPE occurs in secretory granules and that this activity may be mediated by a prohormone convertase-like enzyme.     

Processing of Procarboxypeptidase E into Carboxypeptidase E Occurs in Secretory Vesicles

Abstract: Carboxypeptidase E (CPE) functions in the posttranslational processing of bioactive peptides. Like other peptide processing enzymes, CPE is initially produced as a precursor ("proCPE") that undergoes posttranslational processing at a site containing five adjacent Arg residues near the N-terminus and at other sites near the C-terminus of proCPE. The time course of the N-terminal processing step suggests that this conversion occurs in either the Golgi apparatus or the secretory vesicles. To delineate further the site of proCPE processing, pulse/chase analysis was performed under conditions that block transit out of the Golgi apparatus (brefeldin A, carbonyl cyanide m -chlorophenylhydrazone, or 20°C) or that block acidification of vesicles (chloroquine, monensin, or ammonium chloride). The results of these analysis suggest that efficient proCPE processing requires an acidic post-Golgi compartment. To test whether known processing enzymes can perform this cleavage, purified proCPE was incubated with furin, prohormone convertase 1, or a dynorphin converting enzyme, and the products were analyzed on denaturing polyacrylamide gels. Furin cleaves proCPE within the N-terminal region, although the reaction is not very efficient, requiring relatively large amounts of furin or long incubation times. The other two peptide processing enzymes did not cleave proCPE, whereas a relatively small amount of secretory granule extract was able to convert proCPE into CPE. Taken together, these findings suggest that the conversion of proCPE into CPE occurs primarily in secretory vesicles.     

Reflex Splanchnic Nerve Stimulation Increases Levels of Carboxypeptidase E mRNA and Enzymatic Activity in the Rat Adrenal Medulla

Carboxypeptidase E (CPE; EC 3.4.17.10) is a carboxypeptidase B-like enzyme involved with the biosynthesis of numerous peptide hormones and neurotransmitters, including the enkephalins. Reflex splanchnic stimulation of the rat adrenal medulla, which has previously been found to substantially increase enkephalin mRNA and enkephalin peptide levels, was examined for an influence on CPE mRNA and enzymatic activity. Several hours after insulin-induced reflex splanchnic stimulation, the levels of CPE activity in rat adrenal medulla are reduced to 40-60% of control. CPE activity returns to the control level 2 days after the treatment and then continues to increase, reaching approximately 200% of control 1 week after the treatment. The time course of the changes in CPE activity is different from those of the changes in epinephrine levels and the previously reported changes in enkephalin peptide levels. CPE mRNA is also influenced by the insulin shock, with levels increasing to 155% of the control level after 6 h and 170% after 2 days. The time course of the change in CPE mRNA levels is similar to that previously found for proenkephalin mRNA. However, the magnitude of the change is much different: Proenkephalin mRNA has been reported to increase by 1,600%. The changes in CPE mRNA and enzymatic activity are consistent with the proposal that CPE is not a rate-limiting enzyme in the biosynthesis of enkephalin.     

Cultured Astrocytes and Neurons Synthesize and Secrete Carboxypeptidase E, a Neuropeptide-Processing Enzyme

Carboxypeptidase E (EC 3.4.17.10) is a carboxypeptidase B-like enzyme associated with the biosynthesis of many peptide hormones and neurotransmitters. Media collected from cultured astrocytes contain a carboxypeptidase E-like activity. Cultured astrocytes secrete approximately 73% of their cellular level of carboxypeptidase E per hour, and secretion is not substantially influenced by 35 mM KCl. In contrast, neurons secrete only 29% of their cellular carboxypeptidase E per hour, but secretion increases to 86% on stimulation with 35 mM KCl. Secretion of carboxypeptidase E activity from both neuronal and astrocyte cultures is relatively selective; neither acid phosphatase or acetylglucosaminidase is secreted in appreciable amounts. Cultured neurons and astrocytes express a carboxypeptidase E mRNA of a similar size. The levels of this mRNA differ in astrocytes cultured from different brain regions, with high levels in striatal, cortical, hippocampal, and hypothalamic astrocytes and low levels in cerebellar astrocytes. The level of carboxypeptidase E mRNA in hypothalamic astrocyte cultures is four- to fivefold higher than the level in hypothalamic neuronal cultures. These results indicate that cultured astrocytes express carboxypeptidase E mRNA and enzymatic activity and thus contain one of the enzymes required in the biosynthesis of many peptide hormones and neurotransmitters.     

Enkephalin dipeptidyl carboxypeptidase (enkephalinase) activity: selective radioassay, properties, and regional distribution in human brain

Abstract: The compound [³H-Tyr¹,D-Ala²,Lcu-OH⁵]enkephalin has been synthesised as a potentially selective substrate for enkephalin dipeptidyl carboxypcptidase ( enkephalinase ) activity in brain. lncubations in the presence of homogenates and particulate fractions from rodent and human brain result in the formation of [³H]Tyr-D-Ala-Gly, which can be conveniently isolated by polystyrene bead column chromatography. The enzyme activity responsible for the hydrolysis of the Gly³-Phe⁴ amide bond of this substrate displays close resemblance to that hydrolysing the natural enkephalins at the same level. In addition, enkephalinase activity characterised in postmortem human brain is closely similar to that in rodent brain, with regard to optimal pH and apparent affinities of various substrates and inhibitors, including the potent compound thiorphan. Enkephalinase activity is distributed in a highly heterogeneous fashion among regions of human brain, the highest levels being found in globus pallidus and pars reticulata of the substantia nigra. This distribution is poorly correlated with that of opiate receptor binding sites but displays some resemblance to that of reported Met5-enkephalin levels.     

Regulation of Carboxypeptidase E by Membrane Depolarization in PC12 Pheochromocytoma Cells: Comparison with mRNAs Encoding Other Peptide- and Catecholamine-Biosynthetic Enzymes

PC12 cells, a rat pheochromocytoma cell line, have been found to express carboxypeptidase E (CPE) enzymatic activity and CPE, furin, and peptidylglycine alpha-amidating monooxygenase (PAM) mRNAs. PC12 cells secrete CPE activity in response to depolarization induced by 50 mM KCl. Short-term (1- to 3-h) treatments of PC12 cells with KCl stimulates the secretion of CPE but does not appear to stimulate the synthesis of new CPE protein, based on the measurement of CPE activity and incorporation of [35S]-Met into CPE. Also, CPE mRNA is not altered by 2-h treatments with KCl. In contrast, prolonged treatment (24-48 h) of PC12 cells with 50 mM KCl continues to stimulate the secretion of CPE activity, without altering the cellular level of CPE. Levels of CPE mRNA are significantly elevated after long-term treatment of the cells with KCl, with increases of 35% after 5 h and 55-75% after 24 to 72 h of treatment. The level of PAM mRNA is also elevated approximately 70% after 24 h of stimulation with KCl. In contrast, the mRNA levels of furin and dopamine beta-hydroxylase (DBH) do not change on treatment of PC12 cells with KCl. These findings indicate that long-term depolarization, which leads to a prolonged stimulation of PC12 cells to secrete CPE, also stimulates the synthesis of CPE and PAM but not furin or DBH.     

Subcellular Localization, Partial Purification, and Characterization of a Dynorphin Processing Endopeptidase from Bovine Pituitary

An enzyme capable of cleaving dynorphin B-29 to dynorphin B-13 is present in bovine pituitary, with 40- to 50-fold higher specific activity in the posterior and intermediate lobes than in the anterior lobe. Subcellular fractionation of bovine neurointermediate pituitary shows that this enzyme is present in the peptide-containing secretory vesicles. The enzyme has been purified 2,800-fold from whole bovine pituitaries using ion-exchange and gel filtration chromatography. Purified dynorphin-converting enzyme has a neutral pH optimum, and is subsantially inhibited by the thiol-protease inhibitor p-chloromercuriphenylsulfonic acid, but not by serine or metalloprotease inhibitors. The purified enzyme processes dynorphin B-29 at Arg14, producing both dynorphin B-14 and dynorphin B-13 in a 5:1 ratio. No other cleavages are observed, suggesting that the activity is free from other proteases and is specific for single Arg sequences. Purified enzyme also processes dynorphin A-17 at the single Arg cleavage site, generating both dynorphin A-8 and A-9 in a 7:1 ratio. The tissue distribution, subcellular localization, and substrate specificity of this enzyme are consistent with a physiological role in the processing of dynorphin B-29 and dynorphin A-17, and possibly other peptides, at single Arg residues.     

Activation and membrane binding of carboxypeptidase E

Carboxypeptidase E (CPE) is a carboxypeptidase B-like enzyme that is thought to be involved in the processing of peptide hormones and neurotransmitters. Soluble and membrane-associated forms of CPE have been observed in purified secretory granules from various hormone-producing tissues. In this report, the influence of membrane association on CPE activity has been examined. A substantial amount of the membrane-associated CPE activity is solubilized upon extraction of bovine pituitary membranes with either 100 mM sodium acetate buffer (pH 5.6) containing 0.5% Triton X-100 and 1 M NaCl, or by extraction with high pH buffers (pH greater than 8). These treatments also lead to a two- to threefold increase in CPE activity. CPE extracted from membranes with either NaCl/Triton X-100 or high pH buffers hydrolyzes the dansyl-Phe-Ala-Arg substrate with a lower Km than the membrane-associated CPE. The Vmax of CPE present in extracts and membrane fractions after the NaCl/Triton X-100 treatment is twofold higher than in untreated membranes. Treatment of membranes with high pH buffers does not affect the Vmax of CPE in the soluble and particulate fractions. Pretreatment of membranes with bromoacetyl-D-arginine, an active site-directed irreversible inhibitor of CPE, blocks the activation by NaCl/Triton X-100 treatment. Thus the increase in CPE activity upon extraction from membranes is probably not because of the conversion of an inactive form to an active one, but is the result of changes in the conformation of the enzyme that effect the catalytic activity.     

Carboxypeptidase inhibitor from ripened tomatoes; Purification and properties

A polypeptide which inhibits pancreatic carboxypeptidases has been purified from extracts of tomato fruit (Lycopersicum esculentum). The purification procedure involves chromatography on immobilized carboxypeptidase A and gel filtration, and yields homogeneous inhibitor as judged by disc gel electrophoresis and amino acid analysis. The inhibitor from tomatoes contains 38 amino acid residues and is related to one from potato tubers as determined by a comparison of their amino acid compositions and by cross-reactivity against anti-potato inhibitor antibodies. Inhibition studies indicate that bovine carboxypeptidase A, porcine carboxypeptidase B, and a carboxypeptidase from Streptomyces griseus bind to the inhibitors from tomato and potato with approximately the same free energy of association.     

Characterization of Enkephalin Release from Rat Striatum

Abstract: Using antisera specific for methionine- and leucine-enkephalin, we studied the characteristics of the release of these peptides from rat striatal slices. Only 2–3% of the total tissue stores of enkephalin could be released by potassium depolarization; similar percentages were released from globus pallidus, thalamus, and nucleus accumbens. Enkephalin release from hippocampus could not be detected. The striatal release of both enkephalins was affected similarly by changes in potassium and calcium levels in the superfusion medium. Lithium has no effect on either basal or potassium-stimulated release; tyr-arg did not affect basal release of either peptide. Striatal enkephalin levels were stable during the short-term incubation periods used in these experiments.     

Passage of a δ-Opioid Receptor Selective Enkephalin, [d-Penicillamine2,5]Enkephalin, Across the Blood-Brain and the Blood-Cerebrospinal Fluid Barriers

Abstract: [ d -Penicillamine^2,5]enkephalin (DPDPE) is an enzymatically stable, δ-opioid receptor-selective peptide, which produces analgesia when given intracerebroventricularly. However, because only modest analgesic effects were seen after subcutaneous administration of DPDPE, it has been inferred that it does not cross the blood-brain barrier well. In this present study, a vascular brain perfusion technique in anesthetized rats was used to measure directly whether [³H]DPDPE could cross the blood-brain and/or the blood-CSF barriers. The results indicated that the brain uptake of [³H]DPDPE was significantly greater than that of [¹⁴C]sucrose, a vascular marker ( p < 0.01), and than that of [³H]DPDPE into the CSF ( p < 0.01). Furthermore, HPLC analysis confirmed the integrity of the ³H to DPDPE and demonstrated that intact [³H]DPDPE entered the brain. Although 1 m M leucine-enkephalin failed to inhibit uptake of [³H]DPDPE, unlabeled DPDPE (100 µ M ) caused a significant inhibition of the brain uptake ( p < 0.01) but not the CSF uptake of [³H]DPDPE. These data provide evidence that intact [³H]DPDPE enters the CNS of anesthetized rats by saturable and nonsaturable mechanisms. In addition, the saturable mechanism is likely to be found at the blood-brain barrier, with the blood-CSF barrier playing only a minor role in the brain uptake of this peptide.     

Characterization of Membrane-Bound Aminopeptidases from Rat Brain: Identification of the Enkephalin-Degrading Aminopeptidase

Rat brain aminopeptidase activity was solubilized from membranes by incubation with thiols. This novel procedure resulted in the release of the same two aminopeptidases (MI and MII) previously shown to be solubilized by the nonionic detergent Triton X-100. The solubilized aminopeptidases MI and MII were resolved by ion-exchange chromatography and further purified by hydroxylapatite chromatography. Aminopeptidase MI was shown to hydrolyze only the beta-naphthylamides of arginine and lysine whereas aminopeptidase MII exhibited a broad specificity with respect to amino acid beta-naphthylamides. Only aminopeptidase MII hydrolyzed Leu-enkephalin at a significant rate, indicating that this enzyme can account for the membrane-bound enkephalin aminopeptidase activity. The enkephalin-degrading aminopeptidase is potently inhibited by opioid (alpha-neo-endorphin and dynorphin) as well as nonopioid (substance P, somatostatin, and angiotensin I) peptides in the range of 0.2-2.0 microM. The regional distribution of aminopeptidases MI and MII in rat brain are rather different, with aminopeptidase MII distribution more closely paralleling the distribution of opiate receptors.     

Purification and Characterization of a Membrane-Bound Protease from Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii y-1, newly synthesized chlorophyll a/b-binding apoproteins are degraded when chlorophylls are not present for assembly of stable light-harvesting complexes. A protease was purified from the membrane fraction of degreened y-1 cells, which digested chlorophyll a/b-binding proteins in membranes from C. reinhardtii pg-113, a protease-deficient strain. This protease was active with p-nitroanilides of nonpolar amino acids (Leu and Phe), but not of basic amino acids (Lys and Arg). The apparent molecular weight of the enzyme is 38,000 ± 2,000 as determined by electrophoresis in the presence of sodium dodecyl sulfate. Typical inhibitors of the major classes of proteases were ineffective with this enzyme. Protease activity was constant from pH 7.5 to 9; a plot of log V versus pH suggested that deprotonation of an ionizable group with a pK value of 6.0 to 6.5 is required for activity. The protease was inactivated by diethylpyrocarbonate and by photooxidation sensitized by rose bengal. These results suggested that a histidyl residue is required for catalysis. Although very sensitive to photodynamic conditions in vitro, the enzyme was not inactivated in vivo when cells were exposed to light.     

Purification and Characterization of a Thermostable Carboxypeptidase (Carboxypeptidase Taq) from Thermus aquaticus YT-1

Brassinosteroid (BR) and auxin co-regulate plant growth in a process termed cross-talking. Based on the assumption that their signal transductions are partially shared, inhibitory chemicals for both signal transductions were screened from a commercially available library. A chemical designated as NJ15 (ethyl 2-[5-(3,5-dichlorophenyl)-1,2,3,4-tetrazole-2-yl]acetate) diminished the growth promotion of both adzuki bean epicotyls and Arabidopsis seedlings, by the application of either BR or auxin. To understand its target site(s), bioassays with a high dependence on the signal transduction of either BR (BR-signaling) or auxin (AX-signaling) were performed. NJ15 inhibited the photomorphogenesis of Arabidopsis seedlings grown in the dark, which mainly depends on BR-signaling, while NJ15 also inhibited their gravitropic responses mainly depending on AX-signaling. On the study for the structure–activity relationships of NJ15 analogs, they showed strong correlations on the inhibitory profiles between BR- and AX-signalings. These correlations imply that NJ15 targets the downstream pathway after the integration of BR- and AX-signals.     

Inhibition of a Membrane-Bound Enkephalin-Degrading Aminopeptidase by Bestatin Analogs

Abstract: A variety of bestatin analogs were examined as potent inhibitors of a membrane-bound enkephalin-degrading aminopeptidase that was purified from monkey brain. Bestatinyl amino acid derivatives showed strong inhibition of this enzyme. The most effective was bestatin- l -Arg AcOH, with a K _i value of 0.21 × 10⁻⁸ M with Leu-enkephalin as substrate. It exhibited competitive kinetics and was about 100-fold more potent than bestatin. This compound seems to be useful for pharmacological and other studies.     

Purification from brain of synenkephalin, the N-terminal fragment of proenkephalin

Abstract: The primary sequence of adrenal proenkephalin was recently deduced from the structure of the cloned cDNA that codes for this protein. Several enkephalin-containing proteins with molecular weights between 8,000 and 20,000 daltons were purified from the bovine adrenal medulla. These proteins appear to represent intermediates in the processing of proenkephalin into physiologically active opioid peptides. While the concentrations of these large processing intermediates in the adrenal medulla are quite high, similar proteins have not yet been shown to be present in brain, and there is some question as to whether the brain synthesizes an enkephalin precursor similar to adrenal proenkephalin. We report here the purification from bovine caudate nucleus of synenkephalin, the N-terminal fragment of adrenal proenkephalin. The amino acid composition of synenkephalin indicates that the protein represents residues 1–70 of adrenal proenkephalin. Thus the brain and adrenal glands appear to utilize a similar precursor for enkephalin biosynthesis.     

Properties and Ontogenic Development of Membrane-Bound Histidine Decarboxylase from Rat Brain

Abstract: Histidine decarboxylase (HD) activity was determined in high-speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates. Twenty-eight percent of the HD activity was associated with membranes, and the remaining was soluble. Several properties of the soluble and membrane-bound HD were compared. No significant differences in the values of K _m for histidine and pyridoxal 5'-phosphate were observed. The solubilization of membrane-bound HD with Triton X-100 resulted in an increase of 60% over the nonsolubilized activity with no changes in the K _m for substrate and cofactor. The proportion of free pyridoxal 5'-phosphate-independent activity was identical in both fractions. The soluble and membrane-bound forms of the enzyme differ slightly in their pH-activity profiles, although both enzymes showed an optimum pH near 6.5. The HD activities present in soluble and membrane fractions were determined at different postnatal ages. The soluble activity increased until day 90, whereas the membrane-bound activity became stabilized from day 20.     

Purification and Characterization of Amidase which Participates in Nitrile Degradation

Amidase was purified from the cell-free extract of acetonitrile-grown Arthrobacter sp. J-1 by a procedure involving protamine sulfate precipitation, ammonium sulfate fractionation, and column chromatographies on DEAE-cellulose, hydroxyapatite and Sephadex G-200. The overall purification was 47-fold. The purified enzyme was homogeneous as judged by ultracentrifugal analysis and disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 300,000 and 320,000 by disc gel electrophoresis and gel filtration, respectively. The enzyme was possibly composed of eight identical subunits of a molecular weight of 39,000. The isoelectric point was 3.8. The enzyme catalyzed the stoichiometric hydrolysis of acetamide to form acetic acid and ammonia. The enzyme was active toward acetamide, acrylamide and propionamide and the Km values were 0.97, 23.3 and 8.05 mm, respectively. The enzyme showed acyltransferase activity.