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The kinetics of the previously reported paired basic residue-specific pro-opiomelanocortin-converting enzyme from bovine pituitary intermediate lobe secretory vesicles (Loh, Y. P., Parish, D.C., and Tuteja, R. (1985) J. Biol. Chem. 260, 7194-7205) were studied using human 125I-beta-lipotropin as substrate. The enzyme, at a concentration of 20 ng/100 microliters cleaved human beta-lipotropin to yield gamma-lipotropin, a beta-melanotropin linked to beta-endorphin intermediate and beta-endorphin, whereas at an enzyme concentration of 40 ng/100 microliters, the substrate was completely cleaved to yield beta-endorphin and beta-melanotropin. These products were identified by their immunological properties and size on sodium dodecyl sulfate-polyacrylamide gels. The 125I-beta-endorphin product was further shown by high pressure liquid chromatography to contain two forms; the major form co-eluted with 125I-Arg0-beta h-endorphin and the minor form with 125I-beta h-endorphin. No COOH-terminal shortened forms of beta-endorphin were detected. The products formed indicate cleavages at two of the three pairs of basic residues of human beta-lipotropin, at Lys37-Lys38 and Lys57-Arg58, but not at Lys86-Lys87. The cleavage at Lys57-Arg58 occurred primarily in between these basic residues. The Km values for the cleavage of the Lys37-Lys38 and Lys57-Arg58 pairs were 1.9 and 2.5 microM, respectively. The Vmax values for the cleavage of the Lys37-Lys38 and Lys58-Arg58 pairs were 4.8 nmol/micrograms of enzyme/h and 9.1 nmol/micrograms of enzyme/h, respectively.

Lysates from purified secretory granules of rat anterior pituitary glands were incubated with [3H]phenylalanine or [3H]arginine-labeled toad proopiocortin. The processed products formed were identified by immunoprecipitation with ACTH and beta-endorphin antisera, and by comigration with known markers on acid-urea polyacrylamide gels. Proopiocortin was cleaved by the secretory granule lysate primarily to 21,000 mol wt ACTH, 13,000 mol wt ACTH, 16,000 mol wt NH2-terminal glycopeptide, beta-lipotropin, a beta-endorphin-like peptide, and beta-endorphin. Characterization of the anterior pituitary proopicortin-converting activity shows that it: (1) cleaves specifically at the peptide bond on the carboxy side of the lysine-arginine residues of proopiocortin, (2) has a pH optimum in the acidic range, (3) is present in membrane and soluble fractions of the granule lysate, and (4) is inhibited by leupeptin, pepstatin A, and 2,2' dithiodipyridine, but not by p-chloromercuribenzoate, diisopropyl fluorophosphate, N alpha-p-tosyl-L-lysine chloromethyl ketone hydrochloride, chloroquine, L-1-tosylamide-2-phenylethyl-chloromethyl ketone, or EDTA.

High performance liquid chromatography (HPLC) followed by radioimmunoassay (RIA) of the chromatographic fractions were used to separate and quantify, respectively, the alpha MSH-like peptides stored in the neurointermediate lobe (NIL) of the Xenopus laevis (X. laevis) pituitary gland and released from the X. laevis NIL, in vitro. Immunoreactive (IR) material eluting with a similar HPLC retention time as desacetyl alpha MSH was the major IR peptide in the NIL. Material with a retention time similar to alpha MSH and immunological properties equivalent to alpha MSH was also present in the NIL. However, the retention times of the X. laevis and mammalian alpha MSH-like peptides were not identical, suggesting species difference in these peptides. Following incubation of NILs in the presence of [3H]-acetyl CoA, the X. laevis variant of alpha MSH was the major [3H]-labeled, immunoprecipitable material present. Following an incubation of NILs in the presence of [3H]-amino acids for 21 hours, immunoprecipitable [3H]-alpha MSH was detected in the NILs and the ratio of [3H]-desacetyl alpha MSH to [3H]-alpha MSH was similar to the ratio of IR-desacetyl alpha MSH to IR-alpha MSH. The X. laevis variant of alpha MSH was the major alpha MSH-like peptide released from the NILs into the incubation medium. Dopamine (50 microM) significantly inhibited the release of IR-alpha MSH but not IR-desacetyl alpha MSH. No net increase in total alpha MSH (sum of release and NIL content) was observed in the actively secreting (control) NIL group versus the dopamine-treated group. These results indicate that acetylation of desacetyl alpha MSH occurs intracellularly.

Recent amino acid sequence data suggest that trypsin-like and carboxypeptidase B-like activities are required for the processing of pituitary prohormones--e.g., pro-opiocortin (pro-adrenocorticotropin/lipotropin) and provasopressin in secretory granules. In this study the existence of a carboxypeptidase B activity in purified secretory granules from anterior, intermediate, and neural lobes of rat pituitary has been examined. A carboxypeptidase B activity that cleaved the COOH-terminal -Lys-Lys-Arg residues from the adrenocorticotropin fragment ACTH-(1-17) (a potential hormone product liberated from pro-opiocortin by a trypsin-like enzyme) was detected in anterior and intermediate lobe granules. A similar carboxypeptidase B activity was also present in purified secretory granules from rat pituitary neural lobes that cleaved the -Lys-Arg residues from [Arg8]vasopressin-Gly-Lys-Arg, a potential product cleaved from provasopressin. Secretory granule carboxypeptidase(s) from the three lobes of the pituitary was shown to cleave 125I-[Met]enkephalin-Arg6 to form 125I-[Met]enkephalin as well. 125I-[Met]Enkephalin was used as a model substrate for the quantitative assay of pituitary carboxypeptidase activity. The carboxypeptidase B in secretory granules from all three lobes was shown to be active at pH 5.5, but not at pH 7.4. Inhibition by the zinc metallocarboxypeptidase inhibitors guanidinopropylsuccinic acid, aminomercaptosuccinic acid, benzylsuccinic acid, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and the potato carboxypeptidase B inhibitor, and inhibition by the metal chelators EDTA and 1,10-phenanthroline demonstrate metal ion dependence of the pituitary granule carboxypeptidase activities. However, Co2+ stimulated the secretory granule carboxypeptidase B activities. Thiol protease inhibitors such as Cu2+ and p-chloromercuriphenylsulfonic acid also inhibited the activity. Thus, the secretory granule carboxypeptidase B-like activities in all three lobes of the pituitary appear to be similar thiol-metallopeptidases that differ from other carboxypeptidase activities previously described and may play an exclusive role in hormone biosynthesis in the pituitary.

The prohormone, pro-opiomelanocortin (POMC) is synthesized on ribosomes, subsequently routed to the Golgi apparatus and finally packaged into secretory granules where it is processed to various biologically active hormones (alpha-melanotropin, adrenocorticotropin, beta-endorphin and beta-lipotropin). We report here that in frog and mouse pars intermedia cells, newly synthesized [3H]Arg-labeled POMC is associated with the secretory granule membrane prior to processing. This association with the secretory granule membrane may be related to the intracellular transport and packaging of POMC and/or the facilitation of processing of the prohormone within the organelle.

Pro-opiomelanocortin (POMC) mRNA detected by in situ hybridization and POMC/ACTH (adrenocorticotropin)-containing neurons detected by immunocytochemistry were first observed in the presumptive arcuate nucleus of embryonic mouse brain on gestational day 10.5 (E10.5). Immunostained fibers were also evident on E10.5 in the lateral and dorsal diencephalon. In these areas, a dense network of processes developed by E11.5 and extended into the mesencephalon. Fibers were detected in the myelencephalon at this stage and a day later (E12.5) in the spinal cord. Adult-like patterns of POMC/ACTH fibers were established in the diencephalon, mesencephalon, metencephalon and the myelencephalon between E13.5 and E15.5. POMC-expressing cells in the anterior and intermediate lobes of the pituitary gland appeared on E12.5 and E14.5, respectively. The early expression of POMC and the rapid establishment of dense fiber tracts in the brain is consistent with a role for POMC-derived peptides in the development of the central nervous system.

The biological and behavioral activities of gamma-MSH (gamma-MSH) and alpha-MSH (alpha-MSH) were compared using three different tests: darkening of the skin of Anolis, grooming behavior of rats, and performance of a visual discrimination task by rats. When incubated with the Anolis skin, both peptides cause skin darkening. However, alpha-MSH is much more potent than gamma-MSH. The alpha-MSH effect is not antagonized by coincubation with gamma-MSH. When given intraventricularly to rats, alpha-MSH induces a marked grooming behavior. This effect was not noted upon administration of gamma-MSH. Injection of gamma-MSH with the alpha-MSH did not produce a grooming response significantly different from alpha-MSH alone. In the visual discrimination task, the two peptides had opposite effects on the rate at which rats learned the initial discrimination and a subsequent reversal. The peptides were injected intraperitoneally prior to behavioral testing. Following alpha-MSH, rats learned the discrimination and subsequent reversal faster than the control rats. Following gamma-MSH, rats learned the initial discrimination at approximately the same rate as controls, and learned the reversal much slower. These results are discussed in terms of the similarity and differences in the mechanisms of action of alpha-MSH and gamma-MSH.

The neuropeptides arginine vasopressin and oxytocin are generated from their prohormones in the hypothalamoneurohypophysial system by enzymatic cleavages at paired basic residues (i.e. Lys-Arg). This study describes the purification of an enzyme from bovine neural lobe secretory vesicles, the putative site of this processing, which is capable of cleaving several prohormones at paired basic residues. The enzyme is a glycoprotein of Mr approximately 70,000 and has an acidic pH maximum. It processes the heterologous precursors pro-opiomelanocortin and insulin at paired basic residues in a manner similar to a pro-opiomelanocortin-converting enzyme derived from bovine intermediate lobe secretory vesicles which has been described previously. In addition, the neural lobe-derived converting enzyme cleaves the human vasopressin prohormone in vitro to yield arginine vasopressin-Gly10-Lys11-Arg12 as the major vasopressin cleavage product. This indicates that the enzymatic cleavage in the vasopressin precursor occurred primarily on the carboxyl side of the arginine in the pair of Lys-Arg basic residues separating the vasopressin peptide from the neurophysin moiety in the precursor. The properties of the neural and intermediate lobe-derived enzymes are virtually identical, raising the possibility that a family of similar enzymes may be responsible for cleaving a number of prohormones at paired basic residues in different tissues.

Results of this study demonstrate two distinct forms of acetyltransferase activity which will acetylate alpha-MSH. These acetyltransferases are distinguished by pH optima, subcellular distribution and sensitivity to magnesium and several solubilizing detergents. A general acetyltransferase, as characterized in the rat pituitary anterior lobe and lens, has a pH optima of 7.4 and is inhibited by magnesium. Subcellular fractionation of anterior and neurointermediate lobes revealed that this acetyltransferase is primarily localized in the cytosol fraction of these tissues. An alpha-MSH acetyltransferase (MAT) and a beta-endorphin acetyltransferase (EAT) have a pH optima of 6.0-6.6, are inhibited by detergents, and are specifically localized in the secretory granules of the neurointermediate lobe. Comparative studies of MAT and EAT suggest a single enzyme responsible for the acetylation of opioid and melanotropin peptides, and we term this enzyme opiomelanotropin acetyltransferase (OMAT).

The significance of glycosylation of the ACTH/alpha-MSH-endorphin precursor in the biosynthesis, processing and secretion of its peptide products was examined in the toad neurointermediate (intermediate - posterior) lobe, with the aid of a specific inhibitor of glycosylation, tunicamycin. Tunicamycin did not affect the synthesis of the precursor but prevented its glycosylation. In the presence of tunicamycin the precursor underwent rapid intracellular degradation. Precursor molecules that escaped complete degradation were processed to an ACTH molecule with approximately 19 000 molecular weight and to other atypical peptides, which were released. In vitro studies showed that trypsinization of the non-glycosylated precursor resulted in its random proteolysis while large forms of ACTH were cleaved from the glycosylated precursor. The results indicate that glycosylation of the ACTH/alpha-MSH-endorphin precursor may confer specific conformational properties upon the molecule, thus regulating its limited proteolysis. Turnover and release studies revealed two different pools of ACTH, beta-LPH and alpha-MSH-related peptides in the toad intermediate lobe. One pool contained ACTH, beta-LPH, alpha-MSH and beta-endorphin, which were rapidly synthesized and released, or degraded within 6 h of synthesis if their release was inhibited. The other pool was stored and was stable for at least 10 h, if prevented from being released. Peptides in this stored pool primarily included ACTH, alpha-MSH and beta-LPH; beta-endorphin was a minor component of this pool. The release from both pools of peptides was inhibited by dopamine, while the stored pool was selectively inhibited from release by L-isoprenaline (L-isoproterenol).

Peptide hormones are synthesized from larger precursors by cleavages at paired basic residues. We have isolated a pro-hormone converting enzyme from bovine neural and intermediate lobe secretory vesicles that cleaves pro-vasopressin and pro-opiomelanocortin at Lys-Arg residues to yield vasopressin, and adrenocorticotropin/endorphin-related peptides, respectively. The enzyme from both lobes is an aspartyl protease of approximately 70,000 Da, is a glycoprotein and has an optimum pH range of 4.0-5.0. Present within the same secretory vesicles is an aminopeptidase B-like enzyme which is a metalloprotease that is inhibited by Co2+ and Zn2+. This enzyme may play a role in trimming off the N-terminal extended basic residues from peptides liberated by the pro-hormone converting enzyme.

The effect of arginine vasopressin (AVP) and corticotropin releasing factor (CRF) an adrenocorticotropin (ACTH) secretion, phosphatidylinositol breakdown and cAMP accumulation was examined in primary cultures of mouse anterior pituitary cells. AVP and CRF added alone stimulated ACTH secretion in a dose-dependent manner. At 10(-8) M concentration of peptide, AVP and CRF stimulated ACTH secretion 2.8- and 4.6-fold, respectively. AVP and CRF added in combination at equal doses gave an additive effect. CRF enhanced cAMP accumulation, but AVP had no effect on basal or CRF-induced cAMP accumulation. Both forskolin (10(-5) M) and 8-bromo-cAMP (10(-3) M) increased ACTH secretion in these cells by 2.8- and 1.7-fold, respectively. AVP induced the breakdown of phosphoinositides, and CRF alone, or in combination with AVP did not modify this effect. Phorbol 12-myristate 13-acetate (10(-7) M), dioctanoylglycerol (10(-4) M) and phospholipase C (100 mU/ml) also stimulated ACTH secretion in these cells by 4.2-, 2.4-, and 3.7-fold, respectively. Depletion of intracellular and extracellular Ca2+ decreased ACTH secretion, but had no significant effect on CRF-induced cAMP accumulation. However, AVP-induced phosphoinositide breakdown was dependent on extracellular Ca2+. These results indicate that CRF stimulates ACTH secretion via the cAMP-dependent pathway and AVP via the phosphoinositide breakdown-phospholipase C pathway. In the presence of AVP and CRF, both pathways appear to operate independently to produce an additive effect on ACTH secretion.

Low molecular weight specific proteins in phenotypically distinct, identified neurons of Aplysia californica, have been detected using a high resolution acid-urea polyacrylamide gel system, and the molecular weight of these proteins labeled by in vitro incubation in [3H]leucine were determined by two different methods. The relative mobilities of these proteins on the acid-urea gel differed significantly, even though they appeared to co-electrophorese on SDS gelss. These identified neurons, and the specific proteins that they synthesize and store represent excellent model systems for the study of specific protein regulation in neurons.

Coordinate secretion of two prohormone/proneuropeptide processing enzymes [pro-opiomelanocortin converting enzyme (PCE) and an aminopeptidase B-like enzyme (APBE)] and alpha-melanotropin (alpha-MSH) from bovine intermediate lobe pituitary cells was studied. Stimulation of secretion with 8-bromo-cyclic AMP produced significant increases in levels of immunoreactive alpha-MSH, PCE, and APBE. Treatment of cells with the dopaminergic agonist 2-bromo-alpha-ergocryptine resulted in significant decreases in secretion of alpha-MSH, PCE, and APBE. In neither case were there significant changes in levels of cytosolic lactic dehydrogenase or lysosomal beta-glucuronidase in the medium. The secreted PCE activity was shown to process frog and mouse pro-opiomelanocortin primarily to 23,000-Mr corticotropin (ACTH), 13,000-Mr ACTH, beta-lipotropin, a beta-endorphin-like peptide, and beta-endorphin, products comparable to those synthesized by the mouse and frog intermediate lobe in situ. The secreted enzymatic activity had a pH optimum between 4.0 and 5.0, was strongly inhibited by pepstatin A, and had an inhibitor profile similar to the purified bovine intermediate lobe PCE. The secreted APBE activity cleaved Argo-[Met]-enkephalin to [Met]-enkephalin and had a pH optimum and inhibitor profile similar to that previously reported for an activity from purified secretory vesicle fractions of bovine intermediate and neural lobes. The coordinate regulated secretion of alpha-MSH and enzyme activities (PCE and APBE) strongly indicates their colocalization in the same secretory vesicle compartment within the cell. The characteristics of the two enzymes secreted in the medium paralleled those seen in the tissue and further support their role in pro-opiomelanocortin processing in vivo.

The biosynthesis of corticotropin (ACTH1--39), beta-endorphin [beta(61--91)-lipotropin] and alpha-melanotropin in the toad intermediate lobe was studied by using immunoprecipitation procedures with antisera specific for these peptides. Intermediate lobes were pulse-incubated with [3H]phenylalanine and then chase-incubated for varying periods; the radioactive proteins were immunoprecipitated. Immunoprecipitates were separated by acidic urea or sodium dodecyl sulfate polyacrylamide gel electrophoresis. Evidence from the pulse-chase and sequential immunoprecipitation studies using antisera to ACTH and beta-endorphin suggests that the toad intermediate lobe synthesizes two common precursors (apparent Mr 32,000 and 29,500) containing both the ACTH and beta-endorphin sequences. These precursors are processed to yield several forms of immunoreactive corticotropin (apparent Mr 23,000, 21,000, 13,000, and 4300), immunoreactive endorphin (apparent Mr 11,700 and 3500), and immunoreactive alpha-melanotropin. The 4300 Mr form of corticotropin and the 11,700 and 3500 Mr forms of endorphins were found to comigrate with synthetic ACTH1--39, beta-lipotropin and beta-endorphin, respectively, on both acidic urea and sodium dodecyl sulfate gels.

Many biologically active peptides (e.g., insulin, nerve growth factor, ACTH, endorphin, parathyroid hormone, etc.) appear to be synthesized first as prohormones, which are then converted intracellularly to the biologically active products by various post-translational modifications. Peptides of neuronal origin (e.g., vasopressin and oxytocin) are synthesized by similar mechanisms. The prominent role of post-translational processing in determining the final peptide products allows for the possibility that different peptides will by generated from identical prohormones in different cells.

Increasing the osmotic gradient during hypo-osmotic lysis, by pre-treating Aplysia neurons with 100% ethylene glycol, caused the selective extraction of water-soluble, low molecular weight polypeptides from the cells. Specific 3H-labeled polypeptides in the cells R15, R3-13, and the bag cells which were not extracted by exposure of the cells to distilled water "lysis" were effectively solubilized by this procedure. The possible mechanisms and potential uses of this method are discussed.

Secretory granules (SGs) from rat intermediate lobes (IL) were isolated in a highly purified form by differential centrifugation, followed by sucrose density gradient centrifugation. The purified IL-SGs were lysed by freezing and thawing. The granule lysate was then centrifuged to generate membrane and soluble fractions. Proopiocortin -converting enzyme (PCE) activity was assayed by incubation of [3H]arginine- or [3H] phenylalanine-labeled toad proopiocortin with the total granule lysate, the membrane, or the soluble fraction at pH 5.0. The processed products were identified by immunoprecipitation with ACTH and beta-endorphin antisera, followed by acid-urea-gel electrophoresis. The PCE activity in rat IL-SG lysate cleaved proopiocortin to 21,000 mol wt ACTH, 21,000 mol wt ACTH/lipotropin (LPH), 13,000 mol wt ACTH, beta LPH, beta-endorphin-like peptides, and alpha MSH-like peptides, similar to those synthesized by the toad intermediate lobe in situ. Treatment of the PCE cleavage products with carboxypeptidase B resulted in the liberation of free arginine. This observation together with the nature of the products formed suggest that the PCE activity cleaved at pairs of basic residues of proopiocortin , yielding one or more products that terminated with an arginine or an arginine-lysine. PCE activity was found in membrane and soluble granule fractions, and both activities were inhibited by leupeptin, p-chloromercuribenzoate, dithiodipyridine, and pepstatin A, but not by chloroquine or N-alpha-p-tosyl-L-lysine-chloromethylketone HCl. Diisopropyl fluorophosphate and other thiol protease reagents (p-chloromercuriphenyl sulfonic acid, iodoacetic acid, and HgCl2) had a small inhibitory effect. The products formed by PCE activities in the membrane and soluble fractions were similar to those cleaved by the total granule lysate. The membrane fraction primarily cleaved proopiocortin between ACTH and beta LPH to form 21,000 (21 K) mol wt ACTH and beta-LPH, similar to the first processing step in the IL in situ. The soluble fraction, however, showed a greater tendency to cleave proopiocortin between the 16 K N-terminal glycopeptide and ACTH, to yield twice as much 21 K ACTH/LPH product as the membrane fraction. The membrane-associated PCE activity was found to be easily solubilized by extraction with high salt (1 M NaCl), suggesting that it is not an integral granule membrane protein.

Pro-opiomelanocortin (adrenocorticotropin/endorphin prohormone) is processed to yield active hormones by cleavages at paired basic amino acid residues. In this study, an enzyme that specifically cleaves at the paired basic residues of this prohormone has been purified from bovine pituitary intermediate lobe secretory vesicles, the intracellular processing site of proopiomelanocortin. This enzyme, named pro-opiomelanocortin converting enzyme, has been characterized as a glycoprotein of Mr approximately 70,000. It has an apparent isoelectric point between 3.5 and 4.0. The pH optimum of the pro-opiomelanocortin converting enzyme is between 4 and 5, but the enzyme is highly active at the intravesicular pH of 5.1-5.6. The enzyme specifically cleaved the Lys-Arg pairs of pro-opiomelanocortin to yield Mr = to 21,000-23,000 ACTH, beta-lipotropin, Mr 13,000 and 4,500 ACTH, beta-endorphin, and a Mr = 16,000 NH2-terminal glycopeptide, the products synthesized by the pituitary intermediate lobe in situ. NH2- and COOH-terminal analysis of the products indicated that the pro-opiomelanocortin converting enzyme cleaves the peptide bond either between the Lys and Arg or on the carboxyl side of the Arg at Lys-Arg pairs of pro-opiomelanocortin. The intracellular localization, pH optimum, and cleavage specificity of the enzyme suggest that it may function as a pro-opiomelanocortin processing enzyme in the pituitary intermediate lobe in vivo.

Neurosecretory granules (NSGs) from neural lobes of bovine pituitary glands were isolated in a highly purified form by metrizamide-sucrose gradient centrifugation. The purified NSGs were lysed and centrifuged, and the supernatants were further fractionated by gel filtration on Sephadex G-75. Proopiocortin-converting enzyme activity was assayed by incubation of [3H]arginine- or [3H]phenylalanine-labeled toad proopiocortin with NSG supernatant fractions. The processed products were identified by immunoprecipitation with ACTH and beta-endorphin antisera, followed by acid-urea gel electrophoresis. The optimum pH for the enzyme-mediated conversion was around pH 5.0. Conversion of toad proopiocortin by NSG converting enzyme activity was inhibited by leupeptin, antipain, p-chloromercuribenzoate, and pepstatin A, but not by diisopropyl fluorophosphate, EDTA, or N-alpha-p-tosyl-L-lysine-chloromethyl ketone HCl. The results suggest that the proopiocortin-converting enzyme activity in bovine neurosecretory granules is due to an acid-thiol protease which may contain secondary hydrophobic binding sites that are involved in substrate recognition.

The ability of bovine intermediate lobe secretory vesicle membrane-associated enzyme(s) and purified, soluble paired basic residue-specific, pro-opiomelanocortin converting enzyme (Loh, Y.P., Parish, D. C., and Tuteja, R. (1985) J. Biol. Chem. 260, 7194-7205) to cleave bovine NH2-terminal pro-opiomelanocortin1-77 (N-POMC 1-77) was investigated. Purified pro-opiomelanocortin converting enzyme and an enzyme activity associated with the secretory vesicle membrane were shown to cleave bovine N-POMC1-77 to two major products: N-POMC1-49 and Lys-gamma 3-melanotropin (MSH), and one minor product, gamma 3-MSH. These products were identified by their retention times on high performance liquid chromatography, immunological characteristics, and for Lys-gamma 3-MSH, amino acid composition. The products generated indicate cleavage preferentially between Arg 49-Lys 50 of bN-POMC1-77 (where b indicates bovine), which is identical to the processing pattern found in the bovine intermediate lobe in situ. The membrane converting activity was shown to be stimulated by 5 mM Ca2+ and has a pH optimum of 4-5 and an inhibitor profile characteristic of an aspartic protease. This suggests that the membrane-associated enzyme involved is very similar or identical to the purified, soluble pro-opiomelanocortin converting enzyme, which has previously been reported to be an acidic, aspartic protease responsible for the initial steps of POMC processing. The results of this study lead to the proposal that the lack of processing of the Arg49-Lys50 site in POMC in the anterior lobe versus the intermediate lobe of the pituitary in vivo may be due to other regulatory mechanisms rather than invoking the existence in the intermediate lobe of another enzyme specific for this site, different from pro-opiomelanocortin converting enzyme.

Pro-opiomelanocortin (ACTH/endorphin prohormone) is processed within the secretory vesicles of pituitary intermediate lobe cells to alpha-melanotropin and beta-endorphin. In order to learn more about the microenvironment in which processing occurs, a method was developed to purify large quantities of bovine intermediate lobe secretory vesicles (ILSV) using isoosmolar metrizamide-sucrose gradients. Analysis of alpha-melanotropin and marker enzymes revealed that the gradients provided a 94-fold purification of secretory vesicles with respect to lysosomes and a 15-fold purification with respect to mitochondria. Pro-opiomelanocortin-converting enzyme activity in the ILSVs was assayed and found to be maximally active around pH 5. From measuring the delta pH across the intact ILSV membrane using 9-aminoacridine fluorescence quenching, the internal pH of ILSVs was determined to be less than 5.6, consistent with the operating pH range of the converting enzyme activity. The pH gradient of the ILSVs collapsed in the presence of ammonium sulfate or by using a combination of nigericin and K+, when the external medium pH was 7. Using the voltage-sensitive dye, oxanol VI, Mg2+ ATP was shown to cause a marked change in the ILSV membrane potential with the inside being positive which was reversed by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Thus, the ILSVs appear to have a Mg2+ ATP-dependent electrogenic proton-translocating system.

The modulation of pro-opiomelanocortin (POMC) synthesis in Xenopus laevis pituitary intermediate lobe (IL) during background adaptation and the role of dopamine and cAMP in mediating this effect were examined. Neurointermediate lobes (NILs) were pulselabeled in vitro with [3H]arginine and analyzed for POMC synthesis by acid-urea gel electrophoresis. After black background adaptation of the animal (7 days), POMC synthesis increased 5-6-fold, while after white background adaptation (7 days), POMC synthesis decreased by 76%. Dopamine (50 microM) suppressed POMC synthesis in NILs in culture. In the absence of dopamine, POMC synthesis was stimulated. Several experiments were conducted to determine the category of dopamine receptor in the X. laevis IL. A D-2 dopamine receptor agonist inhibited immunoreactive alpha-MSH release from the NIL in a D-2 antagonist-reversible manner. A D-1 receptor agonist or antagonist did not alter the release of immunoreactive alpha-MSH from the NIL. Dopamine (10 microM) inhibited forskolin-stimulated cAMP accumulation. In addition, dopamine inhibition of POMC synthesis in cultured ILs was reversed by 8-Br-cAMP. These studies suggest that white background adaptation results in stimulation of the X. laevis D-2 receptor, which reduces cAMP production and POMC synthesis. Conversely, during black background adaptation the IL D-2 receptor is not stimulated, leading to increased cAMP production and POMC synthesis.

The 'secretory vesicle hypothesis of precursor processing' states that the initial endopeptidase cleavages which excise the nascent, biologically active peptides from their protein precursors occur primarily in secretory vesicles (or granules). Hence, all the processing steps subsequent to these cleavages must also occur within these organelles. Two types of evidence are presented in support of this view: (1) cell biological studies which implicate the secretory vesicle as the site of precursor conversion to peptides, and (2) enzymological studies which locate and characterize putative processing enzymes in secretory vesicles. The processing enzymes reviewed include the 'prohormone-converting enzymes' which cleave at pairs of basic amino acids, other endopeptidases, carboxypeptidase-B-like enzymes and aminopeptidase, and N-acetylation and alpha-amidation enzymes. The properties of these enzymes in relation to the nature of the processing micro-environment in the secretory vesicles is discussed.

The neurointermediate lobes of dark adapted toads, Xenopus laevis, were incubated for 30 min in [3H]arginine, [3H]arginine plus [14C]glucosamine, or [3H]glucosamine and then chased for various time periods ranging from 1--3 h. The labeled polypeptides synthesized and secreted by the lobes were analyzed by acid-urea polyacrylamide gel electrophoresis. A glycosylated ACTH-endorphin precursor (32,000 mol wt) was synthesized during the pulse and identified by immunoprecipitation by ACTH-(11--24) antiserum. During the chase, this precursor was processed to various glycopeptides and peptides, including ACTH, beta-lipotropin, and alpha-MSH, which were subsequently secreted into the medium. An immunoprecipitable ACTH-related glycoprotein (approximately 150,000 mol wt) and other nonimmunoprecipitable glycoproteins (approximately 80,000--100,000 mol wt) were also synthesized and secreted by the neurointermediate lobe. The secretion of these glycoproteins and peptides was inhibited by dopamine. The significance of glycosylation of the precursor for the biosynthesis, processing, and secretion of the ACTH, beta-lipotropin-, and MSH-related peptides was examined by using a specific inhibitor of glycosylation, tunicamycin. Tunicamycin treatment did not affect the synthesis of the 32,000 mol wt ACTH-endorphin precursor but did prevent its glycosylation. The absence of carbohydrate on the precursor resulted in its rapid intracellular degradation. Precursors that escaped degradation were processed incompletely, leading to the formation and secretion of an unglycosylated intermediate and various other abnormal peptides. The data indicate that glycosylation of the ACTH-endorphin precursor may not be involved in the processes of intracellular transport, packaging, and secretion per se but, rather, may provide specific conformational stability to the precursor as a signal for directed limited proteolysis.

The biosynthesis of alpha-MSH, beta-endorphin and ACTH in the pituitary is reviewed. These neuropeptides are synthesized from a common pro-protein, pro-opiomelanocortin. The pro-protein is cleaved intragranularly, at pairs of basic residues in the molecule to yield the respective peptide products. An unique, thiol protease (pro-opiocortin converting enzyme) and a carboxypeptidase B-like enzyme, both localized within pituitary secretory granules and having a pH optimum of 5-6, appear to be involved in the proteolytic processing of pro-opiomelanocortin.

Pro-opiomelanocortin (POMC) was expressed in CV-1 (green monkey kidney) cells using a vaccinia virus transient expression system [(1986) Proc. Natl. Acad. Sci. USA 83, 8122]. The system involved infection of cells with a recombinant vaccinia virus carrying the T7 RNA polymerase gene and transfection with a plasmid containing the mouse POMC sequence flanked by the T7 RNA polymerase promoter at its 5'-end and the T7 RNA polymerase terminator at its 3'-end. Assay of the medium from transfected cells showed that 1-2 micrograms of immunoreactive ACTH was produced/10(6) cells. Analysis of the same medium by SDS-PAGE/Western blots revealed a band of 30-36 kDa, which was immunostained with both ACTH and beta-endorphin antisera. Labeling the transfected cells with [3H]Arg, followed by immunoprecipitation and SDS-PAGE showed the synthesis of a major peak of POMC, 33 kDa. Purified [3H]POMC expressed by CV-1 cells was cleaved in vitro by bovine intermediate lobe secretory vesicle pro-opiomelanocortin-converting enzyme to ACTH intermediates (19-25 kDa), beta-lipotropin and beta-endorphin. Thus, this work has demonstrated a technique for expressing microgram quantities of prohormones in mammalian cells, suitable for use as substrates for prohormone-converting enzymes in vitro.

In our previous studies, we have purified a unique, paired basic residue-specific, prohormone-converting enzyme from pituitary intermediate lobe secretory vesicles. This enzyme, an aspartyl protease, was shown to cleave the intermediate lobe prohormone, pro-opiomelanocortin (POMC), to adrenocorticotropin, beta-endorphin and a 16 kDa NH2-terminal glycopeptide, in vitro [(1985) J. Biol. Chem. 260, 7194-7205]. To provide some evidence that this enzyme plays a role in prohormone conversion in the intact cell, the ability of pepstatin A, an aspartyl protease inhibitor, to block POMC processing in the mouse intermediate pituitary was investigated. By the use of a radioactive pulse-chase paradigm, [3H]POMC processing was found to be inhibited by 36.4% in pepstatin A-treated intermediate lobes. This result is consistent with the inactivation of pro-opiomelanocortin-converting enzyme by pepstatin A in the intact pituitary and further supports a role of this enzyme in POMC processing in vivo.

Three identified neurons (R14, R15, and L2-6) from Aplysia californica synthesize specific, low molecular weight proteins which are further processed or converted into smaller sized specific proteins. These specific proteins have differential turnover rates, and appear to be selectively transported out of the individual neuronal somata at different rates. The latter transport process can be blocked by colchicine, a well-known blocker of axonal transport. The relationship of these phenomena to the functional activity of the individual neurons is discussed.

A major and several minor trichloroacetic acid (TCA) soluble, bioassayable melanotropic peptides, as well as bioreactive and immunoreactive alpha-MSH have been found in the hypothalamus, olfactory bulb and cerebral cortex of normal and hypophysectomized rats. By employing subcellular fractionation procedures it was demonstrated that alpha-MSH and the major TCA soluble melanotropic peptide (MMPB) were localized in synaptosomes and were released by hypoosmotic shock. The analysis of MMPB by electrophoretic and chromatographic procedures reveales that it is not ACTH4-10, ACTH1-10, ACTH1-24, NAcACTH1-10 or alpha-MSH. MMPB was found to cross-react with an antiserum specific for the Lys-Pro-Val NH2 sequence in alpha-MSH, indicating that this C-terminal sequence of alpha-MSH may be present in its structure. MMPB was also shown to differ electrophoretically from the two major TCA soluble melanotropic peptides found in the neurointermediate lobe of the pituitary. In view of their synaptosomal localizations MMPB and alpha-MSH may play a role in synaptic function in the nervous system.

Pro-vasopressin mRNA, neurophysin and arginine vasopressin (AVP) were assayed in the mouse anterior pituitary gland, in mouse anterior pituitary cells in culture and in the AtT-20 corticotrophic tumour cell line. Northern blot analysis revealed the presence of an approximately 700 base pair pro-vasopressin mRNA in anterior pituitary and AtT-20 cells. Neurophysin, identified by immunoblots, and AVP, identified by high-performance liquid chromatography and cross-reactivity with AVP antiserum, were detected in anterior pituitary cells and AtT-20 cells. Immunocytochemical staining with anti-neurophysin showed that approximately 40-45% of the dissociated anterior pituitary cells in culture and greater than 95% of the AtT-20 cells were stained. Anterior pituitary cells in culture and AtT-20 cells had a basal level of release of AVP in the 0.01-0.1 nM range. These results indicate that anterior pituitary cells and AtT-20 cells have the ability to synthesize and process pro-vasopressin to AVP and neurophysin, endogenously.

The influence of glycosylation of a prohormone, pro-opiocortin, on its processing by intermediate (pituitary) lobe converting enzyme activity in vitro was studied. [3H]-arginine-labeled glycosylated and non-glycosylated pro-opiocortins were isolated from untreated, and tunicamycin treated toad neurointermediate lobes, respectively, after pulse-labeling in [3H]-arginine containing incubation media. These labeled precursors were then incubated at 37 degrees C in the presence of pro-opiocortin converting enzyme activity derived from rat intermediate lobe (pituitary) secretory granule lysates. The rates of conversion of the glycosylated and nonglycosylated pro-opiocortins to smaller peptide products, in vitro, were similar. Analysis of the peptide products by immunoprecipitation with ACTH and beta-endorphin antisera, and subsequent electrophoresis on acid-urea gels, indicate a comparable processing in vitro of the two forms of pro-opiocortin substrate. The only difference was that the normally glycosylated peptide products derived from glycosylated pro-opiocortin (i.e., 13K ACTH, 21K ACTH, and the 16K glycopeptide) differed in their gel electrophoretic mobilities from their counterparts derived from nonglycosylated prohormone, in a manner consistent with the absence of carbohydrate on the latter's peptides. These data show that glycosylation of the prohormone does not influence its processing in vitro by the converting enzyme activity.

Application of an ethylene glycol lysis technique to extract water-soluble, low molecular weight polypeptides in Aplysia neurons, was used in conjunction with microgradient gel electrophoresis and micro-isoelectric focusing, to identify unique polypeptides in specific, identified neurons. The polypeptides found in neurons R15, R3-13, R14, and the bag cells were particularly abundant, consistent with the previously suggested neurosecretory role for these cells. Water extraction of the strongly basic polypeptides (pI 10.7) in R3-13 and R14 required an acidic lysis medium.

Lysates of secretory granules from rat pituitary neurointermediate lobes were incubated with [3H]arginine- or [3H]phenylalanine-labeled toad pro-opiocortin. The processed products formed were identified by immunoprecipitation with adrenocorticotropin (ACTH) and endorphin antisera and by migration behavior on acid/urea/polyacrylamide gels. Pro-opiocortin was cleaved by the proteolytic activity in the secretory granule fraction to approximately 21,000 Mr ACTH, approximately 13,000 Mr ACTH, alpha-melanotropin, 16,000 Mr NH2-terminal glycopeptide, beta-lipotropin, and an endorphin-related peptide. Characterization of this pro-opiocortin-converting activity shows that it (i) is present in membrane and soluble fractions of the granule lysates, (ii) has a pH optimum of 5.0, (iii) appears to cleave at pairs of basic amino acid residues in the precursor, and (iv) is inhibited by leupeptin, pepstatin A, and p-chloromercuribenzoate but not diisopropyl fluorophosphate, N alpha-p-tosyl-L-lysine chloromethyl ketone hydrochloride, chloroquine, or EDTA. These inhibitor studies suggest that the converting-enzyme activity is due to an acid thiol, arginyl protease, distinct from any known cathepsin B-like activity.

Axonal transport of proteins in the Xenopus optic nerve was examined by labeling proteins in the eye with [35S]methionine injected intraocularly and then analyzing the labeled proteins in the eye, nerve, and tectum on linear gradient SDS polyacrylamide gels at different times after the injection. Because the optic nerve in Xenopus is short, in order to distinguish transported proteins from locally synthesized proteins, the optic nerve on one side of the animal was crushed at the orbit (to stop axonal transport) 5-30 min prior to injection and the crushed and normal nerve segments were compared. Proteins in the intact nerve which were absent in the crushed nerve were identified as axonally transported proteins. By such criteria several waves corresponding to transported material moving at greater than or equal to 6 mm/day, 1.6-2.8 mm/day, and approximately 0.2 mm/day were detected in the nerve. The most rapid phases of transport could be further resolved in the optic tectum into 3 additional components at 60-96 mm/day, 30-48 mm/day, and 6-11 mm/day. Analysis of labeled proteins in the crushed nerves distal to the crush, near the injury site, revealed several locally synthesized proteins (mol. wt. 54,000, 48,000, 43,000 daltons) which were not present in normal, uninjured nerves. Such proteins are probably synthesized by glia in response to injury.

Secretory vesicles isolated from the neural and intermediate lobes of the bovine pituitary contained a membrane-bound aminopeptidase activity which cleaved arginine from beta-LPH60-65 (Arg-Tyr-Gly-Gly-Phe-Met) and Arg-MCA. Neither methionine enkephalin (Tyr-Gly-Gly-Phe-Met) nor Substance P, which has an N-terminal arginine followed by a proline, could serve as substrates for this aminopeptidase activity; nor could cathepsin B-like or chymotrypsin-like enzyme activities be detected in the vesicle preparations. Maximal enzyme activity was at pH 6.0, and the activity was inhibited by EDTA, stimulated by Co2+ and Zn2+, but was unaffected by leupeptin, pepstatin A, phenylmethylsulfonyl fluoride and p-chloromercuribenzenesulfonate, suggesting that the enzyme is a metalloaminopeptidase. The presence of this aminopeptidase activity in secretory vesicles suggests that it may be involved in peptide prohormone processing.