Localization of a 16,000-dalton fragment of the common precursor of adrenocorticotropin and beta-lipotropin in the rat and human pituitary gland.

To clearly identify cells and organelles containing the common precursor (31,000 dalton) for both adrenocorticotropin (ACTH) and beta-lipotropin (beta-LPH), an immunohistochemical localization of a fragment (16,000 dalton) of the precursor that is not common to beta-LPH and ACTH was conducted in rat and human pituitary glands. With the help of specific antibodies that do not cross-react with beta-LPH and ACTH, the 16,000-dalton fragment was localized in the cells that also produce ACTH and beta-LPH in both the pars distalis and pars intermedia of the rat pituitary. At the electron microscope level, the secretory granules that contain ACTH were also stained for 16,000-dalton fragment. In the human pituitary, the 16,000-dalton fragment was also observed in all the secretory granules of lipocorticotrophs. These results suggest that, after enzymatic cleavage, fragment(s) of the common precursor and/or the whole common precursor remain packaged within the secretory granules with peptides of known activity.

Coordinate, equimolar secretion of smaller peptide products derived from pro-ACTH/endorphin by mouse pituitary tumor cells.

The secretion of peptide products derived from pro-ACTH/endorphin was examined with several radioimmunoassays and with polyacrylamide gel analyses of immunoprecipitates of radioactively labeled peptides. In studies using a mouse pituitary tumor cell line the accumulation of each of the four molecular forms of adrenocorticotropic hormone (ACTH) in tissue culture medium was shown to be a linear function of time. No evidence for self inhibition of secretion by accumulated, secreted peptides (i.e., ultra-short feedback) was found. Furthermore, synthetic human ACTH and synthetic camel beta-endorphin did not alter secretion of peptides when added to the culture medium at levels up to 10,000 times physiological. Stimulation of the release of ACTH-, endorphin-, lipotropin-, and 16k fragment immunoreactive material by norepinephrine was fully blocked by cobalt; by this criterion, stimulated release was calcium dependent. All the smaller molecules derived from the pro-ACTH/endorphin common precursor were secreted in equimolar amounts under all circumstances tested, within the precision of these studies (+/- 11%). Norepinephrine and cobalt did not significantly alter the secretion of pro-ACTH/endorphin and ACTH biosynthetic intermediate. The stimulation of secretion by norepinephrine and inhibition of secretion by cobalt was restricted to the lower molecular weight products derived from pro-ACTH/endorphin: glycosylated and nonglycosylated ACTH(1-39); beta-lipotropin, beta-endorphin, and gamma-lipotropin; and 16k fragment.

Entry of opioid peptides into the central nervous system.

Cerebrovascular permeability of four modified opioid peptides--[D-Ala2]methionine enkephalin amide, beta-[D-Ala62,14C-Homoarg69]lipotropin 61 -69, alpha-[D-Ala2,14C-Homoarg9]endorphin, and beta-[D-Ala2,14C-Homoarg]endorphin--ranged from 1.4 to 3.9 X 10(-6) centimeters per second in brain regions of the conscous rat. These significant permeabilities should allow the peptides to fill the extracellular brain space with a half time of 3 to 11 minutes, as a result of a step increase in plasma concentration of unbound peptide.

Pituitary intermediate lobe in dog: two cell types and high bioactive adrenocorticotropin content.

The pituitary intermediate lobe of most species is cytologically monotonous, but that of the dog is composed of two immunocytochemically distinct cell types. The predominant A cells are typical pars intermedia cells: they stain immunocytochemically for alpha-melanotropin and, more weakly, for adrenocorticotropin and beta-lipotropin. The B cells are like the corticotrophs of the anterior lobe: they stain intensely for adrenocorticotropin and beta-lipotropin but not for alpha-melanotropin. The B cells may account for the high concentration of bioactive adrenocorticotropin measured in the canine pars intermedia, and may explain why in dogs adenomas causing Cushing's disease through hypersecretion of adrenocorticotropin can arise from the intermediate as well as the anterior pituitary lobe.

Adrenocorticotropin in rat brain: immunocytochemical localization in cells and axons.

By means of antiserum (purified by affinity chromatography) directed against adrenocorticotropin (ACTH) 11-24, cell bodies and beaded axons were visualized in rat brain. The ACTH-like immunoreactivity (ACTH-LI) was primarily located in the hypothalamus (cells and axons). Fibers were scattered throughout thalamus, amygdala, periaqueductal gray area, and reticular formation. There was no change in the distribution of ACTH-LI in rats that had been subjected to hypophysectomy. This distribution of ACTH-LI parallels that of beta-lipotropin and is altered by specific lesions in a similar fashion. The presence of ACTH-LI in cells and beaded axons in brain raises the possibility that it is a neuroregulator functioning as a neurotransmitter, neuromodulator, or neurohormone.

Simultaneous assay of immunoreactive beta-lipotropin, gamma-lipotropin, and beta-endorphin in plasma of normal human subjects, patients with ACTH/lipotropin hypersecretory syndromes, and patients undergoing chronic hemodialysis.

We have studied the relative concentrations of the human immunoreactive (IR) peptides gamma-lipotropin (hgammaLPH, [1-58]hbetaLPH), beta-lipotropin (hbetaLPH), and beta-endorphin (hbetaEND, [61-91]hbetaLPH) using gel exclusion chromatography together with a specific radio-immunoassay (RIA) for hgammaLPH and a RIA that (because hbetaEND is the COOH-terminus of the hbetaLPH molecule) measures both hbetaEND and hbetaLPH on an equimolar basis. In normal subjects, basal plasma IR-hgammaLPH was often undetectable (<12.5 fmol/ml), but ranged up to 21 fmol/ml, and IR-hbetaEND/hbetaLPH was 10.8+/-0.7 fmol/ml; previous studies by others suggest that most of the IR-hbetaEND/hbetaLPH was probably hbetaLPH. Both IR-hgammaLPH and IR-hbetaEND/hbetaLPH were significantly elevated (P < 0.001) in patients undergoing chronic hemodialysis (101.5+/-12.7 and 23.8+/-2.0 fmol/ml, respectively). Their IR-hgammaLPH coeluted with standard hgammaLPH as a single peak, and IR-hbetaEND/hbetaLPH coeluted with hbetaLPH; no distinct peak of IR-hbetaEND was observed. In patients with ACTH/LPH hypersecretion due to Addison's disease, Nelson's syndrome, or ectopic ACTH syndrome, IR-hgammaLPH and IR-hbetaEND/hbetaLPH were both elevated, and IR-hbetaEND/hbetaLPH eluted as two peaks, one coeluting with hbetaLPH and the other with hbetaEND. The molar concentrations of all three peptides were significantly correlated with one another. The lower concentrations of endogenous IR-hbetaEND observed may be due in part to its apparent shorter plasma half-life, as estimated in an Addison's patient given a cortisol infusion. The biologic significance of these three peptides in circulating blood is still unknown. The increased levels of hbetaLPH and hgammaLPH in plasma of patients with chronic renal failure suggest that the kidney may be an important organ for their metabolism.

Immunoreactive beta-endorphin in the rat mammotropic transplantable tumor (MtT-F4).

Raised levels of what appeared to be beta-lipotropin (beta-LPH), beta-melanotropic hormone, and beta-endorphin were detected by radioimmunoassay in the plasma of rats bearing the mammotropic transplantable pituitary tumor MtT-F4. The immunoreactivity to anti-beta-endorphin in the assay was displayed by a substance with the molecular weight of beta-LPH, as determined by gel filtration. Isolated cells of MtT-F4 tumor incubated in vitro released immunoreactive beta-LPH and beta-endorphin, with the expected molecular weights, into the incubation medium. These results suggest that the pituitary transplantable rat tumor MtT-F4 secretes peptides structurally related to beta-LPH.

60 YEARS OF POMC: Lipotropin and beta-endorphin: a perspective.

Many important fields of research had a humble origin. In the distant past, A J P Martin's discovery that amino acids could be separated by paper chromatography and Moore and Stein's use of columns for quantitative amino acid analysis provided the first steps towards the determination of structure in complex biologically active molecules. They opened the door to reveal the essential relationship that exists between structure and function. In molecular endocrinology, for example, striking advances have been made by chemists with their expertise in the identification of structure working with biologists who contributed valuable knowledge and experience. Advantage was gained from the convergence of different background, and it is notable that the whole is greater than the sum. In the determination of structure, it may be recalled that four of the world's great pioneers (Archibald Martin, Rodney Porter, Fred Sanger and Vincent du Vigneaud) were acknowledged for their fundamental contributions when individually they were awarded the Nobel Prize. They foresaw that the identification of structure would prove of outstanding importance in the future. Indeed, study of the structures of ß-endorphin and enkephalin and the different forms of opiate activity they engender has led to a transformation in our understanding of chemical transmission in the brain.

Altered processing of pro-orphanin FQ/nociceptin and pro-opiomelanocortin-derived peptides in the brains of mice expressing defective prohormone convertase 2.

The bioactivity of neuropeptides can be regulated by a variety of post-translational modifications, including proteolytic processing. Here, gene-targeted mice producing defective prohormone convertase 2 (PC2) were used to examine the post-translational processing of two neuroendocrine prohormones, pro-opiomelanocortin (POMC) and pro-orphanin FQ (pOFQ)/nociceptin (N), in the brain. Reversed-phase HPLC and gel-exclusion chromatography were combined with specific radioimmunoassays to analyze the processing patterns of these two prohormones in the hypothalamus and the amygdala. In the case of POMC, the lack of PC2 activity completely prevented carboxy-shortening of beta-endorphins and greatly diminished conversion of beta-lipotropin to gamma-lipotropin and beta-endorphin. Although conversion of beta-lipotropin to beta-endorphin decreased, the lack of PC2 activity caused an increase in beta-lipotropin and beta-endorphin levels in the mutant animals, but no increases in POMC or biosynthetic intermediates were seen. The extent of OFQ/N production was significantly lower in PC2-deficient mice and there was an accumulation of relatively large amounts of pOFQ/N and biosynthetic intermediates. These results demonstrate that PC2 is directly involved in the biogenesis of two brain neuropeptides in vivo and suggest that the specific prohormone and cellular context influences neuropeptide processing by PCs.

The yeast KEX-2-processing endoprotease is active in the Golgi apparatus of transfected NIH 3T3 fibroblasts.

Proteolytic processing of polyprotein precursors at pairs of basic amino acids is a prerequisite for the generation of bioactive peptide hormones. While the mammalian endoproteases responsible for these cleavages are yet to be identified, this function has been unequivocally assigned in yeast to the product of the KEX-2 gene. To study the molecular mechanisms involved in polyprotein processing, we have transfected the yeast KEX-2 gene into mouse NIH 3T3 fibroblasts and established a new cell line (called 2N-DK) where the KEX-2 endoprotease is permanently expressed. Immunofluorescence studies show that the KEX-2 enzyme is retained within the Golgi of the 2N-DK cells. The evidence for this cellular location is supported by measurement of intracellular and extracellular KEX-2 enzyme activity. In this permanently transfected cell line, KEX-2 activity is exclusively intracellular, in contrast to the situation previously described in transiently infected cell lines, where extracellular KEX-2 activity was detected. Furthermore, infection of 2N-DK cells with a recombinant retrovirus expressing a cDNA coding for porcine proopiomelanocortin (POMC) resulted in the synthesis of POMC and its efficient processing into beta-lipotropin and beta-endorphin, two of its physiologically authentic maturation products. These results suggest that in the fibroblast cell line 2N-DK, proteolytic processing of POMC by KEX-2 endoprotease occurs in the Golgi apparatus.

Immunohistochemical localization of beta-lipotropic hormone in the pituitary gland.

Identification of the beta-lipotropic hormone (beta-LPH)-producing cells in several species, including man, was performed with the technique involving use of the unlabeled antibody and peroxidase-antiperoxidase complex. Serial paraffin and ultrathin sections were treated for detection of both beta-LPH and ACTH at the light and electron microscopic levels. It was clearly shown that beta-LPH could be found only in the corticotropic cells located in the pars intermedia and pars distalis of all species studied. At the electron microscope level, it could be established that beta-LPH is contained in all the secretory granules of positive cells. These results suggest that beta-LPH is stored in the same secretory granules as ACTH and that both hormones are released together during granule extrusion.

Disproportionate accumulation of immunoreactive corticotropin, melanotropin, and lipotropin in the brain of the maturing rat.

The accumulation of immunoreactive ACTH (ACTHi), alpha MSH (alpha MSHi), and gamma-lipotropin (gamma LPHi) as a function of age (10-120 days) was determined in three regions of the brain of male rats: the medial basal hypothalamus (MBH), the preoptic anterior hypothalamus (POA), and the thalamus. In each region of the brain, the concentrations of ACTHi, alpha MSHi, and gamma LPHi increased with age. In the MBH, the increase occurred in such a manner that the molar ratio of alpha MSHi to ACTHi remained constant regardless of the age of the animals. In contrast, in the POA and thalamus, the increase occurred disproportionately in favor of alpha MSHi, and thus the molar ratio of alpha MSHi to ACTHi was 3 times higher in the adult (120 days old) than in the young (10 or 21 days old) animals. Nevertheless, the ratio of (ACTHi plus alpha MSHi) to gamma LPHi was constant at a level of about 2 regardless of the age of the animal or the region of the brain. Extracts of the MBH or POA were fractionated on columns of Sephadex G-75 superfine. The gel filtration profiles of ACTHi were indicative of the presence of five molecular weight forms of ACTH: greater than 40K, 30-40K, 20-30K, 5.7K, and 4.5K. We tentatively identified greater than 40K ACTH as a large form of proopiocortin, 30-40K ACTH as proopiocortin, 20-30K ACTH as ACTH biosynthetic intermediate, 5.7K as glycosylated ACTH(1-39), and 4.5K ACTH as ACTH-(1-39). Regardless of the age of the animals, the fractional amount of 30-40K ACTH the age of the animals, the fractional amount of 30-40K ACTH was high in the MBH compared to that in the POA. Moreover, the small fractional amount of 30-40K ACTH in the POA was associated with a large fractional amount of small molecular weight forms of ACTHi. However, the predominant form of ACTHi in the POA changed with age: 20-30K ACTH was the major form in the young, whereas 4.5K ACTH was the major form in the adult. These results support the proposal that the production of proopiocortin increases with age, and there is enhanced processing of proopiocortin to ACTH-(1-39) and alpha MSH in the brain of the maturing rat.

Rate-sensitive glucocorticoid feedback inhibition of adrenocorticotropin and beta-endorphin/beta-lipotropin secretion in rats.

In the present study, we present physiological evidence for rate-sensitive, fast feedback inhibition of secretion of ACTH and beta-endorphin (beta END)-related peptides. We used a 2 min restraint stress to physiologically increase plasma corticosterone, then examined the plasma responses of immunoreactive ACTH and beta END plus beta-lipotropin (beta END/beta LPH) to a subsequent restraint stress. After onset of this stress, plasma corticosterone increased from 2.5-10 min at a rate of 120 nM min-1, then remained at a peak from 10-15 min. A single 2 min restraint stress produced peak plasma levels of ACTH and beta END/beta LPH 2.5 min after onset of the stress, and these plasma concentrations declined after this initial stress at rates of 2.7 and 7.4 pM min-1, respectively. Application of a second restraint stress at the time of the peak corticosterone response produced plasma ACTH and beta END/beta LPH responses similar to those after the first stress. Application of a second stress during the period of significant rate-rise of corticosterone in plasma did not result in decreased incremental responses of plasma ACTH or beta END/beta LPH. However, the rates of decline of plasma ACTH and beta END/beta LPH of 7.6 and 32 pM min-1, respectively, from peak levels, were significantly greater after this second stress applied during the period of significant increase in plasma corticosterone concentration than the corresponding rates of decline observed after the initial stress or after a subsequent stress applied at the peak of plasma corticosterone. These differences in rates of decline of plasma ACTH or beta END/beta LPH appear to reflect differences in secretion rate rather than clearance, since disappearance of [125I]ACTH1-24 was not different after an initial vs. subsequent stress. In contrast to these data from intact rats, initial and subsequent stresses did not show different rates of decline of plasma ACTH or beta END/beta LPH in adrenalectomized rats. In conclusion, the stress-induced rate rise of glucocorticoid provides a negative feedback signal which serves to terminate and limit the duration, but not the peak, of the responses of POMC-derived peptides to subsequent stress.

Corticotropin releasing factor (CRF): effects on the release of pro-opiomelanocortin (POMC)-related peptides by human anterior pituitary cells in vitro.

The effect of synthetic ovine corticotropin-releasing factor (oCRF), alpha-melanotropin (alpha-MSH) and arginine-vasopressin (AVP) on the release of pro-opiomelanocortin (POMC)-related peptides (beta-endorphin, beta-lipotropin, adrenocorticotropin, and the 12K N-terminal) was studied in primary cultures of human anterior pituitary (HAP) cells. The peptides released into the medium were measured by specific radioimmunoassays. HAP cells responded to oCRF at concentrations as low as 10 ng/ml by significantly increasing peptide secretion (p less than 0.005). AVP was also effective, but, it is only 0.1% as potent as oCRF. alpha-MSH was ineffective even at a dose of 10 micrograms/ml. Immunoreactive (IR)-beta EP, IR-beta LPH, IR-ACTH, and IR-HNT were released in approximately equimolar ratios. Partial characterization of the peptides released by HAP cells with gel chromatography on Sephadex G-50 superfine revealed two peaks of IR-beta EP (as beta EP and beta-LPH), two peaks of IR-beta LPH (as beta-LPH and gamma-LPH), one peak of IR-ACTH, and two peaks of IR-HNT. In conclusion, oCRF is a potent secretagogue for the equimolar release of ACTH, HNT and related peptides in the human pituitary gland.

Postnatal development of beta-endorphin-related peptides in rat anterior and intermediate pituitary lobes: evidence for contrasting development of proopiomelanocortin processing.

The concentration of immunoreactive (ir) beta-endorphin (beta-END) in the neurointermediate pituitary lobe was 15-fold higher in adult than in newborn rats; in contrast, that of ir-beta-END in the anterior lobe was twice as high in newborn as in adult animals. Ir-beta-END in the neurointermediate lobe of newborn rats consisted exclusively of beta-END-sized peptides, indicating that at birth rats are capable of processing the opioid peptide precursor proopiomelanocortin (POMC) to beta-END. Moreover, beta-END-related peptides in the neurointermediate lobe of newborn rats were found to be predominantly alpha-N-acetylated and, therefore, inactivated with respect to their opiate-like properties. Further analysis of these alpha-N-acetylated forms on high performance liquid chromatography indicated that newborn rats predominantly contained alpha-N-acetyl-(Ac-)beta-END-(1-31), whereas the major forms in adult rats were Ac-beta-END-(1-27) and -(1-26). Thus, the C-terminal processing of Ac-beta-END-(1-31) to -(1-27) and -(1-26) may not yet be fully active at birth, in contrast to the processing of POMC to beta-END. In the anterior lobe of newborn rats, however, the ratio of beta-lipotropin/beta-END resembled that of adults, and more than 80% of beta-END-sized ir-material was found to consist of nonacetylated (and therefore opiate-active) beta-END-(1-31), as in adults, suggesting that the enzymatic system responsible for processing of POMC to beta-lipotropin and beta-END is already mature at birth. The high concentrations of beta-END in the anterior lobe of newborn rats suggest a possible role of this opioid peptide in perinatal development and/or parturition.

Pretranslational and posttranslational mechanisms for regulating beta-endorphin-adrenocorticotropin of the anterior pituitary lobe.

Stress-induced activation of secretion of ACTH and beta-endorphin (beta-END) from anterior lobe corticotrophs leads to both short term and longer term perturbation of the system. Immediately following an acute stress session, the rate of translation of the ACTH/beta-END precursor proopiomelanocortin appears accelerated by 50% and the rate of conversion of the precursor into products is doubled. These changes appear to take place at the translational and posttranslational level and reflect a better use of the preformed messenger RNA which compensates for the stress-induced peptide depletion. When the animal is subjected daily to the stress session, longer term mechanisms appear to emerge. The ACTH/beta-END stores in the gland are increased, apparently owing to an increase in transcription, as reflected by a small but significant increase in proopiomelanocortin messenger RNA. The posttranslational processing is no longer accelerated after further stress. This longer term mechanism appears to be pretranslational and to supplant the posttranslational mechanisms observed after acute stress. These two levels of control may represent different points in the regulation of this critical peptide system.

Alpha-melanocyte-stimulating hormone and N-acetyl-beta-endorphin immunoreactivities are localized in the human pituitary but are not restricted to the zona intermedia.

The adult human pituitary lacks a well defined intermediate lobe, and it is uncertain whether the POMC cells that remain in the zona intermedia represent melanotropes or corticotropes. In the present study, we investigated whether the N-acetylated beta-endorphin- and alpha-MSH-related peptides that are characteristically produced by melanotropes in the rat and other species are localized in the human pituitary. Sequential gel filtration and ion exchange HPLC analysis revealed that small amounts of alpha-N-acetyl-beta-endorphin-(1-31), as well as beta-endorphin-(1-27) and beta-endorphin-(1-26), were detectable in human pituitary extracts, although beta-endorphin-(1-31) was clearly the major form. Consistent with this analysis, low levels of alpha-MSH, but not N,O-diacetyl-alpha-MSH, were identified by reverse-phase HPLC, although again, the desacetyl form of alpha-MSH predominated. Immunohistochemistry revealed that N-acetyl-beta-endorphin immunoreactivity was colocalized with ACTH and beta-endorphin in a subpopulation of zona intermedia cells. Unexpectedly, immunoreactive N-acetyl-beta-endorphin was also observed in a comparable proportion of corticotropes dispersed throughout the anterior lobe. alpha-MSH immunoreactivity was similarly distributed. These results indicate that N-acetylation is not restricted to the zona intermedia, suggesting that the strict dichotomy between corticotrope and melanotrope POMC processing observed in the rat and other species does not extend to the human pituitary.

The effect of various corticotropin-releasing factor trains on the release of adrenocorticotropin, beta-endorphin, and beta-lipotropin from perifused ovine pituitary cells.

The dynamics of the release of proopiomelanocortin-derived hormones from ovine anterior pituitary cells in response to varying pulse characteristics of ovine corticotropin-releasing factor (CRF) were investigated with an in vitro automated 15-column simultaneous perifusion system. Columns of cells were stimulated continuously or with trains of CRF pulses of varying pulse length (2-16 min), pulse period (20-160 min), and concentration, for 500 min. Ovine ACTH, beta-lipotropin, and beta-endorphin immunoreactivity were measured by unextracted RIA. Each pulse of CRF stimulated clearly defined and highly correlated (r greater than 0.9) pulses of the three pituitary hormones, suggesting similar mechanisms controlling release. In dose-response experiments, the minimum concentration of CRF in a 10-min pulse required to significantly raise the output of ACTH was 200 pM, and initial responses had not attained maximal levels with concentrations of CRF increased to 2 microM. Responses to pulsed CRF stimulation decreased with time with all stimulation patterns selected, although previously unstimulated control columns retained the initial capacity to respond. Multiple linear regression analysis showed that hormone output per pulse of CRF (43 nM) increased with increasing pulse period and pulse length. Output of pituitary hormones per unit of CRF applied decreased with pulse length but increased with pulse period. In summary, the responses of proopiomelanocortin derivatives were shown to be sensitive to abrupt increases in CRF, to reduce output under continued stimulation, and to have an inherent time lag before responding maximally to subsequent pulsed stimulation.

Coordinate regulation of peptide acetyltransferase activity and proopiomelanocortin gene expression in the intermediate lobe of the rat pituitary.

Proopiomelanocortin (POMC) is posttranslationally processed in the intermediate lobe of the pituitary to both N-terminally acetylated and nonacetylated forms of alpha MSH and beta-endorphin (beta END). N-Acetylation substantially modifies the physiological responses produced by both peptides, suggesting that the activity of the peptide acetyltransferase, which posttranslationally acetylates beta END and des-acetyl-alpha MSH, may play an important role in defining the biological activity of the secretory products of the intermediate pituitary lobe. The present results demonstrate that peptide acetyltransferase activity is induced by treating rats chronically with the dopamine receptor antagonist haloperidol. Haloperidol administration produced parallel and essentially equivalent increases in acetyltransferase activity, POMC mRNA levels, and the content and secretion of POMC-derived peptides in the neurointermediate pituitary. Time-course and dose-response studies further demonstrated that acetyltransferase activity covaried with POMC mRNA and peptide levels. Chronic treatment with the dopamine receptor agonist bromocriptine had the opposite effects; it lowered acetyltransferase activity, POMC mRNA levels, and alpha MSH and beta END immunoreactivities. Subcellular fractionation showed that acetyltransferase activity was localized in three subcellular compartments corresponding in density to secretory vesicles, rough endoplasmic reticulum and Golgi apparatus, and cytosol. Haloperidol treatment significantly increased the specific activity of the secretory vesicle-associated acetyltransferase without affecting the specific activity of the enzymes present in the endoplasmic reticulum or cytosol. Together, these data indicate that peptide acetyltransferase activity and POMC biosynthesis are coregulated.

Biosynthesis of proopiomelanocortin-derived peptides in prohormone convertase 2 and 7B2 null mice.

Prohormone convertases (PCs) are thought to represent the major proteinases involved in the biosynthetic processing of peptide hormone precursors to bioactive peptide products. The maturation of PC2 requires the aid of a helper protein, 7B2, in order for the zymogen to become an active enzyme species. The 7B2 and PC2 nulls should thus be functionally equivalent with regard to deficits in precursor processing. In this article, we have examined this proposition through the study of proopiomelanocortin (POMC) biosynthesis and granule content in both null models. RIA data indicate that both PC2 and 7B2 nulls lack pituitary alpha-MSH; interestingly, 7B2 nulls are still able to generate beta-endorphin from beta-lipotropin, whereas PC2 nulls contain little if any beta-endorphin. Labeling experiments demonstrate a build-up of POMC, high molecular weight intermediates, and intact ACTH, as well as the disappearance of alpha-MSH, in both null models. Electron microscopy of neurointermediate lobe melanotrophs reveals the presence of a significantly greater number of secretory granules in both 7B2 and PC2 nulls compared with wild-type controls. However, PC2 null melanotrophs contain twice as many granules as 7B2 null melanotrophs. Another difference between the two null models is a relatively enhanced accumulation of precursors in the PC2 null compared with the 7B2 null; these include not only PC2 substrates, but also presumed PC1 substrates. These data indicate that the two nulls are not phenotypically equivalent.