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.

When isolated rat pars intermedia cells were incubated for 10 min with radioactive amino acids, one major labeled protein with a molecular weight of 30,000 ± 1500 was extracted. This protein was shown to contain in its sequence the antigenic determinants for corticotropin and P-melanotropin by immunoprecipitation. When the radioactivity incorporated into this large molecular weight protein during the first 10 min was chased by a further incubation in presence of an excess of unlabeled amino acid, the initial protein was degraded into several smaller peptides including #-endorphin and ,¢lipotropin.Another 18,000-dalton peptide was also observed and was tentatively identified as a large molecular form of corticotropin. From the kinetics of the maturation of the initial precursor, it is concluded that the initial cleavage of the 30,000-dalton peptide gives rise to f-lipotropin and the 18,000-dalton form of corticotropin. f-Lipotropin is subsequently cleaved to form 0-endorphin. The intermediate lobe of the pituitary has been shown to contain a number of peptide hormones including corticotropin (ACTH) (1-3), a-melanotropin (a-MSH) (4,5), corticotropinlike intermediate lobe peptide (CLIP) (5, 6), f,-lipotropin (13-LPH) (7-9), fl-endorphin (9-11), and f3-MSH (4, 12). a-MSH and CLIP are structurally related to ACTH; ,B-LPH is considered to be the precursor for , .Immunocytochemical studies have shown that ACTH and f3-LPH immunoreactive peptides occur in all of the parenchymal cells of the pars intermedia (7, 9, 11). Furthermore, when these cells were observed under the electron microscope, staining for ACTH and fl-LPH was seen in all the granules and in the structures of the rough endoplasmic reticulum (9, 14). These results agree with the hypothesis of a common precursor for ACTH and fl-LPH, which has recently had two good experimental confirmations (15-17).In both studies, the experimental model was the ACTHsecreting mouse pituitary cell line used a double-immunoprecipitation technique to isolate labeled proteins from cells incubated with radioactive amino acids. Roberts and Herbert (16, 17) prepared a cell-free translation product from a poly(A)-containing mRNA fraction obtained from AtT-20 cells. In both cases, the ACTH/,B-LPH precursor seems to be a 28,000-31,000 dalton protein that is cleaved into f3-LPH, fl-endorphin, and several high molecular weight forms of ACTH (15,18,19).The primary purpose of this study was to investigate the ACTH/,B-endorphin biosynthetic pattern in the intermediate lobe of rat pituitaries. We report here that, during short incubations with labeled amino acids, rat pituitary intermediate lobe cells synthesize predominantly one 30,000 + 1500 dalton protein that bears antigenic determinants for both ,B-MSH and ACTH. Pulse-chase labeling shows that this peptide matures into several products including f,-LPH and ,B-endorphin. METHODSPreparation of Rat Pars Intermedia Cells. Sprague-Dawley male rats (Canadian Breeding Laboratory, St-Constant, Quebec) (150-200 g) were killed by decapit...

In the pars intermedia of rat pituitary glands, two forms of a common precursor for corticotropin (ACTH) and fl-lipotropin with apparent molecular weights of 34,000 and 36,000 were resolved by sodium dodecyI sulfate/acrylamide gradient slab gel electrophoresis. Hi -prformance liquid chromatographic analysis of [asS~methionine-labeled tryptic fragments of the two forms of the precursor revealed that both contained copies of ACTH-1-8) and fl-lipotropin4(61-69) sequences. When biosynthetic studies were performed in the presence of tunicamycin, the 34,000-and 36,000-dalton forms were replaced by a peptide with an apparent molecular weight of 32,000. It was therefore concluded that the 34,000-and 36,000-dalton forms of the precursor represent two glycoprotein variants of similar polypeptides, differing in the number of asparagine-linked carbohydrate moieties. During pulse-chase incubations with , the precursor forms were cleaved into two major groups of labeled products: (i) ,-endorphin and (ii) a mixture of ACTH fragments closely related to a-melanotropin. No ACTH-(1-39) was found at the end of a 2-hr chase period, suggesting that ACTH is not a significant hormone product of the rat pars intermedia.

ABSTRACr Three 3-hr incubations of pars intermedia cells from 40 rat pituitaries with [35Slmethionine, [3H lysine, and [3H]leucine sufficed for the identification and chemical characterization of biosynthesized P-lipotropin, 'y-lipotropin, and ,-endorphin. From the molecular weight, migration on polyacrylamide gels, and sequence Met5, Lys9, Leu14"17, rat a-endorphin was shown to be identical to its shee homologue and no trace of Leu5 fl-endorphin could be detec. Rat 04ipotropin differs from that of sheep in its elution propMies on CM-cellulose, and its sequence shows Leu2'10'14, shows the same NHrterminal sequence as P-lipotropin and is again different from its sheep homologue. The identification of rat f4-lipotropin was confirmed by its selective cleavage into P-endorphin after sin di estion of the citraconylated peptide, a property not observed with rat y-lipotropin. Recently, it was shown that cells of whole bovine pituitaries (1-4) and of bovine pars intermedia (5) actively incorporate radiolabeled amino acids into #-LPH, y-LPH, and #l-endorphin. The pars intermedia of pituitary glands synthesizes relatively large amounts of LPH and corticotropin (ACTH) and their related fragments (6, 7). Immunocytochemical studies showed that immunoreactive 6-LPH and corticotropin are uniformly distributed in all of the secretory cells of the pars intermedia in a number of species (8). Such a rich source of these peptides was thus chosen as a model for study of their in vitro biosynthesis in the rat. The anterior and whole rat pituitaries were recently shown, by an elegant micropurification technique, to contain both fl-LPH (9) and fl-endorphin (10). The fl-endorphin segment was found to be homologous to the corresponding peptide from sheep, bovine, and camel, by amino acid analysis and peptide mapping (10). However, no sequence information is available on rat (l-LPH, nor was y-LPH The isolated cells were harvested and incubated for 3 hr in 2 ml of the above medium as described (5) (5) by using 5 M acetic acid/ phenylmethylsulfonyl fluoride (0.3 mg/ml)/iodoacetate (0.3 mg/ml)/bovine serum albumin (5 mg/ml) containing 5 mmol of the corresponding unlabeled amino acid. The extract was then desalted on a Sephadex G-25 column (1.5 X 60 cm) with 0

Analogues of the endogenous opiate-receptor ligand [5-methionine]enkephalin (H-Tyr-Gly-Gly-Phe-Met-OH) were designed and synthesized for the purpose of testing the proposed similarity in spatial structure between this peptide and morphine derivatives. In the bioassay (inhibition of electrically induced contractions of the mouse vas deferens) [1-O-methyltyrosine,5-methionine]enkephalin, [1-N-methyltyrosine,5-methionine]enkephalin, [4-tryptophan,5-methionine]enkephalin, and [5-methionine sulfoxide]enkephalin possess, respectively, 0.4, 21, 27, and 67% activity of [5-methionine]enkephalin. These morphinomimetic activities correlate well with the opiate receptor affinities determined by displacement of [3H]naloxone in a guinea pig brain membrane preparation. The effects of O-methylation of the tyrosine residue and N-methylation of the terminal amino group on biological activity and receptor affinity support the hypothesis that the latter two moieties in the peptide correspond to the phenol group and the tertiary nitrogen, respectively, in morphine. Determination of the efficiency of energy transfer from tyrosine in position 1 to tryptophan in position 4 in [4-tryptophan,5-methionine]enkephalin from both tyrosine fluorescence quenching and relative enhancement of tryptophan fluorescence by means of a modified procedure permitted the calculation of an average intramolecular tyrosine-tryptophan separation of 10.0 +/- 1.1 A. Inspection of CPK models showed excellent agreement between this value and both the intrafluorophore distance in the 4 leads to 1 and 5 leads to 2 hydrogen bonded betaI-bend models of [4-tryptophan,5-methionine]enkephalin (9-11 A) and the phenol-phenyl separation in the potent morphine derivative 7alpha-(1(R)-hydroxy-1-methyl-3-phenylpropyl)-6,14-endo-ethenotetrahydrooripavine (8-10.5 A). The ensemble of these findings suggests an analogous topography for [5-methionine]enkephalin and morphine-oripavine derivatives.