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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.

beta-Endorphin is a 31 amino acid polypeptide isolated from the pituitary gland of different species of animals. It has strong morphine-like activity. It is formed of amino acid residues 61-91 of beta-lipotropin. Speculation has arisen whether it is biosynthesized in situ or transformed after secretion of beta-lipotropin. The present in vitro studies show that it is found as beta-endorphin in bovine pituitary slices incubated with radioactive amino acid precursor [35S]methionine. Chemical characterization and microsequencing of the newly biosynthesized material proves its identity with isolated unlabeled beta-endorphin and shows that it has a methionine residue at its fifth position, as expected.

A novel glycopeptide has been isolated from human and porcine pituitary glands. This substance is the amino-terminal fragment of pro-opiomelanocortin (POMC). The complete amino acid sequence has been determined. When tested for its steroid stimulating activity on an aldosterone secreting human adrenocortical adenoma, it was found to be more potent than angiotensin and ACTH. These results raise the interesting possibility that primary hyperaldosteronism could be related to a pituitary adrenal dysfunction.

Sheep gamma-lipotropic hormone (gamma-LPH) is a pituitary polypeptide made of 58 amino acids and is formed of the first 58 residues of beta-lipotropic hormone (beta-LPH). The C-terminal portion (41-58) of gamma-LPH is identical with the structure of beta-melanophore-stimulating hormone (beta-MSH). We hypothetized in 1967 that beta-LPH could be the biological precursor of beta-MSH and that gamma-LPH could be an intermediate compound. We demonstrated in 1974 that beta-LPH is actively synthesized in the bovine pituitaries. We now studied the biosynthesis of gamma-LPH by monitoring the incorporation of radioactive amino acids in beef pituitary slices. We separated gamma-LPH from the other radioactive proteins with a method previously described. We characterized the radioactive proteins by ion-exchange chromatography, gel filtration and polyacrylamide gel electrophoresis. Our results show that radioactive gamma-LPH was actively synthesized. This gamma-LPH has all the chemical characteristics of nonradioactive gamma-LPH. However, in the conditions used, we were unable to demonstrate biosynthesis of beta-MSH. These results suggest that gamma-LPH is biosynthesized more slowly than beta-LPH and that the conversion into beta-MSH, if it exists, is a slow or subactive process in the species studied.

The isolation and complete purification of a human glycopeptide representing the major immunoreactive form of the pituitary NH2-terminal segment of pro-opiomelanocortin is presented. The complete sequence of this peptide was determined following CNBr fragmentation and it is shown to be 76 amino acids long. It bears an O-glycosylation site at Thr 45 and an N-glycosidic linkage at Asn 65. Compared to the reported genomic DNA sequence (Chang, A. C. Y., Cochet, M., and Cohen, S. N. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 4890-4894), one variation exists, namely Arg 22 replacing Gly 22. Two disulfide bridges linking Cys 2 to 8 and Cys 20 to 24 have been determined. Based on the sequence and disulfide bridge localization, a large degree of homology exists between the NH2-terminal sequence of the human peptide and all calcitonins, especially porcine calcitonin. The human NH2-terminal peptide is shown to stimulate the release of aldosterone from isolated cells of a human adrenal tumor, in at least equipotency to adrenocorticotropic hormone and the porcine NH2-terminal analogue, which is 100 times more potent than angiotensin II. Finally, this reported sequence completes that of the human DNA which was lacking the first 19 amino acids due to the presence of a 2-kilobase intron.

A new peptide having both lipolytic and melanotropic properties has been isolated from human pituitary glands. It has a molecular weight around 11,000, an amino acid composition different from the known lipolytic-melanotropic hormones, and an isoelectric point of 8.5. Although it is not entirely pure, there is no doubt that it differs from other known lipolytic-melanotropic substances in its total lack of methionine and tryptophan and its unusually high content of lysine and histidine. It has a melanocyte-stimulating hormone activity of 19 units/mug. As a lipolytic agent it is active with rabbit adipocytes, only slightly active with human cells and inactive with adipocytes from the rat.

Synthetic alpha-melanotropin stimulated the release of immunoreactive adrenocorticotropin from primary cultures of rat anterior pituitary cells. The effect of the alpha-melanotropin was dose-dependent. Cells incubated with synthetic arginine-vasopressin and alpha-melanotropin simultaneously produced an amount of adrenocorticotropin that was greater than the sum of the amount that the cells produced in response to each peptide added separately. Other peptides structurally similar to alpha-melanotropin, such as, beta-, gamma 1-, gamma 2-, and gamma 3-melanotropin, were also tested for adrenocorticotropin-releasing activity. Only the gamma 3-melanotropin demonstrated a statistically significant effect. A vasopressin preparation (Pitressin, Parke-Davis) purified from posterior pituitaries and previously shown to contain some alpha-melanotropin was much more potent in releasing adrenocorticotropin than the synthetic vasopressin.

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 beta-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 beta-endorphin and beta-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 beta-lipotropin and the 18,000-dalton form of corticotropin. beta-Lipotropin is subsequently cleaved to form beta-endorphin.

In the pars intermedia of rat pituitary glands, two forms of a common precursor for corticotropin (ACTH) and beta-lipotropin with apparent molecular weights of 34,000 and 36,000 were resolved by sodium dodecyl sulfate/acrylamide gradient slab gel electrophoresis. High-performance liquid chromatographic analysis of [35S]methionine-labeled tryptic fragments of the two forms of the precursor revealed that both contained copies of ACTH-(1-8) and beta-lipotropin-(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 [35S]methionine, the precursor forms were cleaved into two major groups of labeled products: (i) beta-endorphin and (ii) a mixture of ACTH fragments closely related to alpha-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.

Proopiomelanocortin, the common glycoprotein precursor to adrenocorticotropin (ACTH) and beta-lipotropin (beta-LPH), is the most abundant protein synthesized in rat neurointermediate lobes. It represents 30% of the total amount of radioactive proteins obtained after a 1-h pulse incubation with [3H]phenylalanine. Several forms of this protein can be separated by a high-resolution two-dimensional gel electrophoresis technique. The three most abundant species which can be reproducibly characterized by their apparent molecular weights (Mr) and isoelectric points (pI) were called form I (Mr 34 000; pI 8.2), form II (Mr 36 000; pI 8.2), and form III (Mr 35 000; pI 7.3). Additional minor forms, representing together approximately 30% of the total forms I, II, and III combined, are also observed. They have very close molecular weights but differ by their isoelectric points. When glycosylation is prevented by tunicamycin, forms I and II are replaced by a new molecule with the same pI of 8.2 but a slightly lower Mr (32 000). This form is referred to as form T1. Similarly, form III is replaced by form T2 (Mr 33 000; pI 7.3). Forms T1 and T2 are supposed to be nonglycoslyated peptides. They were further characterized by microsequencing and peptide mapping. They both have the same N-terminal amino acid sequence with leucine residues in positions 3 and 11, and they both contain identical [3H]phenylalanine-labeled tryptic fragments, two of them corresponding to the sequences 1-8 of ACTH and 61-69 of beta-LPH. However, a limited digestion with the Staphylococcus aureus (V8 strain) protease generates a collection of peptides different for each form. These results suggest the presence of at least two different gene products corresponding to the major forms of proopiomelanocortin in the rat pars intermedia.

The affinity for antiserum to the multipotent lipotropic hormone (beta-LPH) was tested by immunohistochemical staining of all known cell types in normal and certain abnormal mouse, rat, and human pituitaries. Results indicate that beta-LPH has ACTH, MSH, LH and StH(GH) immunologically cross-reacting determinants. Affinities of anti-LPH for TtH and MtH (prolactin) were not detected in normal pituitaries, but thyrotropic tumor cells reacted with anti-LPH. Absorption experiments confirm that the single polypeptide hormone of the pituitary, beta-LPH, is coded for ACTH and MSH activities. The multi-functional hormone, LPH probably is secreted by the adrenotropes. In addition to ACTH and MSH, it probably contains other antigenic and biologic determinants. Some of these may accentuate its lipotropic activities; others may be incidental. These are points calling for further correlated structural, biologic, and immunologic investigations.

Labeled amino acids were incorporated with proteins during incubations of isolated cells from the pars intermedia of beef pituitary glands. After 3 hr of incubation with methionine and [3H]lysine, approximately equivalent amounts of labeled gamma-lipotropin and beta-endorphins were isolated. They were found to be major synthesis products of the pars intermedia. In contrast, very little labeled beta-lipotropin was recovered. Another major synthesis product of the pars intermedia was also purified. Its partial amino acid sequence and molecular weight were determined and it was concluded that this peptide cannot be identified as any known pituitary hormone or protein fragment.