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In summary, the pituitary glands from at least four species contain betaLPH and and beta endorphins. We have shown that in slices of whole pituitaries betaLPH is actively biosynthesized and transformed into gammaLPH, thus releasing the COOH-terminus portion 61--91, which is now known as beta-endorphin. Newly radioactive biosynthesized beta-endorphin has been clearly and definitely identified. The release of betaMSH and possibly of beta-endorphin could well be under the control of CRF. The intermediate lobe of the pituitary seems to be the tissue that contains most of betaLPH and beta-endorphin, although these are also present in the anterior lobe. We have recently demonstrated its presence in human glands and the structure is completely identical to the COOH-fragment 61--91 of human betaLPH. Thus far, these morphine-like peptides seem not to cross the blood-brain barrier in rats; it is conceivable (neurophysiologists will need to look into it) that an upward circulatory process could bring beta-endorphin into the brain where it is concentrated in different regions as either native or degraded products both of which have similar activities. Until somebody shows that betaLPH and beta-endorphin are actively biosynthesized in other tissues, one can only assume that the pituitary gland is the primary source of the endogenous opiate substance(s) and that betaLPH is its or their biologic precursor. We have worked on the proposed biosynthetic model for many years and we are continuing because all of the experiments, except one from another laboratory, 38indicate that we are moving slowly toward its confirmation. Thus far, there is no reason to believe the contrary, and we are following in some ways Konrad Lorenz's maxim, which appeared in his book Die Acht Todsunden Der Zivilisierten Menscheit, published in French in 1973: "Une bonne hypothése de travail gagne en vraisemblance lorsque, au cours de longues années de recherches, nulle donnée n'est venue la contredire." The major conclusion of our most recent studies on the biosynthesis of betaLPH and its related peptides have led us to the first in vitro biosynthesis of an endogenous morphine-like substance. This constitutes a major step in the comprehension of this exciting new field.

The molecule beta-lipotropin, composed of 91 amino acids (beta-LPH 1-91) has gained considerable importance in recent years. Its double role as the precursor of beta-MSH (beta-LPH 41-58) and beta-endorphin (beta-LPH 61-91) makes this peptide unique in its kind. Results are presented on the role of this molecule and on the complete characterization of two morphine-like peptides from human and sheep pituitaries. The structure-activity relationship of opiate activity is analyzed by scanning for this biological activity of many tryptic and CNBr fragments of beta-lipotropin. The unequivocal localization of one of the important synthesis sites of beta-endorphin in the pituitary neurointermediate lobe is presented. A peptide with partial sequence Met1, Leu8,15 and Lys6,11, 27, 29, 33 has been biosynthesized in large quantities and its ubiquitous nature and conservation of sequence speaks for its importance and possible presence in many living cells. This polypeptide was subsequently identified as ubiquitin, a non-histone fragment of the nuclear protein A-24.

We have demonstrated that beta-lipotropin is a precursor molecule and that during its maturation, it gives rise to beta-endorphin. This model is similar to proinsulin, proparathyroid hormone and proglucagon models. Since beta-endorphin is also found in the hypothalamus, one can foresee that its biosynthesis will be similar if not identical. It is then conceivable to believe that the other hypothalamic factors are issued from larger precursor molecules. When this will be shown, and well established, studies on their control mechanism will be much easier.

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.

Studies on the biosynthesis of beta-LPH on the one hand, and of ACTH on the other, have produced a new concept, that of a single precursor form which contains three active molecules. Thus, it is proper to name such a precursor 'pro-opio-melanocortin.' The concept that beta-LPH was a precursor molecule was first put forward in 1967 and was based on both structural forms and biological activities. The discovery that morphine-like substances are part of the C-terminal fragment of beta-LPH brought an additional important biological side product. That, together with the recent demonstration of ACTH as part of a still larger precursor, constitutes an exciting model for the study of peptide hormone biosynthesis. We have shown unambiguously that beta-endorphin is the result of a maturation process from the large precursor, while beta-LPH is an important and transient intermediary. Since it is also present in the brain, our recent results using pars intermedia cells can be applied to study the fabrication and degradation of these molecules in the brain. We expect to see it established that all other neuropeptides are also biosynthesized as larger precursor molecules whose structure at the site of cleavage could well be constituted of two basic amino acids like in the pro-opio-melanocortin.

Three 3-hr incubations of pars intermedia cells from 40 rat pituitaries with [35S]methionine, [3H]lysine, and [3H]leucine sufficed for the identification and chemical characterization of biosynthesized beta-lipotropin, gamma-lipotropin, and beta-endorphin. From the molecular weight, migration on polyacrylamide gels, and sequence Met5, Lys9, Leu14,17, rat beta-endorphin was shown to be identical to its sheep homologue and no trace of Leu5 beta-endorphin could be detected. Rat beta-lipotropin differs from that of sheep in its elution properties on CM-cellulose, and its sequence shows Leu2,10,14, Lys20. Rat gamma-lipotropin shows the same NH2-terminal sequence as beta-lipotropin and is again different from its sheep homologue. The identification of rat beta-lipotropin was confirmed by its selective cleavage into beta-endorphin after trypsin digestion of the citraconylated peptide, a property not observed with rat gamma-lipotropin.

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.