beta-Endorphin in human plasma: basal and pathologically elevated levels.

β-Endorphin-like immunoreactivity was measured in plasma of normal human subjects and in plasma of patients with pathologically elevated ACTH levels. The antiserum used displayed the same avidity for human β-endorphin and human β-lipotropin (detection limit for both peptides 1–2 fmoles/tube). Gel chromatography of the immunoreactive components in plasma of normal subjects indicated the presence of both β-lipotropin (2.1 -10.1 fmoles/ml) and β-endorphin (3.5–6.4 fmoles/ml). A close correlation between immunoreactive β-endorphin and ACTH was found in plasma of patients suffering from Addison's disease, Cushing's disease and exhibiting Nelson's syndrome. Elevated levels of β-endorphin-like immunoreactivity in plasma of these patients were due to both β-lipotropin and β-endorphin.     

Radioimmunoassay for β-endorphin: Presence of immunoreactive “big-big” β-endorphin (“big” β-lipotropin) in human and rat pituitaries

A sensitive and specific radioimmunoassay for β-endorphin has been developed with an antiserum obtained in a rabbit immunized with β-endorphin contained in crude porcineACTH preparations. The minimal detectable quantity was 5 pg. The antiserum used reacted slightly with ovine β-lipotropin (5.5 %), but showed negligible cross-reactivity with other fragments of β-lipotropin, α-MSH and ACTH. Using this radioimmunoassay we have observed the presence of “big-big” β-endorphin (“big” β-lipotropin) with apparent molecular weights of 37,000 and 31,000 in human and rat pituitaries respectively, in addition to β-lipotropin and β-endorphin, by Sephadex gel-chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Isolation and properties of ovine [1-pyroglutamic acid]-β-lipoprotein

A new form of ovine β-lipotropin has been isolated by partition chromatography on agarose gel. All the evidence is consistent with the conclusion that its structure is identical to that of ovine β-lipotropin with the exception that the glutamic acid residue in position 1 was replaced by a pyroglutamic acid residue. Its lipolytic activity in isolated rabbit fat cells was about one-half that of β-lipotropin.     

Corticotropin/beta-endorphin precursor: concomitant storage of its fragments in the secretory granules of anterior pituitary corticotropin/endorphin cells.

Serial semithin und ultrathin sections of Epoxy resin embedded rat anterior pituitaries were sequentially immunostained for corticotropin, β-lipotropin, β-endorphin and 16k-fragment. The 16k-fragment corresponds to the non-β-lipotropin-non-corticotropin part of the corticotropin/β-endorphin precursor. Immunostaining was performed by the peroxidase-antiperoxidase method. Corticotropin, β-lipotropin, β-endorphin and the 16k-fragment were stored in the same cells of the anterior pituitary. Cross absorption experiments confirmed the specificity of the immunocytochemical reactions. At the ultrastructural level immunoreactivities to all these parts of the corticotropin/β-endorphin precursor were found to be stored together in the same secretory granules. It is concluded that under all stimulatory or inhibitory conditions all fragments will be released together during secretion by granule extrusion.     

Beta-Endorphin: lack of correlation between opiate activity and immunoreactivity by radioimmunoassay.

A sensitive radioimmunoassay for human β-endorphin has been developed. When natural human β-lipotropin and various synthetic analogs of β-endorphin were assayed for their immunoreactivity, lack of correlation was found between opiate activity and immunoreactivity. These data suggest that residues 6–15 of β-endorphin are the antigenic determinant.     

β-lipotropin increases plasma insulin immunoreactivity

Porcine β-lipotropin increased plasma insulin immunoreactivity and glycerol levels in the rabbit. These effects could not be reversed by the opioid antagonist naloxone suggesting that these in vivo effects are not mediated by an opiate receptor. Since serum glucose concentrations remained unaffected despite the increase of insulin a simultaneous release of glucagon is suggested. We conclude that β-lipotropin might be of physiological importance for the regulation of glucose metabolism.     

Isolation of corticotropin-β-lipotropin common precursor from ovine pituitary glands

Ovine corticotropin-β-lipotropin common precursor was purified to homogeneity from commercial frozen ovine pituitary glands. A crude preparation was obtained following a procedure published elsewhere (Lee, T.H. and Lee, M.S. (1977) Biochemistry 16, 2824–2829) and was purified by gel filtration on Sephadex G-200 in the presence of 0.5% SDS and 0.1% 2-mercaptoethanol, and under an atmosphere of nitrogen. The gel filtration was repeated once. The partially purified preparation obtained from the second Sephadex G-200 gel filtration was further fractionated by preparative SDS-acrylamide gel eletrophoresis, using immunoprecipitated and electrophoretically purified [¹²⁵I]corticotropin-β-lipotropin common precursor as a marker. The preparation was judged homogenous by the appearance of a single protein band in analytical SDS-acrylamide gel electrophoresis, which exhibited both corticotropin and β-lipotropin immunoreactivities, and a single symmetrical peak in high-pressure liquid chromatography on a reverse phase C18 column. The isolated ovine corticotropin-β-lipotropin common precursor possessed specific activities of 116 μg of immunoreactive corticotropin and 210 μg of immunoreactive β-lipotropin per mg of protein, equivalent to 89 and 62% of theoretical values, respectively. The amino acid composition of the homogeneous preparation was determined.     

Development and validation of a sensitive radioimmunoassay for naturally occurring beta-endorphin-like peptides in human plasma

β-Endorphin-like peptides in blood plasma of normal human subjects were studied by means of a radioimmunoassay (RIA) and gel filtration. Plasma was extracted with silica gel, which was washed with water and 1 n HCl, and eluted with 50% acetone. Plasma extracts thus obtained and standard synthetic human β-endorphin yielded parallel RIA curves. Total immunoreactivity in normal donors ranged from 1.2 to 10.4 fmol/ml (21 subjects). The immunoreactivity was completely destroyed by treatment with papain. Gel filtration indicated the presence of three components-one of unknown nature at the void volume and the others at elution positions characteristic of β-lipotropin and β-endorphin. Recoveries of human β-endorphin and β-lipotropin added to plasma were 53 and 58%, respectively. Addition of N-ethylmaleimide to plasma or of aprotinin to blood immmediately following collection had no effect on the amount of total immunoreactivity. Furthermore, a large amount of β-endorphin-like immunoreactivity. The above results lead us to conclude that a β-endorphin-like immunoreactive peptide occurs naturally in plasma of normal human subjects.     

β-lipotropin-like material in human pancreas and pyloric antral mucosa

Evidence for the presence of the endogenous opioid precursor β-lipotropin has been found in human pyloric antral mucosa and pancreas by radioimmunochemical displacement curves, Sephadex chromatography, ion exchange chromatography, and immunocytochemistry. Endogenous opioids are not only present in the pituitary gland and nervous tissue but also in the stomach and pancreas, supporting the concept of a common neuro-endocrine system present in brain and gut.     

Conformational properties of central nervous system myelin basic protein, beta-endorphin, and beta-lipotropin in water and in the presence of anionic lipids

Conformational properties have been examined for three proteins which are disordered when dissolved in water but become partially ordered in the presence of anionic lipids. The three proteins, which play important roles in the central nervous system, are myelin basic protein, β-endorphin, and β-lipotropin. When evaluated using matrix methods, the helical content of each protein is predicted to be vanishingly small in water, in agreement with experiment. Unperturbed root-mean-square radii of gyration are also evaluated for these proteins in water using generator matrices, which have seen wide application to synthetic polymers. Agreement between computed and measured dimensions is found to be excellent. Having successfully described the conformations of myelin basic protein, β-endorphin, and β-lipotropin in water, attention is then directed to the changes induced upon interaction with anionic lipids or detergents. Computations predict an increase in helical content, with numerical results being in quite good agreement with experimental observations using several anionic lipids. Examination of the helix-propagation-probability profiles reveals an interesting feature of regions where this probability is high. When folded into a α-helix, these regions show one surface where the only side chains are hydrophobic. Charged side chains (with positive charges predominating) are found on the other surface of the helical segment. The arrangement of side chains on these helices is thus well suited to promote favorable interactions with a membrane containing anionic lipids. Examples of the occurrence of these helices are provided by amino acid residues 13–25 and 130–157 in myelin basic protein and residues 17–29 in β-endorphin.     

Elevated plasma beta-endorphin levels in pregnant women and their neonates.

Using radioimmunoassay technique β-endorphin levels were measured in the plasma of women undergoing labour and partirition and in the plasma of their neonates. The level of immunoreactive β-endorphin in the plasma of women undergoing labour was found to be significantly elevated (mean values: 38–135 fmoles/ml) above the levels found in non-pregnant women (mean values: 5–10 fmoles/ml). After birth, the level of β-endorphin-like immunoreactivity in maternal venous plasma was significantly higher than that in the umbilical vein and artery plasma of the new-borns, but there was no arterio-venous difference in the neonatal plasma. Since the antiserum used displayed the same avidity for human β-endorphin and β-lipotropin chromatographic separation of the immunoreactive components was performed by gelfiltration. Both peptides were found in the plasma of non-pregnant women, in maternal plasma and in the plasma of the neonates. In addition, high amounts of both peptides were found in the fetal pituitary gland showing that the fetus can probably produce its own peptides.     

Cathepsin D generates gamma-endorphin from beta-endorphin.

A crude extract of porcine anterior pituitary was found to contain endopeptidase activity that splits the Leu⁷⁷-Phe⁷⁸ peptide bond of β-lipotropin in a pH range 3.0–7.0. The specificity and susceptibility to pepstatin of pituitary extract were the same as those of cathepsin D (E.C. 3.4.23.5) isolated from calf brain by affinity chromatography. Cathepsin D was shown to split the same Leu-Phe peptide bond of β-endorphin, leading to the formation of γ-endorphin. Based on the above data it is suggested that cathepsin D is a major factor in the generation of endorphins of intermediate size.     

Amino acids and peptides: XLI. A possible active site homology between carbonic anhydrase and β-lipotropin hormone

The hexapeptide l-histidyl-l-phenylalanyl-l-histidyl-l-tryptophanyl-glycyl-l-serine was synthesized because of its possible relationship to the known active-site residues in both carbonic anhydrase and β-lipotropin. The compound possessed weak anhydrase activity.     

Isolation,characterization and opiate activity of β-endorphin from human pituitary glands

The isolation of a 31-amino acid peptide from human pituitary glands has been described. Its amino acid sequence has been proposed to be identical to the sequence of the carboxyl-terminal 31 amino acids of human β-lipotropin. The peptide, designated as β_h-endorphin, possesses significant opiate activity.     

Immunoreactive calcitonin in the intermediate lobe of the pituitary gland.

Immunoreactive-calcitonin was observed in all cells of the intermediate lobe of the rat pituitary gland. It may thus be a fragment of the ≥31, 000 daltons precursor molecule of adrenocorticotropin and β-lipotropin.     

beta-Endorphin: a pituitary peptide with potent morphine-like activity.

The morphine-like peptide, β-endorphin, from pituitary glands of various species including man, has been characterized chemically and biologically. It is a 31-amino acid peptide corresponding to the amino acid sequence of residues 61–91 of β-lipotropin. It possesses potent analgesic activity when injected intracerebroventricularly or intravenously. In addition to analgesic activity, β-endorphin causes behavioral changes in experimental animals. This paper presents current knowledge of this psychopharmacologically active peptide.     

Presence of immunoreactive β-lipotropin and β-endorphin in human placenta

Utilizing a sensitive radioimmunoassay capable of detecting both β-lipotropin (β-LPH) and β-endorphin (β-EP), we demonstrated the existence of immunoreactive β-LPH and β-EP in extracts of human placentas. Gel chromatographic studies revealed that total β-EP immunoreactivity consists of two fractions with elution positions compatible with β-LPH and β-EP, respectively, and a fraction of larger molecular weight. All three fractions showed parallel curves with the standard curve of β-EP in radioimmunoassay. Our observations suggests that β-EP, β-LPH and possibly their precursor exist in human placenta.     

Demonstration of beta-lipotropin activating enzyme in porcine pituitary.

Porcine β-lipotropin was incubated with a crude porcine pituitary homogenate, and the main cleavage products of the hormone were isolated and identified. Our results gave evidence for the enzymic cleavage of the Lys⁴⁶-Met⁴⁷, Arg⁶⁰-Tyr⁶¹, Leu⁷⁷-Phe⁷⁸, and Lys⁷⁹-Asn⁸⁰ bonds of the β-lipotropin structure. The cleavage of the Arg⁶⁰-Tyr⁶¹ peptide bond was accompanied with the concomitant release of opiate activity in the first period of incubation, provided that bacitracin was present in the incubation mixture. The enzyme was differentiated from trypsin or plasmin and appears to be a specific intracellular protease involved in the biosynthesis of pituitary endorphins.     

Cross tolerance between morphine and β-endorphin in vivo

Experiments were performed to establish the development of cross tolerance between morphine and the C-terminal fragment of porcine β-lipotropin /LPH_61–91, β-endorphin/ in rats. Repeated intracerebroventricular /ICV/ or peripheral administration of morphine induced tolerance both to morphine and β-endorphin. The tolerance induced by ICV administration of morphine was more pronounced than in the case of peripheral application. The results give support to the theory that at least in part similar sites and mechanisms are involved in the analgesic activity of morphine and the endorphins.