Camel Beta-endorphin Research Articles

Neurotrophic effects of beta endorphin C-terminal tetrapeptide (MPF)

The ability of newts and other urolele amphibians to regenerate an accidentally-removed or amputated limb is lost after hypophysectomy, but restored following intraperitoneal injection of beta endorphin, or the C-terminal tetrapeptide sequence of human beta endorphin, Lys-Lys-Gly-Glu (=MPF). The closely related tetrapeptide, Lys-Lys-Gly-Gln (the C-terminal sequence of pig, sheep, and camel beta endorphin), the dipeptide Gly-Glu, the enkephalins, and other N-terminal sequences of beta endorphin (eg gamma endorphin) do not elicit the effect.

Binding characteristics of dermorphin, and [dermorphin 1–7]- βc-endorphin in rat brain membranes

Dermorphin and a camel beta-endorphin ( β c-EP) analog in which residues 1–7 correspond to the dermorphin sequence ([Dermorphin 1–7]- β c-EP) have been investigated with respect to their receptor binding characteristics using human and camel β-EP as reference peptides. Tritiated dihydromorphine, [D-Ala 2, D-Leu 5]-enkephalin, ethylketocyclazocine and human β-endorphin were used as primary ligands in the rat brain membrane preparation for radioreceptor assay. Camel β-endorphin was the most potent peptide in all experiments. [Dermorphin 1–7]- β c-EP is significantly less potent towards 3H-ethylketocyclazocine and 3H-[D-Ala 2, D-Leu 5]-enkephalin but is as potent towards 3H-dihydromorphine and 3H-human β-endorphin. Dermorphin itself weakly displaces tritiated dihydromorphine, [D-Ala 2, D-Leu 5]-enkephalin and ethylketocyclazocine (potency relative to camel β-EP, 1–4%) but it is more potent (9%) in competition with tritiated human β-endorphin. Dermorphin and the [Dermorphin 1–7]- β c-EP appear to interact preferentially with μ opiate receptors.

Investigation by photochemically-induced dynamic nuclear polarization and nuclear Overhauser enhancement 1H-NMR of the interaction between beta-endorphin and phospholipid micelles.

The photochemically-induced dynamic nuclear polarization technique has been used to investigate the access of a photoexcited flavin dye to tyrosyl and histidyl residues in [Met]enkephalin and human and camel beta-endorphins, both alone and in the presence of n-dodecylphosphorylcholine micelles. The results indicate that the mode of binding of Tyr-1, but not of residue 27, is similar in the two endorphins and differs from that of Tyr-1 in [Met]enkephalin. In human beta-endorphin, accessibility and mobility of Tyr-27 are strongly reduced in the presence of lipid at physiological pH, whereas in camel beta-endorphin His-27 becomes immobilized only at high pH. Moreover, nuclear Overhauser enhancement experiments suggest a rigidifying influence of the peptide on the polar head groups of the micelles.

The effect of naloxone on vasopressin release from rat neurohypophysis incubated in vitro.

Rat posterior pituitaries were superfused in vitro and stimulated electrically. The concentrations of vasopressin in the superfusion medium were determined by radioimmunoassay. When the pulses were applied in 10 sec trains with 10 sec intervals, vasopressin release per pulse increased progressively over the frequency range of 3-12 pulses/sec applied within the trains. The release was blocked by addition of tetrodotoxin or by removal of calcium ions from the superfusion medium. The opiate antagonist naloxone 1 or 10 microM was introduced into the superfusion medium before a second period of stimulation and enhanced vasopressin release from neurointermediate lobes after phasic stimulation at 9 pulses/sec within the trains, when compared to controls. However, naloxone 10 microM had not effect on vasopressin release from isolated neural lobes (intermediate lobes removed), although the addition of camel beta-endorphin 2 microM inhibited vasopressin release in a naloxone-reversible manner. After continuous stimulation at a frequency of 13 Hz naloxone 10 microM did not influence the release of vasopressin from neurointermediate lobes. We conclude that the evoked release of vasopressin from the neurointermediate lobe is reduced by an endogenous opiate of intermediate lobe origin, possibly beta-endorphin. Appropriate stimulation conditions are necessary for this mechanism to function.

Synthesis and properties of dermorphin and an analog of beta-endorphin containing the dermorphin sequence.

Dermorphin (I) and [D-Ala2, Phe3, Gly4, Tyr5, Pro6]-beta c-EP (II) have been synthesized by the solid-phase method (beta c-EP, camel beta-endorphin). Positions 1 through 7 of II correspond to the sequence of I. Relative potencies of synthetic peptides in the mouse tail-flick test for analgesia by the intracerebroventricular route were: human beta-endorphin, 100; camel beta-endorphin, 164; I, 450; II, 440. The dermorphin was about 670 times more potent than morphine in the assay. Peptide II represents a rare instance where the enkephalin moiety of beta-endorphin has been altered to produce a more potent analgesic.

β‐Endorphin

Human beta-endorphin analogs with various chain lengths have been investigated for their potency in displacing tritiated dihydromorphine and Leu-enkephalin binding in rat brain membrane preparations. It was found that the reduction of chain length from residues 1-31 to 1-5 led to a gradual loss of preference for the morphined receptor. In addition, the extension of the chain length of the Met-ekephalin segment to the COOH-terminal glutamic acid modified the binding of the NH2-terminal sequence to the enkephalin receptor. The fact that camel beta-endorphin is more potent in displacing the two tritiated primary ligands than the human hormone is also reported herein.

Beta-Endorphin: demonstration of a tertiary structure in aqueous solution.

Difference spectra generated during thermolysin digestion of camel beta-endorphin at pH 8.2 or at pH 6.5 indicate rapid blue shifting of the near-UV absorption bands of the NH2-terminal tyrosine. A similar spectral change is not observed for the NH2-terminal tyrosine in [Met]enkephalin when it is digested under similar conditions. These results suggest that enzymatic digestion destroys or alters some structural interaction between the NH2-terminal tyrosyl residue of the endorphin and a residue(s) within the COOH-terminal segment of the molecule. Peptide mapping of the digest as a function of time suggests that cleavage of the bond linking the alanine-21 and isoleucine-22 residues produces most of the observed effect. These data provide evidence for the existence of a tertiary structure for beta-endorphin in aqueous solutions.

Differences in the post-translational processing of beta-endorphin in rat anterior and intermediate pituitary.

The immunoactive beta-endorphin-related material in extracts of rat anterior and intermediate/posterior pituitary was separated by ion exchange chromatography on sulfopropyl-Sephadex (Zakarian, S., and Smyth, D. G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5972-5976). The profile of beta-endorphin immunoactivity in rat anterior pituitary was distinctly different from that found in intermediate/posterior pituitary. In the rat anterior pituitary, most of the immunoactivity co-migrated with synthetic camel beta-endorphin(1-31); in the intermediate/posterior pituitary, only about 10% of the immunoactivity co-migrated with camel beta-endorphin(1-31). In rat anterior pituitary cell suspensions incubated with [3H]tyrosine for 6 h or 48 h, material co-migrating with camel beta-endorphin(1-31) was the major final posttranslational product related to beta-endorphin. This material was reanalyzed by chromatography on sulfopropyl-Sephadex, analyzed by gel filtration on Sephadex G-50 in 6 M guanidine HCl, and digested with pronase and trypsin. In every analysis, the major peak of [3H]tyrosine-labeled anterior pituitary beta-endorphin was indistinguishable from camel beta-endorphin(1-31).

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.

Comparison of rat anterior and intermediate pituitary in tissue culture: corticotropin (ACTH) and beta-endorphin.

The forms of immunoreactive beta-endorphin-sized material in extracts of anterior and intermediate-posterior pituitary from the rat examined by the ion exchange chromatography method of Zakarian & Smyth. The anterior pituitary primarily contained material that co-migrated with synthetic camel beta-endorphin(1-31), whereas the intermediate-posterior pituitary contained relatively little such material. The majority of immunoactive beta-endorphin-sized peptides in the intermediate pituitary eluted at lower concentrations of NaCl than did camel beta-endorphin. Conditions were developed for the stable, long-term tissue culture of dissociated intermediate-posterior pituitary cells. Extracts of cells maintained in tissue culture for 18 h or nine days had the same content of immunoreactive beta-endorphin, 16k fragment, ACTH(18-39) (or CLIP) and ACTH(17-24). Throughout the nine days in culture, characteristic cells that could be immunostained with antibodies to various regions of pro-ACTH/endorphin were present; during the time in culture, non-reactive background cells multiplied rapidly. The major proteolytic processing of pro-ACTH/endorphin remained characteristic of intermediate pituitary tissue throughout the nine days in tissue culture, and did not become similar to the simpler pattern of proteolytic processing found in the anterior pituitary.

Beta-Endorphin: dissociation of receptor binding activity from analgesic potency.

Biological activities of synthetic camel beta-endorphin and human beta-endorphin (beta h-EP) have been measured by the radioreceptor binding assay, using [Tyr27-3H]-beta h-EP as the primary ligand and by the tail-flick test for analgesic potency. Four synthetic analogs of beta h-EP, namely [Gly31]-beta h-EP-Gly-NH2, [Gly31]-beta h-EP-Gly-Gly-NH2, [Gln8,Gly31]-beta h-EP-Gly-Gly-NH2, and [CH3(CH2)4NH231]-beta h-EP, have also been assayed by the same procedures. Results indicate a clear dissociation of radioreceptor binding activity from analgesic potency.

Characterization of mouse tumor cell beta-lipotropin.

Mouse tumor cell beta-lipotropin (beta LPH) and gamma-lipotropin (gamma LPH) were purified from mouse pituitary tumor cell culture medium by ion exchange chromatography and gel filtration. The mouse tumor cell beta LPH was identified by immunoprecipitation with several antisera to beta-endorphin, generation of opioid bioactivity upon brief treatment with trypsin, and its identity with the molecule previously shown to serve as an intermediate in the biosynthesis of beta-endorphin. Mouse tumor cell beta LPH (Mr = 8200 +/- 250) and gamma LPH (Mr = 4600 +/- 200) are significantly smaller than known mammalian beta LPH (Mr = 10,000) and gamma LPH (Mr = 6300) molecules. The beta-endorphin region of mouse tumor cell beta LPH has the same amino acid composition as ovine, bovine, and camel beta-endorphin, and species-specific differences are thus located in the gamma LPH region of the molecule. Mouse tumor cell beta LPH and gamma LPH lack a methionine residue at what had been considered to be a highly conserved site in their beta-melanotropin-like region. A species-specific radioimmunoassay for mouse tumor cell gamma LPH was developed. Rat pituitary beta LPH and gamma LPH were shown to be similar to the corresponding mouse tumor cell molecules in size and lack of methionine in their beta-melanotropin-like segment.

THE PURIFICATION OF PEPTIDES BY PARTITION CHROMATOGRAPHY BASED ON A HYDROPHOBICITY SCALE*

A study of the efficiency of partition chromatography for the purification of peptides as a function of structure has been undertaken. A series of 19 omission analogs of camel beta-endorphin and of some of its partial sequences have been synthesized with each analog missing only a single amino acid. Their chromatographic properties have been examined with use of the Martin hypothesis and the RM concept, and a hydrophobicity scale for the amino acid side chains was obtained. To a first approximation a correlation with the Tanford hydrophobicity scale for amino acids was found. A decrease in hydrophobicity has been observed with increasing chain length and is discussed in terms of column efficiencies required for the purification of synthetic peptides.

Conformation of beta-endorphin and beta-lipotropin: formation of helical structure in methanol and sodium dodecyl sulfate solutions.

Circular dichroic spectra of camel beta-endorphin and ovine beta-lipotropin in water show little, if any, secondary structure. Intrinsic viscosities and sedimentation coefficients of the two peptides also suggest that the molecules are not compact and globular. Methanol or sodium dodecyl sulfate promotes the formation of helical structure to an extent as much as one-half of either peptide molecule. The conformation of the complex between camel beta-endorphin and dodecyl sulfate may be related to the opiate-like function of this peptide hormone.