beta-endorphin: synthesis of analogs with extension at the carboxyl terminus with high radioreceptor binding activity

Four analogs of human beta-endorphin (beta h-EP) have been synthesized: [Gly31]-Beta h-EP-Gly-NH2, [CH3(CH2)4NH231]-beta h-EP, [Gly31]-beta h-EP-Gly-Gly-NH2, and [Gln8, Gly31]-betah-EP-Gly-Gly-NH2. All are more active than beta h-EP in an opiate receptor binding assay. Stepwise extension at the COOH-terminus shows a progressive increase in binding activity. The last analog, which combines extension at the COOH-terminus with elimination of the remaining anionic charge in beta h-EP, is nine times more active than the parent molecule.     

Synthesis and receptor binding activity of elephant beta-endorphin, a beta-endorphin homolog with highly potent analgesic activity.

Elephant beta-endorphin and its analog, elephant beta-endorphin(6-31) were synthesized by standard solid phase method. Receptor binding activity showed that elephant beta-endorphin was five to six times more potent than human beta-endorphin in its ability to bind to opiate receptors on rat brain membrane. In a previous study (Wong, C.-L., Wai, M.-K., Cheng, H.-C., Chung, D. & Yamashiro, D (1990) Clinical and Experimental Pharmacology and Physiology 16, 33-37), tail flick test for intracerebroventricularly administered beta-endorphin showed that the antinociceptive potency of elephant beta-endorphin was seven to eight times higher than that of human beta-endorphin in mice. Results from both studies suggest that elephant beta-endorphin was a much more potent antinociceptive agent than human beta-endorphin in tail flick test and its higher analgesic activity might be due to its higher affinity for opiate receptors in the brain.     

Isolation and primary structure of endorphin from salmon pituitary glands.

Endorphin has been isolated from an acid acetone extract of the pituitary of the salmon $\text{Oncorhynchus}\text{keta}$ by ion exchange chromatography and gel filtration. Sequence analysis revealed it to be a nonacosapeptide with following primary structure: Ac-Tyr-Gly-Gly-Phe-Met-Lys-Pro-Tyr-Thr-Lys-Gln-Ser-His-Lys-Pro-Leu-Ile-Thr-Leu-Leu-Lys-His- Ile-Thr-Leu-Lys-Asn-Glu-Gln-OH. It appears that the amino terminal segment which is necessary for analgesic activity is conserved through the evolution of vertebrate except for the blocking of the amino terminal of salmon endorphin.     

Adrenocorticotropin. 57. Synthesis and biological activity of ostrich and turkey hormones

Synthesis of ostrich and turkey corticotropin (ACTH) has been accomplished by the solid-phase method. Each was identical to the natural hormone in high performance liquid chromatography. Relative potencies in a lipolytic assay in isolated rabbit fat cells were: human ACTH, 100; ostrich ACTH, 53; turkey ACTH, 28. In isolated rat fat cells relative lipolytic potencies were: human ACTH, 100; ostrich ACTH, 2; turkey ACTH, 13. It was concluded that lipolytic potency is sensitive to alterations in structure throughout the entire length of the ACTH sequence in the rat fat cell assay.     

Structural organization of [met]enkephalin and endorphin molecules. III. Theoretical conformation analysis of beta-endorphin

Conformational energy calculations were carried out for beta-endorphin. Its spatial structure can be described by nine low-energy conformations. The calculations yielded the values of all dihedral angles of the backbone and side chains of these forms as well as intra- and inter-residue interaction energies.     

Synthesis,receptor binding and activity of iso and azakainoids

Two syntheses for the production of an unsubstituted azakainoid are described. The 1,3-dipolar cycloaddition of diazomethane with trans-dibenzyl glutaconate yields a 1-pyrazoline, which may be reduced directly to the pyrazolidine. An unexpected trans-cis isomerization is observed during Hg/Al reduction of the 1-pyrazoline NN bond. Alternatively, when TMS diazomethane is used as the dipole, the resulting 2-pyrazoline obtained after desilylation may be reduced with NaCNBH₃ to provide the trans azakainate analog exclusively. The synthesis of an unsubstituted isokainoid via Michael addition is also described. Glutamate receptor binding assays revealed that the azakaniod has a moderate affinity for unspecified glutamate receptors. Membrane depolarization of Aplysia neurons upon application of the azakainoid demonstrates that it is an ionotropic glutamate receptor agonist.     

Development and assessment of radioreceptor binding assays for the detection of saxitoxin binding proteins in biological extracts

Several radioreceptor assays using tritiated saxitoxin ([(3)H]STX) were developed to identify a suitable primary screening method for the detection and characterization of soluble saxitoxin binding proteins from biological extracts. Assays using anion and cation exchange, protein binding, and traditional charcoal radioreceptor methods were compared with two previously reported formats. A protein binding assay incorporating filters of mixed cellulose esters (MCE) outperformed all other assay strategies with maximal signal, low background, exceptional reproducibility, minimal matrix effects, and high throughput. Binding site titrations verified that an increase in total protein in the assay led to a concomitant linear increase in the amount of specifically bound [(3)H]STX within the range of 1-90microg total protein. Saturation binding experiments demonstrated that the binding sites were saturable and that nonspecific binding was linear. The MCE assay was unaffected by 600mM NaCl and 500mM KCl. Likewise, minimal variation of specific binding was observed between pH 5 and pH 9, but inhibition was observed below pH 5.     

Synthesis and analgesic activity of human beta-endorphin analogs substituted at positions 17, 18, or 19

Three peptide analogs of beta-endorphin which are substituted in positions 17, 18 or 19 have been synthesized and their analgesic potencies have been measured by the tail-flick method in mice. The results showed that the replacement of Phe-18 or Lys-19 by alanine reduced the potency to 15% whereas the replacement of Leu-17 by alanine reduced the analgesic potency to 68%.     

Refinement of a radioreceptor binding assay for nicotinic acid adenine dinucleotide phosphate

The measurement of changes in nicotinic acid adenine dinucleotide phosphate (NAADP) levels in cells has been, and remains, key to the investigation of the functions of NAADP as a Ca2+ -releasing second messenger. Here we provide details of how to isolate NAADP from cells by extraction with perchloric acid and then measure the NAADP using a radioreceptor assay. We demonstrate that NAADP is neither generated nor broken down during sample processing conditions and that radioreceptor assay is highly selective for the detection of NAADP under cell extract conditions. Furthermore, a number of improvements, such as solid-state detection of the radioactivity, are incorporated to enhance the safety of the procedure. Finally, we have developed a new method to prevent the endogenous metabolism of NAADP by chelating Ca2+ with bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), thereby reducing the difficulty of catching a small transient rise in NAADP levels. In summary, we have refined and improved a method for measuring NAADP levels and presented it in a manner accessible to a wide range of laboratories. It is expected that this will enhance research in the NAADP field.     

Primary structure and morphine-like activity of human beta-endorphin.

The complete amino acid sequence of human beta-endorphin was obtained by automatic sequencing of a sulfonyl isothiocyanate derivative of this peptide, in combination with peptide mapping of a tryptic digest of the native molecule. It was found to be identical with the carboxy-terminal portion 61-91 of human beta-lipotropin (beta-LPH). The morphine-like activity of beta-endorphin is comparable both in the mouse vas deferens bioassay and in the opiate receptor binding assay. However, beta-LPH is not active up to concentrations of 10(-6) M.     

Opioid and non-opioid binding of beta-endorphin to bovine adrenal medullary membranes

Binding of human beta-endorphin (beta h-EP) to bovine adrenal medullary membranes was characterized using [125I]Tyr27-beta h-EP [( 125I]beta h-EP) as a primary ligand. The specific binding of [125I]beta h-EP was time-dependent, saturable and stereospecific. Analysis of a saturation isotherm revealed two apparent classes of specific binding sites with dissociation constants of 2.4 and 34 nM. The extent of maximum inhibition of specific [125I]beta h-EP binding by either levorphanol, morphine, naloxone, dynorphin A (1-13) or D-Ala2-D-Leu5-enkephalin was similar to each other and remained partial (60-70%). Levorphanol eliminated the high affinity component but showed no effect on the low affinity component of [125I]beta h-EP binding. beta h-EP(1-31) displaced completely the [125I]beta h-EP binding. However, beta h-EP(1-23) only partially (approximately 80%) inhibited the [125I]beta h-EP binding. beta h-EP(6-31) showed inhibitory activity on [125I]beta h-EP binding. These results suggest that [125I]beta h-EP binding to bovine adrenal medullary membranes consists of a high affinity opioid-sensitive component and a low affinity non-opioid component. The non-opioid component of [125I]beta h-EP binding may be related to COOH-terminal of the beta h-EP molecule.     

Synthesis and binding activity of endomorphin-1 analogues containing beta-amino acids

Endomorphin-1 (Tyr-Pro-Trp-PheNH₂) has been proposed as the most potent endogenous ligand of the μ-opioid receptors. In this paper, we describe the synthesis of some endomorphin-1 based tetrapeptides in which a residue of the sequence Tyr-Pro-Trp-PheNH₂ is replaced by the corresponding β-isomer. These novel peptides showed different affinities for the opioid receptors labeled with [³H]-DAMGO in rat brain membranes, depending on the β-amino acid. In particular, the tetrapeptide containing β-Pro (Tyr-β-(R)-Pro-Trp-PheNH₂) displayed a higher affinity than endogenous endomorphin-1, as revealed by their K_i values (0.33 and 11.1 nM, respectively).     

Synthesis and protein kinase C binding activity of benzolactam-V7.

Benzolactam-V7 (3a), a simplified analogues of (-)-indolactam-V with twist-form conformation, was synthesized and evaluated as a new protein kinase C modulator. Both 3a and its-7-substituted analogue 3c showed weak binding activity to displace PDBU binding from recombinant PKCalpha.     

Synthesis,DNA binding and anti-leukemic activity of an aminoacyl nucleolipid

The synthesis and characterization of a new class of DNA binding molecule exhibiting potent and selective anti-leukemic activity is described. The synthesis of an aminoacyl nucleolipid was developed from an efficient EEDQ coupling strategy, in which a series of seven bioconjugates were synthesized in yields of 53–78%. Guanosine bioconjugate 7, was used as building block for the synthesis of a target aminoacyl nucleolipid 14. Its GRP78 DNA binding affinity was confirmed by gel shift assay, CD spectroscopy, T_m measurements and dynamic light scattering experiments. Moreover, in a single dose (10 μM) screen against a panel of 60 cancer cell lines, aminoacyl nucleolipid 14 was found to selectively trigger greater than 90% cell death in a SR human leukemia cancer cell line. The reported aminoacyl nucleolipid represents a useful model for a new class of DNA binding molecules for the development of potent and selective anti-cancer agents.     

beta-endorphin: synthesis and biological activity of shortened peptide chains

Three analogs of beta-endorphin have been synthesized by the solid-phase method: betac-endorphin-(1--5)-(28--31), betac-endorphin-(6--31) and betah-endorphin-(1--5)-(16--31). The analgesic activities of these synthetic peptides relative to that of the parent molecule are reported. All three peptides at high doses exhibit either no or much weaker analgesic activity than beta-endorphin. These data suggest that the entire beta-endorphin molecule is necessary for full in vivo analgesic activity.     

Development of radioimmunoassayable beta-endorphin and methionine-enkephalin binding sites in regions of rat brain

The development profiles of [3H]methionine-enkephalin ([3H]Met-enk) binding sites and radioimmunoassayable (RIA) beta-endorphin in regions of rat brain were determined. The amount [3H]Met-enk bound reached its maximum in the 1st week after birth in the cerebellum, in the brainstem at the 2nd week, and in the whole forebrain at the 3rd week, that of RIA beta-endorphin reached its highest level at day 2 postpartum (p.p.) in the cerebellum. at days 7-15 p.p. in the whole forebrain, and at days 17-21 p.p. in the brainstem. These findings suggest that both the development of RIA beta-endorphin and [3H]met-enk sites in rat brain follow a caudal to rostral sequence. Also, the close interrelationship between the elevation and decline in the amount of [3H]Met-enk bound and RIA beta-endorphin levels in each brain region suggests that these two components are important entities of the central nervous system.     

Preparation and characterization of fully deacetylated chitosan

A new method for N-deacetylation of chitosan is proposed in which a polymer free of N-acetyl groups is obtained without much decrease in molecular weight. Different methods for determining the degree of acetylation were used, mainly i.r. spectroscopy and conductimetry. Average molecular weights were determined by membrane osmometry and light scattering; a polydispersity <2 was found. This well defined amino-polymer can be used for investigation of chelating properties.     

Comparative calcium binding and conformational studies of turkey and rabbit skeletal troponin C

Troponin C from turkey skeletal muscle has been compared with its chicken counterpart in terms of amino acid composition and fragmentation patterns and with rabbit TN-C by Ca2+ binding and conformational response to Ca2+ as monitored by CD and fluorescence. Cyanogen bromide and tryptic digestion mixtures of chicken and turkey TN-C have been separated by reversed-phase HPLC. The similarity of the elution profiles, along with the almost identical amino acid compositional data, suggest that the sequences are essentially equivalent. Both turkey and rabbit TN-C bound 2 mol Ca2+/mol protein at pH 5.3, while at pH 6.8, this figure was raised to 4 mol/mol protein. Circular dichroism and fluorescence measurements indicated that the conformations of the two proteins responded in a very similar manner to the presence of Ca2+.     

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.