Chemical characterization and opioid activity of an exorphin isolated from in vivo digests of casein

The in vivo formation of an opioid peptide (exorphin) derived from beta-casein has been proved for the first time. It was isolated from duodenal chyme of minipigs after feeding with the milk protein casein. The exorphin has been identified as a beta-casein fragment by end-group determinations and qualitative amino acid analysis of the purified peptide. This peptide, named beta-casomorphin-11, displayed substantial opioid activity in an opiate receptor-binding assay.     

Purification and sequence of an opioid peptide derived from ovine proenkephalin

An enkephalin-containing peptide originating from ovine adrenal proenkephalin has been purified and sequenced. The sequence of the peptide is: GLY-GLY-GLU-VAL-LEU-GLY-LYS-ARG-TYR-GLY-GLY-PHE-MET (preproenkephalin 128-140) which represents a portion of peptide F (preproenkephalin 107-140). This peptide has a sequence identical to that of bovine preproenkephalin 128-140 while it differs from the corresponding human sequence in positions 129, 131 and 133.     

NMR structure of the N-SH2 of the p85 subunit of phosphoinositide 3-kinase complexed to a doubly phosphorylated peptide reveals a second phosphotyrosine binding site

The N-terminal src homology 2 (SH2) domain of the p85 subunit of phosphoinositide 3-kinase (PI3K) has a higher affinity for a peptide with two phosphotyrosines than for the same peptide with only one. This unexpected result was not observed for the C-terminal SH2 from the same protein. NMR structural analysis has been used to understand the behavior of the N-SH2. The structure of the free SH2 domain has been compared to that of the SH2 complexed with a doubly phosphorylated peptide derived from polyomavirus middle T antigen (MT). The structure of the free SH2 domain shows some differences from previous NMR and X-ray structures. In the N-SH2 complexed with a doubly phosphorylated peptide, a second site for phosphotyrosine interaction has been identified. Further, line shapes of NMR signals showed that the SH2 protein-ligand complex is subject to temperature-dependent conformational mobility. Conformational mobility is also supported by the spectra of the ligand peptide. A binding model which accounts for these results is developed.     

Anti-insulin-like (diabetogenic) peptides in pituitary extracts: role of oxytocin

A peptide has been extracted and characterized from whole bovine pituitaries that has anti-insulin-like activities when assayed in rat adipocytes. This peptide has been purified approximately 100,000-fold, is homogeneous by thin-layer chromatography in three separate solvent systems, and shows a single peak by reverse-phase high-performance liquid chromatography. By these chemical criteria, as well as biological activity criteria (14CO2 production from D-[U-14C]glucose and D-[U-14C]glucose incorporation into glycogen in rat adipocytes], the peptide is indistinguishable from oxytocin. It reacts with anti-oxytocin antibody, and has an amino acid composition indistinguishable from purified oxytocin. The relationship between this material and other previously described anti-insulin or diabetogenic peptides is discussed, but it was not possible to conclude that this peptide, which has been purified to homogeneity and constant specific activity, is related to these previously described factors.     

Functional roles of peptide cotransmitters at neuromuscular synapses inAplysia

Neuromuscular synapses inAplysia have been used as model systems to study peptidergic cotransmission. Here we describe neuromuscular preparations in which it has been possible to investigate the physiological consequences of peptide transmitter release in detail. In the first preparation, the release of peptide cotransmitters from identified motor neuron B15 has been shown to be sensitive to the pattern of stimulation. High frequencies and long burst durations evoke peptide release that modulates muscle contractions in a manner similar to that produced by exogenous cotransmitter. By contrast, the release of the same peptide transmitters from motor neuron B1 show little dependence on pattern. We conclude that there are no stimulation patterns that are prerequisites for peptide release. Peptide cotransmitter release from motor neuron B47 has also been studied. B47, depending on the stimulation pattern, uses either ACh, which acts as a conventional inhibitory transmitter, or Ach plus neuropeptides, which act as excitatory modulatory cotransmitters. Thus, neuropeptide cotransmitters have the capability to greatly increase synaptic plasticity at neuromuscular synapses.     

The neurohypophysial hormones of the egg-laying mammals: identification of arginine vasopressin in the platypus (Ornithorhynchus anatinus)

Two neurohypophysial peptides have been purified from acetone desiccated posterior pituitary glands of the platypus (Ornithorhynchus anatinus) by molecular sieving and high-pressure liquid chromatography. A single pressor peptide, having an amino acid composition and a chromatographic retention time identical to those of arginine vasopressin, has been identified. A single oxytocic peptide has been isolated that ressembles oxytocin by its chromatographic retention time, but lack of material has prevented to obtain a correct amino acid composition. The pressor peptide is roughly four times more abundant than the oxytocic peptide. Neurohypophysial hormones of platypus seem similar to those of echidna, the other living prototherian, and to those of most placental mammals.     

Structure of the smooth muscle myosin light-chain kinase calmodulin-binding domain peptide bound to calmodulin

The interaction between the peptide corresponding to the calmodulin-binding domain of the smooth muscle myosin light-chain kinase and (Ca2+)4-calmodulin has been studied by multinuclear and multidimensional nuclear magnetic resonance methods. The study was facilitated by the use of 15N-labeled peptide in conjunction with 15N-edited and 15N-correlated 1H spectroscopy. The peptide forms a 1:1 complex with calcium-saturated calmodulin which is in slow exchange with free peptide. The 1H and 15N resonances of the bound have been assigned. An extensive set of structural constraints for the bound peptide has been assembled from the analysis of nuclear Overhauser effects and three-bond coupling constants. The backbone conformation of the bound peptide has been determined using these constraints by use of distance geometry and related computational methods. The backbone conformation of the peptide has been determined to high precision and is generally indicative of helical secondary structure. Nonhelical backbone conformations are seen in the middle and at the C-terminal end of the bound peptide. These studies provide the first direct confirmation of the amphiphilic helix model for the structure of peptides bound to calcium-saturated calmodulin.     

Analysis of a crosslinked peptide from calf bone collagen: evidence that hydroxylysyl glycoside participates in the crosslink

A crosslinked, double-chained peptide has been isolated from calf bone collagen after digestion with crude bacterial collagenase. Initially, the ³H-labelled peptide was isolated from collagen that had been treated with [³H]-NaBH₄, but an almost identical peptide was also isolated from collagen without prior reduction. After periodate oxidation of the reduced peptide the two component chains were resolved by further chromatography. Amino acid compositions showed that the peptide probably derived from an intermolecular crosslink between a carboxyterminal sequence of the collagen molecule and a sequence near the aminoterminus that previously has been shown to be the site of a glycosylated hydroxylysine residue. The crosslinking compound in the reduced peptide, hydroxylysinohydroxynorleucine, appeared to have derived mainly by reduction with borohydride of hydroxylysinooxonorleucine, the keto-amine rearranged form of the dehydro crosslink. The remaining hydroxyl group of the crosslink, the one not derived by reduction of the keto group, appeared to be glycosylated.     

Isolation and primary structure of the C-peptide of proinsulin from the European eel (Anguilla anguilla)

1. The primary structure of the C-peptide of proinsulin from the European eel has been established as: DVEPLLGFLSPKSGQENEVDDFPYKGQGEL. The peptide was isolated from the extract of eel pancreas in a yield that was approximately equimolar with insulin. A comparison with the predicted structures of C-peptides from other teleost fishes has identified a domain in the central region of the peptide that has been more highly conserved than the rest of the molecule.     

Control of function of a small peptide by a protein

A peptide that functions only in the presence of a protein has been developed using reaction-based selection from peptide phage libraries. The peptide was not functional in the absence of the protein, but formed enaminones with 1,3-diketone derivatives when bound to the protein.     

Isolation and characterization of a soluble, immunoactive peptide of glial fibrillary acidic protein

A soluble immunoactive peptide with a molecular weight of 16 000 was isolated and purified from the cyanogen bromide digest of the insoluble 50 000 dalton glial fibrillary acidic protein by Sephacryl S-200 gel filtration followed by DEAE-Bio-gel A chromatography. The homogeneity of the peptide was established by SDS-polyacrylamide gel electrohporesis and isoelectric focusing. The peptide from several species showed immunocrossreaction with rabbit antibody to intact glial fibrillary acidic protein. The peptide has a pI value of 5.32. The amino acid sequence of 28 residues from the amino terminus of the calf peptide has been determined.     

Isolation of less than Glu-Gly-Leu-Arg-Trp-NH2 (Antho-RWamide II), a novel neuropeptide from sea anemones

Using a radioimmunoassay for the peptide sequence Arg-Phe-NH2 (RFamide), a novel peptide has been purified from acetic acid extracts of the sea anemone Anthopleura elegantissima. This peptide has the structure less than Glu-Gly-Leu-Arg-Trp-NH2, and was named Antho-RWamide II. Antho-RWamide II is a neuropeptide. Its structure is closely related to an earlier characterized neuropeptide from Anthopleura less than Glu-Ser-Leu-Arg-Trp-NH2 (Antho-RWamide I).     

Amyloid Angiopathy of Alzheimer''s Disease: Amino Acid Composition and Partial Sequence of a 4,200-Dalton Peptide Isolated from Cortical Microvessels

The cardinal lesions of Alzheimer's disease are neurofibrillary tangles, senile neuritic plaques, and vascular amyloid, the latter generally involving cortical arteries and small arterioles. All three lesions are composed of amyloid-like, beta-pleated sheet fibrils. Recently, a 4,200-dalton peptide has been isolated from extraparenchymal meningeal vessels, neuritic plaques, and neurofibrillary tangles. The assumption of N-terminal homogeneity in vascular amyloid has been used as an argument for a neuronal (versus blood) origin of the peptide. However, intracortical microvessels from Alzheimer's disease have not been previously isolated. The present studies describe the isolation of a microvessel fraction from Alzheimer's disease and control fresh autopsy human brain. Alzheimer's disease isolated brain microvessels that were extensively laden with amyloid and control microvessels were solubilized in 90% formic acid and analyzed by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The arteriole fraction from the Alzheimer's subject with extensive amyloid angiopathy contained a unique 4,200-dalton peptide, whereas the arterioles or capillaries isolated from two controls and two Alzheimer's disease subjects without angiopathy did not. This peptide was purified by HPLC and amino acid composition analysis showed the peptide is nearly identical to the 4,200-dalton peptide recently isolated from neuritic plaques or from neurofibrillary tangles. Sequence analysis revealed N-terminal heterogeneity. The N-terminal sequence was: Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr, which is identical to the N-terminal sequence of the 4,200-dalton peptide isolated previously from extraparenchymal meningeal vessels and neuritic plaques.(ABSTRACT TRUNCATED AT 250 WORDS)     

The arginine-rich peptide from cytosol of Guerin epithelioma.

Arginine-rich peptide from cytosol of Guerin epitheliomas has been isolated and characterized. It contains 212.0 arginine residues, 77.1 lysine and 36.9 histidine residues per 1000 residues of amino acids. The ratio of basic to the acidic amino acids amounted to 1.23. The peptide has a cationic character with an isoelectric point of 8.6. No carbohydrate component was found in this peptide. It is suggested that this peptide originates from decomposition of arginine-rich basic protein stated previously in cytosol of this tumor.     

Solution NMR studies of colicin E1 C-terminal thermolytic peptide. Structural comparison with colicin A and the effects of pH changes

The aqueous solution structure of the C-terminal thermolytic peptide of colicin E1 has been investigated using both one- and two-dimensional NMR techniques. The NMR data are consistent with a fold for the peptide very similar to that reported for the colicin A C-terminal peptide in the crystalline state, although some differences have been noted. The one-dimensional NMR spectrum of the peptide has been used to follow changes in both the structure and dynamics of the peptide on changing pH. The in vitro functionally competent form of the peptide (present in solution only below pH 6) does not differ in structure significantly from the higher pH form. However, small local conformational changes are observed together with an increase in mobility in some of the more hydrophilic regions. This suggests that the effect of lower pH is to change the ease with which the major conformational changes during insertion into a membrane can occur.     

Purification and primary structure of ostrich pancreatic polypeptide

Pancreatic polypeptide has been isolated from ostrich pancreas by gel filtration, ion exchange chromatography and high pressure liquid chromatography. The ostrich peptide contains 36 amino acids and has an amino acid composition similar to pancreatic polypeptide of other avian species. The primary structure of ostrich pancreatic polypeptide differs from that of the chicken peptide only at residues 3 and 18 where the ostrich peptide contains an alanine and a valine residue compared to the serine and isoleucine residues found in those positions in the chicken peptide.     

The primary structure of the N-terminal region of mature alkaline phosphatase is critical for secretion and function of the enzyme

The export signal has been assumed to be localized not only in the signal peptide of a secreted protein precursor, but also in the N-terminal region of the mature polypeptide chain. Mutant alkaline phosphatases with amino acid substitutions of two positively charged residues (Lys or Arg) in this region at different distances from the signal peptide have been studied to test this assumption. The efficiency of secretion has been shown to decrease in mutant proteins with amino acid substitutions in the region of 16-18 amino acid residues; the closer to the signal peptide is the substitution, the greater is the decrease. A change in the primary structure of the N-terminal domain results also in an increase in the Michaelis constant, which is greater the farther is the amino acid substitution from the signal peptide, suggesting a change in the enzyme function as well.     

The primary structure of a PYY-related peptide from chicken intestine suggests an anomalous site of cleavage of the signal peptide in preproPYY.

Although the amino acid sequence of members of the pancreatic polypeptide (PP)-family of regulatory peptides has been poorly conserved during vertebrate evolution, the overall length of the peptides (36 amino acid residues) has remained constant. Nucleotide sequence analysis of cloned cDNAs and/or genomic fragments has shown the PP-related sequence immediately follows the signal peptide in the prepropeptides. A peptide tyrosine-tyrosine (PYY)-related peptide with 37 residues has been isolated from the chicken intestine, and its primary structure was established as: Ala-Tyr-Pro-Pro-Lys-Pro-Glu-Ser-Pro-Gly10-Asp-Ala-Ala-Ser-P ro-Glu-Glu-Ile-Ala-Gln20-Tyr-Phe-Ser-Ala-Leu-Arg-His-Tyr-Il e-Asn30-Leu-Val-Thr-Arg-Gln-Arg-Tyr.CONH2. The presence of an additional alanine residue at the NH2-terminus of the peptide suggests that the site of cleavage of the signal peptide in chicken preproPYY is different from the site of cleavage in other PP-family prepropeptides.     

Isolation and characterization of a diuretic peptide from Acheta domesticus. Evidence for a family of insect diuretic peptides.

A diuretic peptide (Acheta-DP) has been isolated from extracts of whole heads of the house cricket, Acheta domesticus. The native peptide increases both cyclic AMP production and the rate of fluid secretion by isolated Malpighian tubules in vitro to an extent comparable with those responses obtained with supra-maximal amounts of crude extracts of corpora cardiaca. The primary structure of Acheta-DP was established as a 46-residue amidated peptide: TGAQSLSIVAPLDVLRQRLMNELNRRRMRELQGSRIQQNRQLLTSI-NH2. Acheta-DP has 41% sequence identity with a diuretic peptide isolated from Manduca sexta, providing direct evidence for the presence of a family of diuretic peptides in insects.