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).     

Isolation of the neuropeptide less than Glu-Trp-Leu-Lys-Gly-Arg-Phe-NH2 (Pol-RFamide II) from the hydromedusa Polyorchis penicillatus.

Using a radioimmunoassay for the sequence Arg-Phe-NH2 (RFamide), we have isolated the peptide less than Glu-Trp-Leu-Lys-Gly-Arg-Phe-NH2 (Pol-RFamide II) from acetic acid extracts of the hydromedusa Polyorchis penicillatus. This peptide is a neuropeptide and constitutes a peptide family together with less than Glu-Leu-Leu-Gly-Gly-Arg-Phe-NH2 (Pol-RFamide I), the first neuropeptide isolated from Polyorchis, and less than Glu-Gly-Arg-Phe-NH2 (Antho-RFamide), a neuropeptide isolated from sea anemones and sea pansies.     

Brain natriuretic peptide-32: N-terminal six amino acid extended form of brain natriuretic peptide identified in porcine brain

Brain natriuretic peptide (BNP) is a newly identified peptide of 26 residues, which has a remarkable homology to but is distinct from atrial natriuretic peptide. The peptide exerts natriuretic-diuretic activity as well as potent chick rectum relaxant activity. By using radioimmunoassay specific to BNP and immunoaffinity chromatography, we have isolated from porcine brain a novel peptide of 32 residues carrying a BNP structure at the C-terminus. The amino acid sequence of this peptide was determined to be: Ser-Pro-Lys-Thr-Met- Arg-Asp-Ser-Gly-Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly- Leu- Gly-Cys-Asn-Val-Leu-Arg-Arg-Tyr. This peptide is an N-terminal six amino acid extended form of BNP and henceforth is designated BNP-32. BNP and BNP-32 are found to be major forms of BNP family in porcine brain.     

ANF-like peptide(s) in the peripheral autonomic nervous system

The recent demonstration of the atrial natriuretic factor (ANF) within the brain has been extended in the present study by the additional localization of ANF-like activity in the peripheral nervous structures. Using a sensitive radioimmunoassay, it was possible to detect ANF-like immunoreactive peptide(s) in crude and chromatographically separated extracts of parasympathetic rat ganglia. The partially purified ANF-like peptide exhibited a biological action similar to cardiac ANF. This finding supports a possible involvement of ANF in the regulation of both, central and peripheral neuronal activities.     

Cardiac troponin T, a sarcomeric AKAP, tethers protein kinase A at the myofilaments

Efficient and specific phosphorylation of PKA substrates, elicited in response to β-adrenergic stimulation, require spatially confined pools of PKA anchored in proximity of its substrates. PKA-dependent phosphorylation of cardiac sarcomeric proteins has been the subject of intense investigations. Yet, the identity, composition, and function of PKA complexes at the sarcomeres have remained elusive. Here we report the identification and characterization of a novel sarcomeric AKAP (A-kinase anchoring protein), cardiac troponin T (cTnT). Using yeast two-hybrid technology in screening two adult human heart cDNA libraries, we identified the regulatory subunit of PKA as interacting with human cTnT bait. Immunoprecipitation studies show that cTnT is a dual specificity AKAP, interacting with both PKA-regulatory subunits type I and II. The disruptor peptide Ht31, but not Ht31P (control), abolished cTnT/PKA-R association. Truncations and point mutations identified an amphipathic helix domain in cTnT as the PKA binding site. This was confirmed by a peptide SPOT assay in the presence of Ht31 or Ht31P (control). Gelsolin-dependent removal of thin filament proteins also reduced myofilament-bound PKA-type II. Using a cTn exchange procedure that substitutes the endogenous cTn complex with a recombinant cTn complex we show that PKA-type II is troponin-bound in the myofilament lattice. Displacement of PKA-cTnT complexes correlates with a significant decrease in myofibrillar PKA activity. Taken together, our data propose a novel role for cTnT as a dual-specificity sarcomeric AKAP.     

Porcine brain natriuretic peptide-like immunoreactivity in rat tissues

The presence of immunoreactive porcine brain natriuretic peptide in rat tissues was studied with a specific radioimmunoassay for porcine brain natriuretic peptide-26. The cross-reactivity of the antiserum used was less than 0.001% with rat atrial natriuretic peptide, rat brain natriuretic peptide-32 and rat brain natriuretic peptide-45. Immunoreactive porcine brain natriuretic peptide was detectable in various tissues of the rat, and high concentrations of immunoreactive porcine brain natriuretic peptide were found in the brain and cardiac atrium, with the highest level in the hypothalamus (159±30 fmol/gram wet tissue, mean±SEM, n=4). Reverse phase high performance liquid chromatography showed that the immunoreactive porcine brain natriuretic peptide of the whole brain and heart extracts eluted mainly at an identical position to synthetic porcine brain natriuretic peptide-26. These findings indicate that porcine brain natriuretic peptide-like substance, distinct from rat brain natriuretic peptide, is present in high concentrations in the rat brain and cardiac atrium.     

Ghrelin and des-acyl ghrelin: two major forms of rat ghrelin peptide in gastrointestinal tissue

Ghrelin, a novel peptide purified from stomach, is the endogenous ligand for the growth hormone secretagogue receptor and has potent growth hormone-releasing activity. The Ser3 residue of ghrelin is modified by n-octanoic acid, a modification necessary for hormonal activity. We established two ghrelin-specific radioimmunoassays; one recognizes the octanoyl-modified portion and another the C-terminal portion of ghrelin. Using these radioimmunoassay systems, we found that two major molecular forms exist-ghrelin and des-n-octanoyl ghrelin. While ghrelin activates growth-hormone secretagogue (GHS) receptor-expressing cells, the nonmodified des-n-octanyl form of ghrelin, designated as des-acyl ghrelin, does not. In addition to these findings, our radioimmunoassay systems also revealed high concentrations of ghrelin in the stomach and small intestine.     

Identification and distribution of immunoreactive peptide YY in the human, canine, and murine gastrointestinal tracts: species-related antibody recognition differences

A radioimmunoassay using two antisera (antibody 80 and antibody 213) from rabbits immunized with porcine peptide YY has been characterized for both sensitivity and specificity. To determine the distribution of peptide YY in the gut, fresh tissue specimens from the human and canine gut were separated into mucosal-submucosal and muscularis externa layers by microdissection. These tissues and transmural specimens from murine gut were acid-extracted and neutralized, followed by radioimmunoassay using each antiserum. Immunoreactive peptide YY in canine and murine gut was present in similar concentration and distribution using each antiserum, with highest concentrations in the mucosal-submucosal layer of the descending colon. Using antibody 213, immunoreactive peptide YY throughout the human gut was measured only at the lower detection limit of the radioimmunoassay. By contrast, using antibody 80, peptide YY in human gut was present in a distribution similar to canine and murine gut. Using antibody 80, one major immunoreactive species was identified with C18 reverse-phase high-performance liquid chromatography in extracts of human, canine, and murine colon. These results suggest species-related antibody recognition differences. The similar concentrations of peptide YY in canine and murine gut determined with the two antisera are consistent with the hypothesis that the amino acid sequences of canine and murine peptide YY are similar to porcine peptide YY. Using antibody 213, the low concentrations of immunoreactive peptide YY found in human gut are consistent with the hypothesis that human and porcine peptide YY have different amino acid sequences. Antisera prepared by immunization with porcine PYY must therefore be carefully characterized prior to studies using human sera or human tissue extracts.     

Isolation of Leu-Pro-Pro-Gly-Pro-Leu-Pro-Arg-Pro-NH2 (Antho-RPamide), an N-terminally protected, biologically active neuropeptide from sea anemones.

Using a radioimmunoassay against the C-terminal sequence Arg-Pro-NH2 (RPamide), we have isolated the peptide Leu-Pro-Pro-Gly-Pro-Leu-Pro-Arg-Pro-NH2 (Antho-RPamide) from an extract of the sea anemone Anthopleura elegantissima. Antho-RPamide is located in neurons of sea anemones. Application of low concentrations of Antho-RPamide to tentacle preparations of sea anemones strongly increased the frequency and duration of spontaneous contractions, suggesting that this peptide is involved in neurotransmission. Antho-RPamide has a free N-terminus, yet its X-Pro-Pro sequence makes it relatively resistant to degradation by nonspecific aminopeptidases. Thus, we have discovered another strategy by which sea anemones protect the N-termini of their bioactive neuropeptides.     

Occurrence of a novel cardiac natriuretic peptide in rats

We established a specific radioimmunoassay for the ring structure of "iso-ANP" and detected iso-ANP[23-46]-like immunoreactivity (-LI) in the rat atrium (2.76 +/- 0.5 micrograms/g) and ventricle (13.9 +/- 5.7 ng/g). High performance-gel permeation chromatography revealed that iso-ANP[23-46]-LI in the rat heart was composed of two components with molecular weights of 10K and 5K. In reverse phase-high performance liquid chromatography, the retention times of these components were clearly different from that of synthetic iso-ANP. The 5K peptide was demonstrated to be present in the perfusate from isolated rat hearts and possessed binding ability to ANP receptors. This natriuretic peptide was, however, not detectable in other tissues including the brain. We conclude that the novel cardiac natriuretic peptide distinct from iso-ANP and ANP occurs in the rat heart and is secreted from the heart.     

Isolation and identification of C-type natriuretic peptide in chicken brain

C-type natriuretic peptide (CNP) has recently been identified in porcine brain as a third member of the mammalian natriuretic peptide family (1). Using a radioimmunoassay system for porcine CNP, we found a significant concentration of immunoreactive (ir-) CNP in chicken brain, from which a new peptide was isolated. By microsequence analysis, the amino acid sequence of the peptide was determined to be Gly-Leu-Ser-Arg-Ser-Cys-Phe- Gly-Val-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys. Based on its high homology to porcine CNP, the peptide was designated chicken C-type natriuretic peptide. Chicken CNP also elicits pharmacological effects highly similar to porcine CNP, suggesting that CNP functions as a neuropeptide in the chicken central nervous system.     

The presence of brain natriuretic peptide of 12,000 daltons in porcine heart

Brain natriuretic peptide (BNP) and its N-terminally six amino acid extended form (BNP-32) have been identified in porcine brain. These peptides exert diuretic-natriuretic and hypotensive effects, and have remarkably high sequence homology to atrial natriuretic peptide (ANP). We have set up a radioimmunoassay system specific to BNP and surveyed immunoreactive (ir-) BNP in peripheral tissue. In porcine cardiac atrium, we found the highest concentration of ir-BNP. By using gel filtration and reverse phase high performance liquid chromatography, ir-BNP was characterized. Most of ir-BNP in the atrium was found to exist as a high molecular weight form of 12,000 daltons; less than 15% of the total ir-BNP exist as low molecular weight forms such as BNP and BNP-32. These results suggest that BNP functions as a circulating hormone in addition to the neuropeptide function in brain.     

Met-enkephalyl-Arg6-Phe7 immunoreactivity in a human neuroblastoma cell line: effect of dibutyryl 3':5'-cyclic AMP and reserpine

The carboxy terminal part of the proenkephalin A sequence is the 31 amino acid peptide B, which has as its final seven amino acids the sequence of the opioid peptide Met-enkephalyl-Arg6-Phe7. Using a radioimmunoassay which recognises both these peptides we have investigated the relative amounts of peptide B and Met-enkephalyl-Arg6-Phe7 in a human neuroblastoma cell line. We show that these cells contain peptide B-like immunoreactivity but not its heptapeptide fragment. This may be due to lack of proteolytic activity cleaving Met-enkephalyl-Arg6-Phe7 from its precursor, peptide B. On treatment with dibutyryl cyclic AMP the level of immunoreactivity approximately doubles, due to increased amounts of peptide B-like immunoreactivity. Treatment with reserpine, which increases conversion of peptide B to the heptapeptide in bovine chromaffin cells in culture does not stimulate the accumulation of Met-enkephalyl-Arg6-Phe7 in the human neuroblastoma cells. The results are discussed with respect to peptide processing.     

N-terminally extended form of C-type natriuretic peptide (CNP-53) identified in porcine brain

C-type natriuretic peptide of 22 residues (CNP-22) is very recently identified in porcine brain as a third member of the mammalian natriuretic peptide family (1). Using a radioimmunoassay system newly established for CNP-22, we searched for CNP-related peptides in porcine brain. In addition to CNP-22, one major form of immunoreactive CNP was detected in porcine brain extracts, being isolated by immunoaffinity chromatography and reverse phase high performance liquid chromatography. By microsequence analysis, the peptide was deduced to be a 53-amino acid peptide carrying a CNP-22 sequence at the C-terminus, and was designated C-type natriuretic peptide-53 (CNP-53). CNP-53 was found to be a major molecular form of CNP in porcine brain.     

Distribution and characterisation of neuropeptide Y immunoreactivity in the brain and gastrointestinal tract of the rainbow trout,Oncorhynchus mykiss

1. Neuropeptide Y (NPY) immunoreactivity has been localised cytochemically in neuronal somata and fibres in rainbow trout brain, nerve fibres and mucosal epithelial endocrine cells within the gastrointestinal tract and in endocrine cells within pancreatic islets.2. Using a C-terminal specific NPY radioimmunoassay, immunoreactivity was detected in extracts of brain (519 pmol/g), cardiac stomach (37.9 pmol/g), pyloric stomach plus pancreas (37.9 ol/g) and intestine (29.2 pmol/g).3. Gel permeation and reverse-phase HPLC analysis of brain and intestinal extracts resolved a single NPY immunoreactive peptide.     

Characterization of the C-terminal flanking peptide of human beta-preprotachykinin

The nucleotide sequence of cDNA encoding the common biosynthetic precursor of substance P, neurokinin A and neuropeptide K (beta-preprotachykinin) predicts that, in the human, the precursor contains a C-terminal flanking peptide of 19 amino acid residues [beta-preprotachykinin(111-129)-peptide]. Using an antiserum raised against synthetic human beta-preprotachykinin(117-126)-peptide in radioimmunoassay, we have demonstrated that an extract of a human neuroendocrine tumor of the adrenal medulla contained approximately equimolar concentrations of C-terminal preprotachykinin immunoreactivity (C-PPT-IR), substance P and neurokinin A. The C-terminal preprotachykinin flanking peptide was purified to homogeneity and its primary structure was determined. The amino acid sequence of the peptide, Ala-Leu-Asn-Ser-Val-Ala-Tyr-Glu-Arg-Ser-Ala-Met-Gln-Asn-Tyr-Glu, indicates identity with beta-preprotachykinin(111-126)-peptide. The data suggest that the C-terminal flanking peptide, like the tachykinins, is packed into secretory storage vesicles but the Arg127-Arg128-Arg129 residues in human beta-preprotachykinin are removed from the peptide by the action of endogenous processing enzyme(s).     

Atrial natriuretic polypeptides (ANP): rat atria store high molecular weight precursor but secrete processed peptides of 25-35 amino acids

A radioimmunoassay was developed for rat atrial natriuretic polypeptides. Using the radioimmunoassay and gel filtration in reducing and dissociating conditions, extracts of rat atria were found to contain mainly a 15000-dalton immunoreactive material, probably corresponding to pronatriodilatin. However, when isolated beating atria were incubated in plasma-free conditions, the secreted immunoreactive material consisted almost exclusively of 2500-3500-dalton peptide(s). These results show that rat atrial cells secrete a low molecular weight natriuretic peptide which is highly active, but store the less active large molecular weight form(s).     

Leumorphin is a novel endogenous opioid peptide in man

Porcine leumorphin, a putative opioid peptide corresponding to amino acid residues 228-256 of preproenkephalin B has been demonstrated to exist in the porcine neurointermediate pituitary. A recent study on the sequence analysis of genomic DNA of human preproenkephalin B has shown that human leumorphin differs in 3 amino acid residues from porcine leumorphin. In order to clarify whether leumorphin is an endogenous opioid peptide in man, we have studied its existence in the human brain using a radioimmunoassay and its opioid activity by a bioassay with the guinea-pig ileum myenteric plexus-longitudinal muscle preparation. High performance gel permeation chromatography and reverse-phase high performance liquid chromatography coupled with the radioimmunoassay for leumorphin have revealed that human leumorphin exists in water extracts of the human striatum. In the guinea-pig ileum assay, synthetic human leumorphin exhibited potent opioid activity, with the concentration of 3nM to give 50 per cent inhibition. These results indicate that leumorphin is a novel endogenous opioid peptide in man.     

Antibodies to FMRF amide, and the related pentapeptide LPLRF amide, reveal two groups of immunoreactive peptides in chicken brain

A radioimmunoassay has been developed for the chicken brain peptide, Leu-Pro-Leu-Arg-Phe-amide (LPLRF amide); this peptide was originally discovered because it reacts with antibodies to the molluscan neuropeptide FMRF amide. The present antibody to LPLRF amide reacts about twenty times less well with FMRF amide compared with LPLRF amide. Using radioimmunoassays employing antibodies raised against LPLRF amide and FMRF amide we have separated by gel filtration and HPLC several different immunoreactive peptides in acid alcohol extracts of chicken brain. When LPLRF amide was used as the assay standard one group of peptides reacted similarly with the two types of antibody; the other group, which was represented by a single major component, reacted at least 50 times better with FMRF amide antibodies compared with LPLRF amide antibodies. It seems, therefore, that in the avian central nervous system, and probably other vertebrates, there are several different groups of peptides immunochemically related to FMRF amide.     

Determination of a putative phosphate-containing peptide in calreticulin.

Calreticulin is an abundant endo/sarcoplasmic reticulum (ER/SR) protein that may carry out multiple functions inside cells. Except for calreticulin, all of the major ER/SR Ca2+-binding proteins are substrates for protein kinase CK2 in vitro, which led us to hypothesize that native calreticulin might exist in the phosphorylated form. To investigate this possibility, we purified calreticulin from cardiac microsomes and verified its identity by immunoblot analysis and sequencing of tryptic peptides. Purified calreticulin, like cardiac calsequestrin, contained endogenous phosphate as determined by a Malachite green assay for phosphate. Previous analyses of cardiac calsequestrin have localized phosphate to a single tryptic peptide containing serine phosphate on sites phosphorylated by protein kinase CK2. Using a similar procedure, we analyzed calreticulin tryptic peptides with Malachite green, localizing phosphate binding to a single calreticulin peptide 367LKEEEEDKK. As this peptide contains no phosphorylatable residues, our results suggest that calreticulin may tightly bind phosphate or a phosphate-containing molecule at this site.