Isolation of neuronal parvalbumin by high-performance liquid chromatography. Characterization and comparison with muscle parvalbumin

Neuronal parvalbumin has been isolated from rat brain and purified to homogeneity by high-performance liquid chromatography (HPLC) on reverse-phase supports. This procedure includes four consecutive chromatographic steps with an overall protein recovery of 74% and a 26 400-fold purification. The concentration of parvalbumin was found to be approximately 10 mg/kg wet weight in brain tissue, which is about 100 times lower than that in rat muscle. The physical properties of brain parvalbumin are described and compared with those of the muscle counterpart. These proteins were identical in their molecular weights (12 000), isoelectric points (4.9), retention times on C-18 reverse-phase HPLC columns, Ca2+ content (two per molecule), amino acid compositions, and immunological properties. A comparison of the tryptic peptide maps of brain and muscle parvalbumin by analytical HPLC also revealed identity and showed that the isolation method described here did not alter the chemical structure of the protein.     

Angiotensin II-Like Material Extracted from the Rat Brain Is Distinct from Authentic Angiotensin II

Specific radioimmunoassay and radioreceptor assay for angiotensin II (A II) were used for the possible identification of this peptide in the rat brain. An A II-like material (A II-LM) was detected with both assays applied to acidic extracts of various brain structures. The regional distribution of A II-LM was uneven, but absolute levels (in A II equivalents) could not be accurately determined, as they were highly dependent on the assay used. Partial purification of A II-LM by Sep-Pak C 18 chromatography and affinity chromatography using anti-A II antibodies bound to Ultrogel gave a compound coeluting with authentic A II in reverse-phase HPLC. However, gel filtration through Sephadex G-25 and TSK Spherogel 3000 SW as well as anion exchange HPLC demonstrated that A II-LM did not correspond to authentic A II. Partial characterization of A II-LM indicated that this compound was probably a peptide with an apparent molecular weight of 5,000-7,000 (instead of 1,046 for A II) and more polar but less positively charged than A II. Whether A II-LM is, in fact, the endogenous ligand of A II binding sites in brain remains an interesting hypothesis for further investigations.     

Recombinant expression, purification, and characterization of scorpion toxin BmαTX14

Long-chain and cysteine-rich scorpion toxins exhibit various pharmacological profiles for different voltage-gated sodium channel subtypes. However, the exploration of toxin structure-function relationships has progressed slowly due to the difficulty of obtaining synthetic or recombinant peptides. We now report that we have established an effective expression and purification approach for the novel scorpion toxin BmαTX14. BmαTX14 was over-expressed as inclusion bodies in Escherichia coli. The insoluble pellet was successfully transformed into active peptide by using a refolding procedure. One-step purification by reverse-phase HPLC was sufficient to generate chromatographically pure peptide. The yield of recombinant toxin reached 4mg from 1L LB medium. The pharmacological data further showed that BmαTX14 selectively inhibited the fast inactivation of mNa(v)1.4 (EC(50)=82.3±15.7nM) rather than that of rNa(v)1.2 (EC(50)>30μM), which indicates that BmαTX14 is a new α-like toxin. This work enables further structural, functional, and pharmacological studies of BmαTX14 and similar toxins.     

Discovery of a natriuretic peptide family and their clinical application

The identification of atrial natriuretic peptide (ANP) induced an explosive series of studies on the new peptide involved in control of the circulation, both in the basic and clinical fields. During the first decade of ANP research surprising progress has been made, revealing that the heart is an endocrine organ regulating the circulation system. ANP has been developed as a diagnostic tool and as a therapeutic drug for cardiac failure. In the second decade, brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were identified, unveiling new profiles of this peptide family. Although BNP is also a circulating hormone that shares a common receptor with ANP, it is different from ANP in its' synthesis and secretion. Plasma concentration of BNP reflects the severity of heart failure in patients in a dramatic fashion, much moreso than ANP. Thus, BNP has been developed as a powerful diagnostic tool for cardiovascular diseases. The third congener, CNP, having a receptor of its own, was initially thought to function only in the brain. CNP was subsequently found to be produced from vascular endothelial cells and macrophages, indicating that CNP is a local regulator and also an antiproliferative factor in the vascular cell system, rather than a circulating hormone. Trials for the clinical application of CNP have also been discussed.     

Urotensin I- and CRF-like peptides in Catostomus commersoni brain and pituitary—HPLC and RIA characterization

Two distinct neuronal systems, containing urotensin 1-like immunoreactivity (UI-LI) and CRF-LI respectively, have previously been demonstrated immunocytochemically in the brain and pituitary of the teleost Catostomus commersoni. In the present studies, we used HPLC followed by UI- and CRF-RIA to further characterize these UI- and CRF-LI substances. HPLC of Catostomus brain extracts (including extracts modified by dilute H₂O₂ oxidation or dilute acid cleavage) suggests that the major form of brain UI-LI is very likely identical to the previously characterized urophysial UI; the small amount of pituitary UI-LI is not yet fully characterized. The behavior of brain CRF-LI on reversed phase (RP) extraction suggests that it may exist largely in precursor form. Pituitary CRF-LI, however, behaved as expected for a peptide on RP extraction, and eluted from HPLC as essentially a single (though somewhat broad) peak. The HPLC behavior of pituitary CRF-LI, and its crossreactivity with various CRF antisera, suggest that it represents authentic fish CRF, apparently similar to (but not identical with) human/rat CRF.     

Receptor autoradiography as mean to explore the possible functional relevance of neuropeptides: focus on new agonists and antagonists to study natriuretic peptides, neuropeptide Y and calcitonin gene-related peptides

Over the past 20 years, receptor autoradiography has proven most useful to provide clues as to the role of various families of peptides expressed in the brain. Early on, we used this method to investigate the possible roles of various brain peptides. Natriuretic peptide (NP), neuropeptide Y (NPY) and calcitonin (CT) peptide families are widely distributed in the peripheral and central nervous system and induced multiple biological effects by activating plasma membrane receptor proteins. The NP family includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). The NPY family is composed of at least three peptides NPY, peptide YY (PYY) and the pancreatic polypeptides (PPs). The CT family includes CT, calcitonin gene-related peptide (CGRP), amylin (AMY), adrenomedullin (AM) and two newly isolated peptides, intermedin and calcitonin receptor-stimulating peptide (CRSP). Using quantitative receptor autoradiography as well as selective agonists and antagonists for each peptide family, in vivo and in vitro assays revealed complex pharmacological responses and radioligand binding profile. The existence of heterogeneous populations of NP, NPY and CT/CGRP receptors has been confirmed by cloning. Three NP receptors have been cloned. One is a single-transmembrane clearance receptor (NPR-C) while the other two known as CG-A (or NPR-A) and CG-B (or NPR-B) are coupled to guanylate cyclase. Five NPY receptors have been cloned designated as Y(1), Y(2), Y(4), Y(5) and y(6). All NPY receptors belong to the seven-transmembrane G-protein coupled receptors family (GPCRs; subfamily type I). CGRP, AMY and AM receptors are complexes which include a GPCR (the CT receptor or CTR and calcitonin receptor-like receptor or CRLR) and a single-transmembrane domain protein known as receptor-activity-modifying-proteins (RAMPs) as well as an intracellular protein named receptor-component-protein (RCP). We review here tools that are currently available in order to target each NP, NPY and CT/CGRP receptor subtype and establish their respective pathophysiological relevance.     

Development and potential of non-peptide antagonists for calcitonin-gene-related peptide (CGRP) receptors: evidence for CGRP receptor heterogeneity

Calcitonin-gene-related peptide (CGRP) is a 37-amino-acid vasodilatory peptide, of which two isoforms, alpha CGRP and beta CGRP, have been described. The use of C-terminal fragments of CGRP peptide, such as human alpha CGRP-(8-37), has led to the pharmacological subdivision of CGRP receptors into CGRP-1 [high potency for binding of human alpha CGRP-(8-37)] and CGRP-2 (lower potency) receptors. We have recently developed BIBN4096BS, the first non-peptide CGRP antagonist, which has sub-nanomolar affinity for primate CGRP receptors. The use of BIBN4096BS has led to the discovery of further functional CGRP receptor heterogeneity in rat tissues. To further exploit BIBN4096BS as a pharmacological tool, we used BIBN4096BS in pig left anterior descending coronary vessels and cerebral basilar arteries, and compared functional with molecular data, characterizing CGRP receptor components. Our data show that, apart from a subdivision into CGRP-1 and -2 receptors, BIBN4096BS reveals additional functional differences between the actions of alpha CGRP and beta CGRP. However, evidence for CGRP receptor heterogeneity on a molecular level is scarce.     

Determination of agmatine in brain and plasma using high-performance liquid chromatography with fluorescence detection

Decarboxylated arginine, agmatine, is a neurotransmitter candidate for imidazoline receptors. A method is described to measure agmatine in rat brain and human plasma by isocratic high-performance liquid chromatography (HPLC) with flourescence detection and o-phthalaldehyde derivatization. Quantitation is based on the method of additions of internal agmatine spikes. This assay has sensitivity in the low picomole range and a detection limitof 100 fmol. The correlation coefficient for the agmatine standard curve was 0.999±0.001 S.D., and intra- and inter-assay C.V.s were less than 8%. The accuracy of our isocratic method compared favorably with a gradient HPLC protocol, originally developed for bacterial agmatine, which we modified for use with tissues. Agmatine concentrations in rat brain were proportioned similarly to the regional distribution of imidazoline-1 receptors. These methods can be used as reliable research tools in various biological samples.     

C-type natriuretic peptide (CNP): a new member of natriuretic peptide family identified in porcine brain

Two types of natriuretic peptide, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), very similar to each other in structure and in pharmacological effect, are known to be present in mammalian heart and brain. In our present survey for unidentified peptides in porcine brain extracts, we found a new peptide of 22 amino acid residues, eliciting a potent relaxant activity on chick rectum. The amino acid sequence determined for the peptide shows remarkable similarity to those of ANP and BNP, especially in the 17-residue sequences flanked by two cysteine residues. The peptide shows a pharmacological spectrum similar to ANP and BNP. Thus, the peptide was designated "C-type natriuretic peptide (CNP)", the third member to join the natriuretic peptide family. In contrast to ANP and BNP, CNP terminates in the second cysteine residue, lacking a further C-terminal extension.     

Locustakinin, a novel myotropic peptide from Locusta migratoria, isolation, primary structure and synthesis.

The isolated hindgut of the cockroach, Leucophaea maderae is a very efficient bioassay tool for the monitoring of certain structural types of insect myotropic peptides during HPLC purification. Using this detection system, a six residue peptide has been isolated from an extract of 9000 brain corpora cardiaca-corpora allata suboesophageal ganglion complexes of Locusta migratoria. Amino acid composition and sequence analysis combined with enzymatic digestion data established the structure of the novel peptide as Ala-Phe-Ser-Ser-Trp-Gly-amide. The chromatographic and biological properties of the synthetic peptide were the same as those of the native peptide, thus confirming structural analysis. The carboxy-terminal pentamer sequence is the active core of leucokinins II, V and VII and of achetakinin III (myotropic neuropeptides isolated from Leucophaea m. and from Acheta domesticus; Holman et al., 1990). Furthermore, the octapeptide leucokinin VII contains the novel sequence as its carboxy-terminal hexamer and Achetakinin V (AFHSWGamide) differs from it by one residue. This new peptide designated as locustakinin I (locusts) may therefore represent an evolutionary molecular link between leucokinin VII (cockroaches) and achetakinin V (crickets). Using synthetic locustakinin, physiological studies will be performed in the locust. In view of the known effects of leucokinins, locustakinin may be important in the stimulation of ion transport and inhibition of diuretic activity in Malpighian tubules. This study indicates that the AFXSWGamide sequence appears to have been well conserved and that members of this peptide family may be widely distributed among insects and posses a number of functions.     

Isolation and sequence determination of frog C-type natriuretic peptide

A new bioactive peptide was isolated from frog brain using a bioassay for chick rectum relaxant activity. Amino acid sequence of this peptide was determined to be Gly-Tyr-Ser-Arg-Gly-Cys-Phe-Gly-Val-Lys-Leu-Asp-Arg-Ile-Gly-Ala-Phe-Ser- Gly- Leu-Gly-Cys, in which two cysteines were linked by a disulfide bond. The peptide was found to belong structurally to the natriuretic peptide family and to exert diuretic-natriuretic activity as well as hypotensive activity when injected into rats. The peptide showed a high homology to recently identified porcine C-type natriuretic peptide (CNP) and a pharmacological spectrum highly similar to porcine CNP. Thus, the peptide was designated frog C-type natriuretic peptide (frog CNP). Frog CNP may participate in the central control of body fluid homeostasis, since its tissue concentration is high in brain.     

Soluble Combinatorial Libraries: Extending the Range and Repertoire of Chemical Diversity

The generation and use of libraries made up of millions of chemical entities and the ability to identify the active compound in such libraries are at the forefront of a revolution in drug discovery and basic research. Such libraries, when made up of peptide sequences, offer a fundamental, practical advance in the study of interactions between peptides and their biochemical or pharmacological targets. The utility of soluble peptide libraries ranging from three to eight amino acids in length, and made up of mixtures from 361 tripeptides to 200 billion decapeptides, is described. These are readily usable in virtually all in vitro (and even in vivo) assay systems. The examples presented illustrate the utility of soluble peptide libraries for the study of antibody/antigen interactions, the identification of highly active opioid peptides in receptor binding studies using crude rat brain homogenates, and in vivo studies, in which the peptide mixtures making up the library are administered intravenously to determine peptide sequences that affect heart rate and blood pressure. A new class of library is also described, termed a modified peptide library, which is used to determine potent anti-Staphylococcus aureus compounds.     

Biogenic amines in the brain of Manduca sexta during larval-pupal metamorphosis

1. The extracts of brains (cerebral ganglion) of the tobacco hornworm larvae contain octopamine, dopamine and 5-hydroxytryptamine, but not norepinephrine via analysis using a 16-channel electrochemical array HPLC system.2. A comprehensive daily analysis reveals the patterns of biogenic amine levels in the brain during larval—pupal metamorphosis.3. Monoamine oxidase-based amine metabolites are not present at detectable levels. N-β-Alanyldopamine is the predominant metabolite of dopamine present in the brain during the postwandering phase of the last larval stadium.     

Automated in-solution protein digestion using a commonly available high-performance liquid chromatography autosampler

A completely automated peptide mapping liquid chromatography/mass spectrometry (LC/MS) system for characterization of therapeutic proteins in which a common high-performance liquid chromatography (HPLC) autosampler is used for automated sample preparation, including protein denaturation, reduction, alkylation, and enzymatic digestion, is described. The digested protein samples are then automatically subjected to LC/MS analysis using the same HPLC system. The system was used for peptide mapping of monoclonal antibodies (mAbs), known as a challenging group of therapeutic proteins for achieving complete coverage and quantitative representation of all peptides. Detailed sample preparation protocols, using an Agilent HPLC system, are described for Lys-C digestion of mAbs with intact disulfide bonds and tryptic digestion of mAbs after reduction and alkylation. The automated procedure of Lys-C digestion of nonreduced antibody, followed by postdigestion disulfide reduction, produces both the nonreduced and reduced digests that facilitate disulfide linkage analysis. The automated peptide mapping LC/MS system has great utility in preparing and analyzing multiple samples for protein characterization, identification, and quantification of posttranslational modifications during process and formulation development as well as for protein identity and quality control.     

11C-labeling and preliminary evaluation of vortioxetine as a PET radioligand

Vortioxetine is a new multi-modal drug against major depressive disorder with high affinity for a range of different serotonergic targets in the CNS. We report the ¹¹C-labeling of vortioxetine with [¹¹C]MeI using a Suzuki-protocol that allows for the presence of an unprotected amine. Preliminary evaluation of [¹¹C]vortioxetine in a Danish Landrace pig showed rapid brain uptake and brain distribution in accordance with the pharmacological profile, all though an unexpected high binding in cerebellum was also observed. [¹¹C]vortioxetine displayed slow tracer kinetics with peak uptake after 60 min and with limited wash-out from the brain. Further studies are needed but this radioligand may prove to be a valuable tool in unraveling the clinical effects of vortioxetine.     

Separation and isolation methods for analysis of the active principles of Sho-saiko-to (SST) oriental medicine

Sho-saiko-to (SST) was introduced into Japan as an oriental classical medicine from China approximately 1500 years ago, and it is currently the most representative Kampo medicine (traditional Japanese medicine). SST is manufactured in Japan as an ethical drug on a modern industrial scale in which the quality of ingredients is standardized with Good Manufacturing Practices (GMP) regulation. SST is widely used for the treatment of chronic hepatitis. Experimental and clinical studies including multi-center, placebo-controlled, double-blind studies have demonstrated the various pharmacological effects of SST. SST is prepared from the hot water extraction of seven raw materials, therefore many kinds of constituents are included. Three-dimensional (3D) HPLC analysis is useful for obtaining many kinds of constituents, especially low molecular ultraviolet (UV) quenching compounds, contained in SST as well as its fractions. Fingerprint pattern provided by 3D HPLC analysis makes possible to identify the overall-viewing of SST. Databases of UV spectra of the components of medicinal herbs obtained by reversed-phase (RP) HPLC using a photodiode array (PDA) and fingerprint patterns of crude drugs made by 3D HPLC analysis facilitate the identification, analysis and quality of herbal drugs. Studies using both PDA HPLC and an amino acid analysis with a fluorometric detector have found that SST contains fifteen major low molecular compounds (i.e. baicalin, wogonin-7-O-glucuronide, liquiritin, their three aglycons, liquiritin apioside, glycyrrhizin, saikosaponin b1, saikosaponin b2, ginsenoside Rg1, ginsenoside Rb1, (6)-gingerol, (6)-shogaol and arginine). These compounds have various pharmacological actions, and are assumed to be responsible, at least partly, for the pharmacological effects of SST. Although there have only been a few investigations on high molecular compounds with pharmacological actions contained in SST, several kinds of polysaccharides have been isolated from constituent herbs of SST. This review paper summarizes analytical methods of separation, isolation and identification of compounds with biological activities from SST, which is a mixture drug of medicinal herbs. Accordingly, this paper would not focus on methods of separation, isolation and analysis of particular compounds from each constituent herb of SST.     

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.     

Identification of a 29-amino acid natriuretic peptide in chicken heart

Morphological and pharmacological observations have suggested that chicken atrial natriuretic peptide (ANP) is different from mammalian ANP. The present survey for the as yet unidentified ANP in chicken heart was performed by monitoring the relaxant effect on chick rectum. From the low molecular weight component of rectum relaxant activity observed in acid extracts of chicken ventricle, a novel 29-amino acid peptide was purified. The identical peptide was also isolated from acid extracts of chicken atrium. The peptide elicited a pharmacological spectrum very similar to that of mammalian ANP, including diuretic-natriuretic and hypotensive activity. Thus, the peptide was designated "chicken alpha-ANP (alpha-chANP)". The complete amino acid sequence determined for the peptide showed remarkable homology with that of mammalian alpha-ANP. However, maximum homology was observed when the peptide was compared with a recently identified porcine brain natriuretic peptide (BNP).     

A simple method for measuring intracellular activities of glutamine synthetase and glutaminase in glial cells

Here we report and validate a simple method for measuring intracellular activities of glial glutamine synthetase (GS) and glutaminase (GLNase) in intact glial cells. These enzymes are responsible for glutamate and glutamine recycling in the brain, where glutamate and glutamine transport from the blood stream is strongly limited by the blood-brain barrier. The intracellular levels of glutamate and glutamine are dependent on activities of numerous enzymatic processes, including 1) cytosolic production of glutamine from glutamate by GS, 2) production of glutamate from glutamine by GLNase that is primarily localized between mitochondrial membranes, and 3) mitochondrial conversion of glutamate to the tricarboxylic cycle intermediate α-ketoglutarate in the reactions of oxidative deamination and transamination. We measured intracellular activities of GS and GLNase by quantifying enzymatic interconversions of L-[(3)H]glutamate and L-[(3)H]glutamine in cultured rat astrocytes. The intracellular substrate and the products of enzymatic reactions were separated in one step using commercially available anion exchange columns and quantified using a scintillation counter. The involvement of GS and GLNase in the conversion of (3)H-labeled substrates was verified using irreversible pharmacological inhibitors for each of the enzymes and additionally validated by measuring intracellular amino acid levels using an HPLC. Overall, this paper describes optimized conditions and pharmacological controls for measuring GS and GLNase activities in intact glial cells.     

Bioassays in modern peptide research

Bioassays have been used in pharmacology since its beginning, nearly one hundred years ago. With the recent development of new techniques such as radioreceptor assays, radioimmunoassays, high pressure liquid chromatography (HPLC) and recombinant DNA technology, it might be asked if bioassays are still up-to-date in a fast moving field like peptide research? This brief review describes the possible uses of bioassays as well as the basic criteria used to characterize a bioassay. Recent results obtained with bioassays for various peptides will demonstrate the importance of this technique in modern peptide research.