ABSTRACT
With the continuous development, in the last decades, of analytical techniques providing complex information at single cell level, the study of cell heterogeneity has been the focus of several research projects within analytical biotechnology. Nonetheless, the complex interplay between environmental changes and cellular responses is yet not fully understood, and the integration of this new knowledge into the strategies for design, operation and control of bioprocesses is far from being an established reality. Indeed, the impact of cell heterogeneity on productivity of large scale cultivations is acknowledged but seldom accounted for. In order to include population heterogeneity mechanisms in the development of novel bioprocess control strategies, a reliable mathematical description of such phenomena has to be developed. With this review, we search to summarize the potential of currently available methods for monitoring cell population heterogeneity as well as model frameworks suitable for describing dynamic heterogeneous cell populations. We will furthermore underline the highly important coordination between experimental and modeling efforts necessary to attain a reliable quantitative description of cell heterogeneity, which is a necessity if such models are to contribute to the development of improved control of bioprocesses.
Subject(s)
Cell Biology , Cell Physiological Phenomena , Cytological Techniques , Models, Biological , Systems BiologyABSTRACT
Synthesis and derivatization of a series of substituted tetrahydrofluorenone analogs giving potent, ERbeta subtype selective ligands are described. Several analogs possessing ERbeta binding affinities comparable to 17beta-estradiol but with greater than 75-fold selectivity over ERalpha are reported.
Subject(s)
Estrogen Receptor beta/drug effects , Fluorenes/chemical synthesis , Fluorenes/pharmacology , Cell Line , Crystallography, X-Ray , Estrogen Receptor alpha/chemistry , Estrogen Receptor alpha/drug effects , Estrogen Receptor beta/chemistry , Fluorenes/classification , Humans , Ligands , Models, Molecular , Molecular Structure , Stereoisomerism , Structure-Activity RelationshipABSTRACT
BACKGROUND: Estrogens exert their effects on target tissues by binding to a nuclear transcription factor termed the estrogen receptor (ER). Previous structural studies have demonstrated that each class of ER ligand (agonist, partial agonist, and SERM antagonist) induces distinctive orientations in the receptor's carboxy-terminal transactivation helix. The conformation of this portion of the receptor determines whether ER can recruit and interact with the components of the transcriptional machinery, thereby facilitating target gene expression. RESULTS: We have determined the structure of rat ERbeta ligand binding domain (LBD) in complex with the pure antiestrogen ICI 164,384 at 2.3 A resolution. The binding of this compound to the receptor completely abolishes the association between the transactivation helix (H12) and the rest of the LBD. The structure reveals that the terminal portion of ICI's bulky side chain substituent protrudes from the hormone binding pocket, binds along the coactivator recruitment site, and physically prevents H12 from adopting either its characteristic agonist or AF2 antagonist orientation. CONCLUSIONS: The binding mode adopted by the pure antiestrogen is similar to that seen for other ER antagonists. However, the size and resultant positioning of the ligand's side chain substituent produces a receptor conformation that is distinct from that adopted in the presence of other classes of ER ligands. The novel observation that binding of ICI results in the complete destabilization of H12 provides some indications as to a possible mechanism for pure receptor antagonism.
Subject(s)
Estradiol/chemistry , Estrogen Antagonists/chemistry , Receptors, Estrogen/chemistry , Animals , Binding Sites , Crystallography, X-Ray , Dimerization , Estradiol/analogs & derivatives , Estrogen Receptor beta , Ligands , Models, Molecular , Molecular Structure , Polyunsaturated Alkamides , Protein Structure, Quaternary , Protein Structure, Tertiary , RatsABSTRACT
Here we summarise the results that have emerged from our structural studies on the oestrogen receptor (ER) ligand-binding domain. We have investigated the conformational effects of a variety of ligands on the structures of both ER isoforms. Each class of ligand (agonists, partial agonists and selective oestrogen receptor modulators) induces a unique conformation in the receptor's ligand-dependent transcriptional activation function. Together these studies have broadened our understanding of ER function by providing a unique insight into ER's ligand specificity and the structural changes that underlie receptor agonism and antagonism.
Subject(s)
Receptors, Estrogen/chemistry , Estradiol/pharmacology , Humans , Ligands , Receptors, Estrogen/agonists , Receptors, Estrogen/antagonists & inhibitors , Selective Estrogen Receptor Modulators/pharmacologyABSTRACT
We have determined the three-dimensional structures of both alpha- and beta-forms of the ligand-binding domain of the oestrogen receptor (ER) in complexes with a range of receptor agonists and antagonists. Here, we summarize how these structures provide both an understanding of the ER's distinctive pharmacophore and a rationale for its ability to bind a diverse range of chemically distinct compounds. In addition, these studies provide a unique insight into the mechanisms that underlie receptor activation, as well as providing a structural basis for the antagonist action of molecules, such as raloxifene.
Subject(s)
Receptors, Estrogen/agonists , Receptors, Estrogen/antagonists & inhibitors , Receptors, Estrogen/chemistry , Animals , Binding Sites , Ligands , Models, Molecular , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Raloxifene Hydrochloride/pharmacology , Selective Estrogen Receptor Modulators/pharmacologyABSTRACT
The overall architecture of the ligand binding domain (LBD) of members of the nuclear receptor superfamily are similar. There are now standard procedures to express and purify these proteins. A rapid and sensitive method for the structural analysis of these proteins is nano-electrospray tandem mass spectrometry. In the present study we have analysed the LBD of the human thyroid hormone receptor-beta-1 (TR-beta) by quadrupole time-of-flight tandem mass spectrometry. The intact protein was analysed in a carboxymethylated form in an attempt to identify which cysteine residues are located on the surface. The protein molecular weight (31 652.5 Da) was determined with an accuracy of +/-1 Da, while masses of tryptic fragments were determined with an accuracy of at least 75 ppm. The sequence coverage of the tryptic peptide mass map was 93.2 %. Tryptic peptides were subjected to collision-induced dissociation (CID) and the resulting product ions were mass measured with an accuracy of about 100 ppm. When accurate mass measurements were made with internal calibration, mass accuracies were improved to +/-2 ppm in mass spectra, and +/-20 ppm in CID spectra. From these data it was possible to determine the presence of post-translational modifications, locate the sites of carboxymethylation and, in addition, confirm the amino acid sequence of the expressed protein. To the best of our knowledge, this is the first characterisation of the TR-LBD-beta at the protein level.
Subject(s)
Receptors, Thyroid Hormone/chemistry , Thyroid Hormones/chemistry , Amino Acid Sequence , Binding Sites , Humans , Ligands , Mass Spectrometry , Molecular Sequence Data , Receptors, Thyroid Hormone/metabolism , Thyroid Hormones/metabolismABSTRACT
Oestrogens exert their physiological effects through two receptor subtypes. Here we report the three-dimensional structure of the oestrogen receptor beta isoform (ERbeta) ligand-binding domain (LBD) in the presence of the phyto-oestrogen genistein and the antagonist raloxifene. The overall structure of ERbeta-LBD is very similar to that previously reported for ERalpha. Each ligand interacts with a unique set of residues within the hormone-binding cavity and induces a distinct orientation in the AF-2 helix (H12). The bulky side chain of raloxifene protrudes from the cavity and physically prevents the alignment of H12 over the bound ligand. In contrast, genistein is completely buried within the hydrophobic core of the protein and binds in a manner similar to that observed for ER's endogenous hormone, 17beta-oestradiol. However, in the ERbeta-genistein complex, H12 does not adopt the distinctive 'agonist' position but, instead, lies in a similar orientation to that induced by ER antagonists. Such a sub-optimal alignment of the transactivation helix is consistent with genistein's partial agonist character in ERbeta and demonstrates how ER's transcriptional response to certain bound ligands is attenuated.
Subject(s)
Ligands , Receptors, Estrogen/agonists , Receptors, Estrogen/antagonists & inhibitors , Receptors, Estrogen/chemistry , Amino Acid Sequence , Animals , Crystallography, X-Ray , Estrogen Receptor beta , Genistein/chemistry , Humans , Models, Molecular , Molecular Sequence Data , Raloxifene Hydrochloride/chemistry , Rats , Sequence Homology, Amino Acid , Tamoxifen/chemistryABSTRACT
Oestrogens are involved in the growth, development and homeostasis of a number of tissues. The physiological effects of these steroids are mediated by a ligand-inducible nuclear transcription factor, the oestrogen receptor (ER). Hormone binding to the ligand-binding domain (LBD) of the ER initiates a series of molecular events culminating in the activation or repression of target genes. Transcriptional regulation arises from the direct interaction of the ER with components of the cellular transcription machinery. Here we report the crystal structures of the LBD of ER in complex with the endogenous oestrogen, 17beta-oestradiol, and the selective antagonist raloxifene, at resolutions of 3.1 and 2.6 A, respectively. The structures provide a molecular basis for the distinctive pharmacophore of the ER and its catholic binding properties. Agonist and antagonist bind at the same site within the core of the LBD but demonstrate different binding modes. In addition, each class of ligand induces a distinct conformation in the transactivation domain of the LBD, providing structural evidence of the mechanism of antagonism.
Subject(s)
Receptors, Estrogen/agonists , Receptors, Estrogen/antagonists & inhibitors , Binding Sites , Crystallography, X-Ray , Estradiol/chemistry , Estradiol/metabolism , Estrogen Antagonists/chemistry , Estrogen Antagonists/pharmacology , Humans , Models, Molecular , Piperidines/chemistry , Piperidines/pharmacology , Protein Conformation , Raloxifene Hydrochloride , Receptors, Estrogen/chemistry , Receptors, Estrogen/metabolismABSTRACT
Functional rat estrogen receptor beta ligand binding domain (rER beta LBD, aa 210-485) and human estrogen receptor alpha ligand binding domain (hER alpha LBD, aa 301-553) were expressed in Escherichia coli. Hormone binding assays revealed that both ER beta and ER alpha LBDs bound the natural ligand estradiol (E2) with similar affinity (Kd approximately 100 pM). Competitive binding experiments were carried out with ICI 164384, 4-hydroxytamoxifen, 16 alpha-bromo-estradiol, and genistein employing [3H]E2 as a tracer. No significant differences in responses of ER alpha and ER beta LBDs to ICI 164384 and 4-hydroxytamoxifen were observed, 16 alpha-Bromo-estradiol and genistein discriminated between the ER subtypes and acted as ER alpha and ER beta selective ligands, respectively. Final purification of recombinant proteins was achieved on an E2 affinity column, where they were subjected to in situ carboxymethylation. The partially carboxymethylated proteins actively bound E2. The carboxymethylated rER beta LBD had a molecular mass of 32251.6 Da, equivalent to the calculated mass with the addition of three carboxymethyl groups. No other proteins (of lower or higher molecular mass) were detected, so the LBD was considered structurally authentic and pure. By using a combination of intact protein mass spectrometric fragmentation and trypsin proteolysis (98% sequence coverage), it was established that rER beta cysteine-289 and -354 were not carboxymethylated on the affinity column, suggesting that they were shielded from alkylation in the E2-bound conformational state. Concurrent analysis of hER alpha LBD showed that under the same experimental conditions, the two equivalent ER alpha cysteines were not alkylated (alpha C381 and alpha C447). These data support close structural relationship between the E2-bound ER alpha LBD and ER beta LBD proteins.
Subject(s)
Escherichia coli/genetics , Gene Expression , Mass Spectrometry , Peptide Fragments/chemistry , Receptors, Estrogen/chemistry , Alkylation , Amino Acid Sequence , Animals , Binding Sites , Binding, Competitive , Estradiol/analogs & derivatives , Estradiol/metabolism , Estrogen Antagonists/metabolism , Genistein/metabolism , Humans , Molecular Sequence Data , Peptide Fragments/genetics , Peptide Fragments/metabolism , Polyunsaturated Alkamides , Rats , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Sequence Alignment , Tamoxifen/analogs & derivatives , Tamoxifen/metabolismABSTRACT
Electrospray ionization mass spectrometry (ESMS) of the estrogen receptor ligand binding domain (ER LBD) in its estradiol-binding form was performed. A dimeric ER LBD was observed, with a greatly reduced capacity for protonation (major charge state for dimer +16 vs. +23 for a monomer). Peak broadening (probably due to heterogeneity resulting from salt and water adduct formation) adversely affected our ability to distinguish between multiple discreet dimeric species and thus prevented us from establishing an accurate average mass for the dimerized domain. A mixture of species with molecular masses between 57,240 Da and 57,900 Da was observed, which would compare to 57,274 Da, 57,546 Da, and 57,818 Da for the calculated masses of the dimer without estradiol, or with one or two bound ligand molecules, respectively. Hence, nonliganded ER LBD dimer appeared to constitute the major species. The presence of low levels of a singly liganded ER LBD dimer cannot be ruled out, but the data argue against the possibility of the ER LBD dimer carrying two molecules of estradiol. Allowing for current limitations in the technology, our data demonstrate that ESMS on a quadrupole mass spectrometer of limited mass range (4000 Da for singly charged ions) has potential utility for studying ligand-binding proteins. In particular, in future it might be possible to compare spectra obtained from agonist- and antagonist-bound receptors and determine from subtle changes in protonation state possible differences in the higher order structure of those noncovalent protein complexes.
Subject(s)
Mass Spectrometry/methods , Receptors, Estrogen/chemistry , Receptors, Estrogen/metabolism , Binding Sites , Estradiol/chemistry , Estradiol/metabolism , Hydrogen-Ion Concentration , Ligands , Models, Molecular , Protein Conformation , Protein Denaturation , SolventsABSTRACT
Experiments were carried out to determine the degree of solvent and reagent accessibility of the cysteines in the ligand-binding domain of the human estrogen receptor (hER LBD). The cysteine residues were alkylated when human ER LBD was present in its ligand (estradiol)-bound conformation. Direct electrospray ionization mass spectrometry (ESMS) as well as liquid chromatography coupled with ESMS, and matrix-assisted laser ionization desorption time-of-flight mass spectrometry were used to determine the location and the yield of the derivatized residues after proteolysis with trypsin. We observed that the cysteine 447 was protected against alkylation under these conditions, whereas cysteines 381, 417, and 530 were fully derivatized.
Subject(s)
Cysteine/metabolism , Estradiol/metabolism , Iodoacetates/chemistry , Peptide Fragments/chemistry , Receptors, Estrogen/metabolism , Alkylation , Amino Acid Sequence , Binding Sites , Chromatography, High Pressure Liquid , Humans , Iodoacetic Acid , Mass Spectrometry/methods , Methylation , Molecular Sequence Data , Peptide Fragments/metabolism , Peptide Mapping , Protein Conformation , Protein Denaturation , Receptors, Estrogen/chemistryABSTRACT
A peptide hormone has been isolated from porcine intestine. Its primary structure was found to consist of 36 amino acid residues in a sequence identical to that of the porcine pancreatic polypeptide, previously not isolated from intestines or a tissue other than pancreas. The gut polypeptide significantly suppresses glucose-induced insulin secretion in vitro. Using an immunohistochemical technique, we also identified cells in the porcine gastrointestinal tract that were immunoreactive with pancreatic polypeptide. The immunoreactivity disappeared after absorption with the isolated gut polypeptide or synthetic human pancreatic polypeptide.
Subject(s)
Intestine, Small/chemistry , Pancreatic Polypeptide/isolation & purification , Peptides/isolation & purification , Animals , Chromatography, Gel , Chromatography, High Pressure Liquid , Immunohistochemistry , Mass Spectrometry/methods , Pancreatic Polypeptide/chemistry , Peptides/chemistry , SwineABSTRACT
Anion-exchange chromatography of solubilized pig liver cell membranes on DEAE-Sepharose gave a fraction with high affinity binding proteins for VIP and glucagon distinct from each other. Scatchard analysis indicated the presence of one binding site for VIP (Kd 1.5 +/- 0.6 nM and Bmax 1.3 +/- 0.4 pmol/mg). The order of potency for VIP-related peptides to inhibit [125I]VIP binding was: VIP > peptide histidine isoleucine amide (PHI) > rat growth hormone releasing factor (rGRF) > secretin. GTP-gamma-S inhibited [125I]VIP binding and reduced the affinity of VIP binding sites to 6.5 nM. In the same isolated fraction, [125I]glucagon binding was displaced by glucagon preferentially to oxyntomodulin, and GTP did not affect this [125I]glucagon binding. Scatchard analysis indicated the presence of one binding site for glucagon (Kd 0.08 +/- 0.03 nM and Bmax 0.31 +/- 0.01 pmol/mg). A low-affinity VIP binding protein (IC50 0.7 microM) was detected in a fraction eluting later and exhibited a peptide specificity: rGRF > VIP > VIP(10-28) > secretin > PHI. This rGRF-preferring protein (18 kDa) was purified and had a partial amino-acid sequence identical to that of calmodulin. Its [125I]VIP binding was competitively inhibited by VIP and calmidazolium in a manner similar to that for pig brain calmodulin. Thus we have co-solubilized VIP and glucagon high affinity receptors from pig liver cell membranes and separated them from VIP-binding calmodulin.
Subject(s)
Calmodulin/metabolism , Glucagon/metabolism , Receptors, Gastrointestinal Hormone/metabolism , Vasoactive Intestinal Peptide/metabolism , Amino Acid Sequence , Animals , Cholic Acids , Chromatography, DEAE-Cellulose , Detergents , Electrophoresis, Polyacrylamide Gel , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Molecular Sequence Data , Receptors, Gastrointestinal Hormone/drug effects , Receptors, Glucagon , Receptors, Vasoactive Intestinal Peptide , Solubility , SwineABSTRACT
Cysteine is a relatively infrequent constituent of proteins, which in its thiol or half-cystine form contributes in a special manner to their three-dimensional structure. We show that in small cystine-containing peptides, the Cys content is always higher than the average in proteins in general. This observation makes it possible to search for new peptides by monitoring only their Cys content. We have developed a chemical assay for the detection of cyst(e)ine-rich peptides in tissue extracts. Using this assay we have isolated from porcine intestine a novel cysteine-rich peptide, which we denote ZF-1. ZF-1 is homologous to a single zinc-finger motif and has an acetylated N-terminus. This is the first demonstration of the existence of a processed single zinc-finger-like structure. The structural homology of ZF-1 to the zinc-finger motif, present in several metal-binding and DNA-binding proteins, suggests an important role of this peptide in metal transport and/or modulation of gene expression.
Subject(s)
Intestines/chemistry , Proteins/chemistry , Zinc Fingers , Amino Acid Sequence , Animals , Cysteine/chemistry , Molecular Sequence Data , Proteins/isolation & purification , Sequence Homology, Amino Acid , SwineABSTRACT
Two different forms of the monomeric NADP(+)-linked prostaglandin dehydrogenase/carbonyl reductase were purified from human placenta and shown to differ by the modification of a lysine residue. The modified and the unmodified proteins were reproducibly recovered in a ratio of approximately 1:3, and both were chemically stable. The modified form was more acidic (pI approximately 7.4 versus pI approximately 7.7) but indistinguishable from the unmodified form in specificity and activity. Amino acid analysis, sequence analysis, mass spectrometry, and chemical synthesis identified the modified residue as N6-(1-carboxyethyl)lysine with C-2 of propionic acid attached to the side-chain N of Lys-238. This compound can be formed from the lysine residue and pyruvate via a Schiff base and subsequent reduction. The enzyme and its NAD(+)-dependent counterpart are distantly related (23% residue identity) and have the same family assignment to short-chain dehydrogenases. Alignments and model-building into the tertiary structure of 3 alpha/20 beta-hydroxysteroid dehydrogenase show that carbonyl reductase has an extra loop (positions 149-189) that forms a separate extension and replaces a backbone C-terminal beta-strand. This change affects the substrate pocket, explaining the different substrate specificities but conserves residues of known functional importance. Carboxyethyllysine at position 238 corresponds to a proteolysis-sensitive position in several short-chain dehydrogenases, less well-defined in the model but close to a surface, and is compatible with the accessibility and enzyme properties observed.
Subject(s)
Alcohol Oxidoreductases/classification , Alcohol Oxidoreductases/isolation & purification , Isoenzymes , Lysine/analogs & derivatives , Prostaglandins/metabolism , Alcohol Oxidoreductases/chemistry , Aldehyde Reductase , Aldo-Keto Reductases , Amino Acid Sequence , Female , Humans , Hydroxysteroid Dehydrogenases/chemistry , Lysine/isolation & purification , Models, Molecular , Molecular Sequence Data , Placenta/enzymology , Protein Conformation , Protein Structure, Secondary , Sequence Analysis , Sequence Homology, Amino AcidABSTRACT
In a peptide concentrate, prepared from acid extracts of porcine brain, several galanin-like immunoreactive peptides were detected and two of these were purified. Characterization of the peptides by sequence analysis, mass spectrometry, and capillary zone electrophoresis identified them as a N-terminally nine residue elongated form of galanin, preprogalanin(24-61) amide, and as an N-terminally four residue truncated form of galanin corresponding to preprogalanin(37-61) amide. The former finding suggests that the removal of the signal peptide in preprogalanin occurs by enzymatic cleavage between glycine-23 and leucine-24. The presence of truncated galanin might refer to a mechanism, where galanin is inactivated by removal of functionally important amino acid residues from the N-terminus.
Subject(s)
Brain Chemistry/physiology , Neuropeptides/isolation & purification , Peptides/isolation & purification , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Galanin , Molecular Sequence Data , Radioimmunoassay , Sequence Homology, Amino Acid , SwineABSTRACT
Liver alcohol dehydrogenase of the ethanol-active type ('class I enzyme') from the lizard, Uromastix hardwickii, was purified and screened for relationships with other vertebrate forms of the enzyme. Two different acetylated N-termini (acetyl-Gly and acetyl-Ser) and further positional differences already in the N-terminal segments establish the presence of two types of protein chain. The multiplicity is different from that hitherto detected within vertebrate class I alcohol dehydrogenase isozymes but typical of that which would be expected for subunits of different classes. In particular, relationships to class II or to class II-related forms appear likely. This may indicate yet further vertebrate alcohol dehydrogenase multiplicity or discovery of a class II non-mammalian enzyme. The results give prospects of defining gene duplications corresponding to more than one alcohol dehydrogenase class split to at an early vertebrate stage.
Subject(s)
Alcohol Dehydrogenase/metabolism , Isoenzymes/metabolism , Alcohol Dehydrogenase/chemistry , Alcohol Dehydrogenase/classification , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid , Isoenzymes/chemistry , Liver/enzymology , Lizards , Mammals , Molecular Sequence Data , Sequence AlignmentABSTRACT
We recently isolated from pig intestine and characterized a 31-residue antibacterial peptide named cecropin-P1 with activity against Escherichia coli and several other Gram-negative bacteria. The isolation involved a number of batch-wise steps followed by several chromatography steps. The continued investigation of these antibacterial peptides has now yielded another antibacterial peptide with high activity against both E. coli and Bacillus megaterium. Amino acid analysis showed a very high content of proline (49 mol%) and arginine (26 mol%), an intermediate level of phenylalanine and low levels of leucine, tyrosine, isoleucine, and glycine. The primary structure was determined by a combination of Edman degradation, plasma desorption mass spectrometry and C-terminal sequence analysis by carboxypeptidase Y degradation using capillary zone electrophoresis for detection of liberated residues. The calculated molecular mass was 4719.7 Da, which is in excellent agreement with 4719 Da obtained by plasma desorption mass spectrometry. The peptide was named PR-39 (proline-arginine-rich with a size of 39 residues). The lethal concentration of the peptide was determined against six Gram-negative and four Gram-positive strains of bacteria.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Antimicrobial Cationic Peptides , Intestine, Small/chemistry , Peptides/isolation & purification , Amino Acid Sequence , Animals , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Chromatography, Thin Layer , Escherichia coli/drug effects , Microbial Sensitivity Tests , Molecular Sequence Data , Peptide Fragments/isolation & purification , Peptides/chemistry , Peptides/pharmacology , SwineABSTRACT
Sorbin has been isolated from extracts of porcine upper intestine, and the biological activity in absorbing water and electrolytes utilized to monitor the purification procedure. Pure sorbin was obtained in a yield of about 1 mg/Mg boiled intestine. The protein chain has 153 amino acid residues and the primary structure was determined by analyses of CNBr-cleaved fragments and four enzymatic digests. The protein has a free N-terminal Met and an amidated C-terminal Ala. No structural similarity was observed with other known proteins in data bases, but several segments have special properties and the C-terminal half is rich in Pro and Arg.
Subject(s)
Intestines/chemistry , Peptides/isolation & purification , Absorption , Amino Acid Sequence , Amino Acids/analysis , Animals , Cyanogen Bromide , Gallbladder/drug effects , Gallbladder/metabolism , Guinea Pigs , Molecular Sequence Data , Peptide Fragments/chemistry , Peptides/chemistry , Peptides/pharmacology , Swine , Water/metabolismABSTRACT
We have isolated galanin from chicken intestine by monitoring for the N-terminal glycyltryptophan, which constitutes a conserved part characteristic of the peptide. This monitoring method complements that previously used for C-terminal amide detection and proves chemical monitoring of specific structures to be useful. The isolation allowed determination of the structure, which was found to be unidentical to any of the known galanins. However, N-terminal pentadecapeptide parts are identical, showing this segment to be of special importance. In addition to common substitutions at positions 16, 18, 23, 26 and 29, chicken galanin has phenylalanine at position 28, where all known mammalian galanins have leucine.