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A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.

The crystal structures of the ligand-binding domain (LBD) of the oestrogen receptor (ER) in complex with the endogenous oestrogen, 17-beta-oesradiol, and the selective antagonist raloxifene give a molecular basis for the clearly recognizable pharmacophore of the ER and its catholic binding properties. Agonist and antagonist show different binding modes, even though they bind at the same site within the core of the LBD. It appears that the antagonist properties of raloxifene are based on its ability to inhibit the formation of a transcriptionally competent AF-2 conformation.

The NADP+ -linked prostaglandin dehydrogenase exists inseveral molecular forms. Two isoforms of the enzyme were purified from human placenta and characterized to determine the basis of molecular multiplicity. The results showed that the two isoforms differed in the presence of a carboxyethylated lysine residue at postion 238. The NADP+ -linked enzyme was also related to the NAD-linked form through alignments and model-building.

A novel form of gastric inhibitory polypeptide (GIP), later also retorted to as glucose-dependent insulinotropic polypeptide, has been isolated from bovine tipper intestine. The purification was monitored by a recently developed radioreceptor assay, specific for GIP, using membrane preparations from hamster β-cell tumors, A combination of ion-exchange and reverse-phase high-performance liquid chromatography was used in the isolation which resulted in homogeneous bovine GIP. Bovine GIP is, like porcine GIP, composed of 42 amino acid residues. The sequence is: Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-Arg-Gln-Gln-Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Ser-Asp-Trp-Ile-His-Asn-Ile-Thr-Gln, which differs from that of the previously characterized porcine GIP by having isoleucine instead of lysine at position 37. Upon proteolytic digestion of GIP with the staphylococcal V8 protease and with enterokinase, two fragments are formed in each case, corresponding to GIP1-3, GIP4-42, and GIP1-16, GIP17-42, respectively. [ABSTRACT FROM AUTHOR]

A novel form of the polypeptide termed PHI (peptide HI with N-terminal histidine and C-terminal isoleucine amide) has been isolated from bovine upper intestine. This bovine peptide was obtained in a 40 times higher yield than the corresponding polypeptide isolated from porcine intestine. Bovine PHI is, like porcine PH1, composed of 27 amino acid residues. The complete amino acid sequence of the bovine peptide is His - Ala - Asp - Gly - Val - Phe - Thr - Ser - Asp - Tyr - Ser - Arg - Leu - Leu - Gly - Gln - Leu - Ser - Ala - Lys - Lys - Tyr - Leu - Glu - Ser - Leu - Ile - NH2. This sequence differs from porcine PHI at position l0 and from human PHI at positions 10, 12 and 27. The amino acid residue exchange between porcine and bovine PHI makes the latter more similar to the vasoactive intestinal polypeptide (VIP), gastric inhibitory polypeptide (GIP), glucagon and the growth-hormone-releasing factor (GRF). [ABSTRACT FROM AUTHOR]

The polypeptide termed somatomedin A (SMA) was isolated from outdated human plasma by a new purification procedure, not using acid ethanol extraction. Fractions containing SMA were monitored by a placenta radioreceptorassay and a radioimmunoassay for SMA. The purification method utilized a microcomputer-controlled chromatography system, yielding both SMA (identified as insulin-like growth factor 1 (IGF-1) or a deamidated derivative) and insulin-like growth factor 2 (IGF-2). The first step of CM-Affigel blue adsorbed at neutral pH the majority of somatomedins detectable by the radioreceptorassay for SMA. Exclusion chromatography on Sephadex G-50 in 0.1 M acetic acid separated this active material from albumin and NaCl. Separation between SMA and IGF-2 was achieved on two different cation-exchange columns, but not in the final high-performance liquid chromatography step. The isoelectric points, determined by chromatofocusing, were 8.0 for SMA and 6.2 for IGF-2. The amino acid compositions of the two isolated peptides were indistinguishable from the known compositions of IGF-1 and IGF-2. Sequence analysis up to position 39 of the peptide with a pI of 6.2 also proved identity with IGF-2 for all positions examined. The peptide with a pI of 8.0, corresponding to SMA, was degraded directly as well as after CNBr cleavage. The results show that it is identical to IGF-1, with the possible exception of an acid/amide assignment, which could correspond to a deamidation. if occurring in the native preparation before analysis, it could explain the chromatographic properties and isoelectric point of SMA versus IGF-1 isolated by other techniques. [ABSTRACT FROM AUTHOR]

Chicken secretin has been isolated and its structure determined. It is composed of 27 amino acid residues, and has an amidated C terminus. The amino acid sequence is His-Ser-Asp-Gly-Leu-Phe-Thr-Ser-Glu-Tyr-Ser-Lys-Met-Arg-Gly-Asn-Ala-Gln-Val-Gln-Lys-Phe-Ile-Gln-Asn-Leu-Met-NH2. The structure shows distinct similarities to that of porcine secretin, amino acid identities occur at 14 positions (residues 1 – 4, 6 – 9, 11, 14, 17, 20, 24 and 26) but considerable differences are also present among the remaining 13 positions. Chicken secretin further shows a clear structural similarity to the vasoactive intestinal peptide (37% identical positions), the gastric inhibitory peptide (30% identical positions) and to glucagon (52 % identical positions), suggesting wide evolutionary and functional relationships. [ABSTRACT FROM AUTHOR]

We describe the structure of a gene expressed in the salivary gland cells of the dipteran Chironomus tentans and show that it encodes 1 of the approximately 15 secretory proteins exported by the gland cells. This sp115,140 gene consists of approximately 65 copies of a 42-bp sequence in a central uninterrupted core block, surrounded by short nonrepetitive regions. The repeats within the gene are highly similar to each other, but divergent repeats are present in a pattern which suggests that the repeat structure has been remodeled during evolution. The 42-bp repeat in the gene is a simple variant of the more complex repeat unit present in the Balbiani ring genes, encoding four of the other secretory proteins. The structure of the sp115,140 gene suggests that related repeat structures have evolved from a common origin and resulted in the set of genes whose secretory proteins interact in the assembly of the secreted protein fibers. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Receptors for the 29-amino-acid peptide, galanin, in membranes from the rat ventral hippocampus were examined using chloramine-T-iodinated porcine galanin as ligand. The equilibrium binding of 125I-galanin showed the presence of a high-affinity binding site (Kd = 1.91 ± 0.40 nM). The concentration of the high-affinity-binding sites was 107 ± 15 fmol/mg membrane protein. The on rate constant was estimated to be 2.6 ± 0.1 M-1 min-1 at 37°C. The affinity of rat galanin (differing in three amino acid residues from the porcine protein) was equal to that of porcine galanin. The 125I-galanin-binding site is a trypsin-sensitive membrane protein, which is heatdenaturated at 60°C within 5 min. The effect of GTP and its analogs and of pertussis-toxin-catalyzed ADP ribosylation on the binding of 125I-galanin suggest that the galanin receptor is coupled to an inhibitory G protein (Gi protein). 127I-galanin was shown to be a ligand with affinity equal to that of galanin in displacing 125I-galanin. The 125I-galanin-binding site in the ventral hippocampus recognizes as a ligand the tryptic fragments 1-20 and 21-29 of rat galanin and the synthetic fragments 12-29, 18-29 and 21-29 of porcine galanin. None of these afforded full inhibition of the binding of fragment 1-29 of 125I-galanin at a concentration of 1 µM. [ABSTRACT FROM AUTHOR]

A sensitive method for the rapid identification of the C-terminally amidated amino acid in peptides is described. Peptides containing the α-amide group at the C-terminus were cleaved with endopeptidases. The fragments released (oligopeptides, amino acids and the C-terminally amidated residue) are coupled to phenylisothiocyanate. The phenylthiocarbamoyl derivative of the amino acid α-amide is selectively extracted from the mixture by alkaline butyl acetate and identified by a high-performance liquid chromatography system that enables rapid and complete separation of the derivatives of 17 amino acid amides at a detection limit of 20 - 50 pmol. The C-terminal α-amides of neurokinin-A (Met-NH2), mammalian secretin (Val-NH2), pancreatic polypeptide (Tyr-NH2) and peptide HI (IIe-NH2) are unequivocally determined at a level of 0.5 - 2 nmol per peptide. This method was used to characterize a crude peptide fraction prepared from porcine brain. Cholecystokinin-58 was identified in this fraction by detection of phenylthiocarbamoyl-phenylalaninamide. The method is suitable for the identification of the Cterminal α-amidated residue of purified peptides, but can also be used as a screening strategy to isolate from complex biological extracts novel peptides containing an α-amidated amino acid at the C-terminus. [ABSTRACT FROM AUTHOR]

The total composition, the N-terminal amino acid sequence, and the amino acid sequences of four internal regions have been determined for the ribonucleotide reductase large subunit, protein B1. prepared from a recombinant λ-lysogenic Escherichia coli K strain, which overproduces the enzyme 30 - 50-fold. The data have been compared with those previously reported for B1 prepared from a thymin-starved E. coli B strain and with the indirectly derived primary structure of B1 recently reported from the nucleotide sequence of the E. coli K nrdA gene. Two major differences to these results were found. First, the B1 polypeptides started with initiator Met-1 (45%), Asn-2 (30%) or Gln-3 (15%), demonstrating a different type of N-terminal heterogeneity than that found earlier. Secondly, the total amino acid composition as derived from hydrolyzed protein B1 differed substantially from the amino acid composition derived from the nucleotide data. This has the consequence that Cys, Arg, Thr and possibly Val and Ser appear more frequently whereas Asx, Glx, Tyr and possibly Gly appear less frequently in the nucleotide-derived data as compared to direct protein hydrolysates. We suggest usage of other reading frames in the approximate area of residues 630 - 700 of the primary structure of the nrdA gene to compensate for these discrepancies and for the relatively high incidence of uncommon codons in the reading frame proposed for this area of the gene. Such changes have implications on the previously assigned putative active-site region of protein B1. [ABSTRACT FROM AUTHOR]

An immunosorbent method using antiglutaredoxin-Sepharose was developed for purification of glutaredoxin in high yield from a mutant strain of Escherichia coli K 12 lacking thioredoxin reductase (C 10–17). The primary structure of the protein was determined by analyses of [14C]carboxymethylated glutaredoxin and its proteolytic fragments obtained by digestions with trypsin, clostripain, chymotrypsin and staphylococcal Glu-specific extracellular protease. The single active-center disulfide has the structure-Cys-Pro-Tyr-Cys, with the half-cystine residues located at Positions 11 and 14 in the polypeptide chain. In total the protein was deduced to have 85 residues corresponding to a Molecular weight of 9674 for the reduced form of glutaredoxin, making it one of the smallest known enzymes (a glutathione-disulfide transhydrogenase). The half-cystines are identically spaced and similarly positioned in the N-terminal part of the protein when Compared with a corresponding functionally active disulfide/dithiol in thioredoxins. Glutaredoxin is also distantly Homologous with thioredoxins from phage T4 and E. coli, but extensive differences, even around the redox-active disulfide, distinguish glutaredoxin from the thioredoxins. Allowing for deletions in the glutaredoxin sequence (or insertions in T4 thioredoxin sequence) t four places, there are identical residues at 25 positions of the 77 compared (=32% identity). The results establish that glutaredoxin belongs to the same superfamily of small redox proteins as the thioredoxins. The structures are, however, subject to large changes, only four positions have residues identical among all presently analyzed forms. The fluorescence of reduced and oxidized glutaredoxin demonstrates an increase in the quantum yield of the tyrosine emission upon reduction with dithiothreitol. Differences in the spectra support the presence of tyrosine adjacent to the redox-active disulfide bridge. They also confirm that glutaredoxin lacks the disulfide-adjacent tryptophan residues of E. coli thioredoxin. There are known to be great differences between the bacterial E. coli and phage T4 forms of thioredoxin. The glutaredoxin structure is most similar to the phage type, both with respect to size of the polypeptide chain and to actual sequence. From the structural results and the previously known functional similarities it appears possible that the phage thioredoxin may have evolved from an early glutaredoxin gene. The mixed properties are compatible with this conclusion, the superfamily assignment, and the differences in biological activity. [ABSTRACT FROM AUTHOR]

Sorbitol dehydrogenase was characterized as a homogeneous protein on affinity chromatography and ion- exchange chromatography. Tests of stability, sensitivity to inhibitors, and protection by coenzyme suggest that the enzyme has essential cysteine, metal, and probably histidine. The native enzyme has a molecular weight around 140000 and a subunit around 35000- 40000, suggesting a tetrameric quaternary structure. Subunits are highly similar if not identical as judged by characterization of one unique 4S-residue sequence containing a single reactive cysteine residue. Properties resemble those of mammalian and yeast alcohol dehydrogenases, and the sequence determined for the region around the reactive cysteine residue is homologous to that around one of the zinc-liganding cysteine residues at the active site of horse liver alcohol dehydrogenase. Sorbitol dehydrogenase thus reveals an unexpected relationship to alcohol dehydrogenases, from which ancestral connections and functional mechanisms in this group of enzymes may be further elucidated. [ABSTRACT FROM AUTHOR]