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S A Carr

Publications and source records attributed to S A Carr.

At least 73 records · Page 4Linked to original sources
PubMed1987

Structural characterization of sulfated glycosaminoglycans by fast atom bombardment mass spectrometry: application to heparin fragments prepared by chemical synthesis.

We report herein the results of f.a.b.-m.s. experiments conducted on synthetic fragments of glycosaminoglycans, one of them representing the pentasaccharidic sequence present in heparin and responsible for the binding to antithrombin III, and the others being related to this sequence. The results indicate that f.a.b.-m.s. can be very useful for the structural analysis of sulfated glycosaminoglycans. The relatively small amounts of sample required enable molecular characterization at physiologically significant levels. In contrast to the chondroitin sulfates, the heparin saccharides analyzed and reported here do not provide sequence information. The data indicate that glycosidic rupture is not a process competing with the much more facile loss of N-sulfite residues. Dominating the spectra are a series of molecular-weight-related ions (distributed to indicate the associated countercation composition), and fragments related directly to sulfite elimination. This f.a.b.-induced, facile loss of sulfite may impose limitations in molecular-weight analysis for the larger oligomers.

Carbohydrate Conformation↗
PubMed1987

Parvodicin, a novel glycopeptide from a new species, Actinomadura parvosata: discovery, taxonomy, activity and structure elucidation.

An extensive taxonomic investigation identified strain SK&F-AAJ-271 as a new species, designated Actinomadura parvosata. Fermentations of this organism produce a complex of acidic, lipophilic glycopeptide antibiotics, the parvodicins. Structures for seven of the isolated components were derived from a combination of mass spectral, high-field NMR and chemical techniques. The O-acetyl functionality present in two of the isolated components is a structural feature unique among the known members of this class of antibiotics. The parvodicins are active in vitro against a range of Gram-positive bacteria. The most active parvodicin, C1, produces high serum levels in vivo and has the potential for a long duration of action.

Animals↗
PubMed1986

Complete primary structure of prostatropin, a prostate epithelial cell growth factor.

Bovine brain prostatropin is a potent and essential mitogen for prostate epithelial cell growth. The major form of prostatropin contains 154 amino acid residues in a single amino terminally blocked chain corresponding to a molecular weight of 17,400. The amino acid sequence of the 150 carboxy-terminal residues of prostatropin was derived by Edman degradation of overlapping peptides primarily generated by cleavage at lysyl and glutamyl residues. Analysis of the amino-terminal tetradecapeptide by fast atom bombardment mass spectrometry identified the blocking group as an acetyl moiety, and tandem mass spectrometry provided the sequence of the first 12 residues. Prostatropin residues 15-154 contain the sequence of bovine brain polypeptides recently described as acidic fibroblast growth factor and class I heparin-binding growth factor. The sequence of the first 25 residues of prostatropin is acetyl-Ala-(Gly, Glu)-Glu-Thr-Thr-Thr-Phe-Thr-Ala-Leu-Thr-Glu-Lys-Phe-Asn-Leu-Pro-Leu-Gly -Asn-Tyr-Lys-Lys-Pro. Reduced and carboxymethylated prostatropin exhibits mitogenic activity, suggesting that disulfide bonds among cysteine residues 30, 61, and 97 are not functionally essential. These results demonstrate by rigorous structural analysis that the brain-derived polypeptide previously described only as a mesenchymal and neuroectodermal cell mitogen is also an epithelial cell growth factor that may be involved in support of prostate hyperplasia and adenocarcinoma.

Amino Acid Sequence↗
PubMed1986

Carbohydrate mapping by mass spectrometry: a novel method for identifying attachment sites of Asn-linked sugars in glycoproteins.

A new method is described for locating the specific sites of attachment of Asn-linked carbohydrates in glycoproteins. The molecular weights of peptides released from the glycoprotein with proteases of known specificity are determined by fast atom bombardment mass spectrometry and fitted to the known or DNA-derived sequence. Oligosaccharides attached to Asn are released either before or after proteolysis with a glycosidase, usually peptide: N-glycosidase F, an enzyme that cleaves the beta-aspartylglycosylamine linkage of all known types of Asn-linked sugars and converts the attachment-site Asn to Asp. New peaks appearing in the mass spectra after treatment with glycosidase correspond to formerly glycosylated sites. Conversely, signals which disappear after glycosidase treatment correspond to glycopeptides. The differences in mass between these sets of signals define the composition of the carbohydrate at the given site in terms of deoxyhexose, hexose, N-acetylhexosamine, and sialic acid content. The extent of glycosylation at a given site can be estimated from the ratio of the peak heights corresponding to the Asn- vs Asp-containing peptides which differ by 1 Da in mass. This rapid and sensitive (low nmol) technique is illustrated here for ribonuclease B and for tissue plasminogen activator, a multiply glycosylated glycoprotein.

Asparagine↗
PubMed1985

Fluorescent labeling of carbohydrates and analysis by liquid chromatography. Comparison of derivatives using mannosidosis oligosaccharides.

To enhance resolution and detectability of carbohydrates by liquid chromatography, two fluorescent labels, introduced into oligosaccharides by reductive amination, were compared by use of standard sugars and a complex, biological sample of D-mannose oligomers obtained from the urine of a mannosidosis patient. Both labels, 2-aminopyridine and 7-amino-1-naphthol, improved the chromatographic efficiency and detection sensitivity. However, reductive amination with the pyridinylamine derivative was incomplete. The Schiff-base intermediates left in the mixtures were only partially resolved by chromatography and complicate the patterns. In contrast, the naphtholamine derivatives were completely reduced and, in addition, possess enhanced fluorescence.

Aminopyridines↗
PubMed1985

Enhancement of structural information in FAB ionized carbohydrate samples by neutral gas collision.

Fast atom bombardment (FAB) ionization and two coupled analyzers (BE-EB) have been combined with neutral gas collision (C) to enhance structural information in the mass spectra of oligosaccharides. (B and E are abbreviations for magnetic and electric sectors respectively.) FAB ionization and the first analysers (BE) have provided parent ions free from biological and liquid matrix contaminants. Structural detail of these products were observed after collision and daughter ion analysis in a second coupled analyser (EB). Starting from complex mixtures, this instrumental approach, BE-C-EB, has provided specific oligomeric sequence information which was not observed in the normal FAB mass spectra. Collision spectra obtained from isomeric linear and branched oligosaccharides show unique fragments that can be directly related to structure.

Asparagine↗
PubMed1985

Structural characterization of glycopeptide antibiotics related to vancomycin by fast atom bombardment mass spectrometry.

A series of glycopeptide antibiotics related to the vancomycin-ristocetin family have been successfully analyzed by fast atom bombardment mass spectrometry (FABMS). The FAB mass spectra of glycopeptides weighing up to 2,100 daltons exhibit intense molecular ions and fragment ions from which information concerning carbohydrate composition and sequence are readily obtained. Careful adjustment of the FABMS experimental conditions has enabled the accurate masses of the glycopeptides to be determined by high resolution FABMS with an accuracy of better than six ppm. Comparison of the observed molecular ion cluster pattern with calculated isotope distributions reveals the precise number of chlorine atoms in these molecules, which, together with the accurate mass data, can be used to restrict the number of possible elemental compositions to a meaningfully small value. These techniques have been used to characterize several glycopeptides of known structure including ristocetin, actinoidin, avoparcin, vancomycin and A35512B, as well as aridicins A, B and C which are three new, novel members of the vancomycin class.

Anti-Bacterial Agents↗
PubMed1984

Identification of the NH2-terminal blocking group of NADH-cytochrome b5 reductase as myristic acid and the complete amino acid sequence of the membrane-binding domain.

The NH2-terminal blocking group of the membrane-binding domain of NADH-cytochrome b5 reductase has been deduced as myristic (n-tetradecanoyl) acid. This fatty acid was identified by gas chromatography of the digest of the NH2-terminal tetrapeptide of cytochrome b5 reductase. Fast atom bombardment and direct chemical ionization mass spectroscopy of the underivatized NH2-terminal tetrapeptide confirmed the presence of myristic acid, identified its linkage to the NH2 terminus and established CH3(CH2)12-CO-Gly-Ala-Gln-Leu as the NH2-terminal sequence. In addition, the complete amino acid sequence of the membrane-binding domain of cytochrome b5 reductase is also reported. The finding of a myristic acyl chain on the NH2-terminal segment, comprised of hydrophobic amino acid residues, implies that the function of the myristate group may be other than simply to anchor the reductase to the microsomal membrane. This post-translational modification, presumably in the endoplasmic reticulum, may selectively stabilize a particular membrane structure and orientation that optimally facilitates electron transport on the cytosolic surface of this membrane organelle.

Amino Acid Sequence↗
PubMed1984

Incorporation of 18O2 into thymidine 5'-aldehyde in neocarzinostatin chromophore-damaged DNA.

Strand scission of DNA by the chromophore of neocarzinostatin converts the 5'-hydroxyl of deoxyribose to a 5'-aldehyde. The origin of the aldehydic oxygen has now been elucidated by mass spectrometry. DNA-associated thymidine 5'-aldehyde produced by treatment of DNA with neocarzinostatin chromophore in 2H218O/16O2 or in 2H216O/18O2 was reduced, liberated by nuclease treatment, permethylated, and analyzed by gas chromatography-mass spectrometry. The data clearly show that molecular oxygen is the only source of the 5'-aldehydic oxygen. The addition of molecular oxygen at C-5', possibly via a reactive form of neocarzinostatin chromophore, must be involved; a carbonium ion intermediate at C-5' is ruled out.

Animals↗
PubMed1984

Structural characterization of glycosphingolipids by direct chemical ionization mass spectrometry.

This report describes the use of direct chemical ionization mass spectrometry with ammonia as the reagent gas (NH3-DCI) for structure analysis of underivatized, permethylated and permethylated and reduced glycosphingolipids. In contrast to ionization by electron impact, the NH3-DCI mass spectra exhibit intense molecular and carbohydrate sequence-related ions using microgram amounts of sample. Underivatized glycosphingolipids with up to two sugar residues yield abundant protonated and ammonia-cationized molecular ions and structurally significant fragments. Permethylation in conjunction with NH3-DCI can be used to obtain molecular weight as well as oligosaccharide sequence and branching information on neutral, acidic and complex-type glycosphingolipids with up to five sugar residues. Reduction of the permethylated derivatives gives rise to several new, structurally significant fragments in the corresponding NH3-DCI mass spectra which enable fatty acid and base compositions to be determined. Isotopically labeled reagent gases have been used to confirm the assignment of fragment structures and to demonstrate that the ions observed are unique to the NH3-DCI mass spectra.

G(M1) Ganglioside↗
PubMed1984

Citramalic acid in cerebrospinal fluid of patients with bacterial meningitis.

Cerebrospinal fluid (CSF) from uninfected patients and from patients with bacterial and viral meningitis was analyzed by gas-liquid chromatography, with use of a flame ionization detector, and by gas chromatography-mass spectrometry. The resulting profiles were consistent and reproducible. Hydroxy acids were the compounds found in greatest abundance in both normal and infected CSF. Control experiments to establish the sensitivity and efficiency of the extraction and derivatization methods are also presented. Constituents of CSF from patients with bacterial meningitis differed quantitatively and qualitatively from those of CSF from uninfected patients or patients with nonbacterial infections. CSF from seven of eight patients with bacterial meningitis contained citramalic acid, a compound not previously identified in either normal or infected CSF. The implications of these findings are discussed.

Adolescent↗
PubMed1984

Binding site specificity of gamma-radiation-induced crosslinking between phenylalanine and a phenylalanine-containing tetrapeptide.

The binding site specificity of crosslinking mediated by the hydroxyl radical has been investigated in a simple model system: a tetrapeptide, Gly-Gly-Phe-Leu, and 14C-labeled phenylalanine. Crosslinking leads to the tetrapeptide-phenylalanine adduct which has been isolated by gel filtration. The amino acid analysis of these adducts compared with those of gamma-radiation-induced dimers of the tetrapeptide and of the dipeptide, Gly-Phe, shows that only the phenylalanine residue is affected and that the same new peaks appear in each case. Spectrophotometric measurement indicates that the extinction coefficient at 260 nm of dimeric tetrapeptide is four times higher than that of monomeric, as is dimeric phenylalanine compared to monomeric. These observations suggest a common crosslinking mechanism in all three cases that involves the aromatic ring of phenylalanine. The appearance of several radioactive peaks in the gel filtration separation of the acid hydrolysate of the adduct suggests that the crosslinking involves more than one possible modification of the phenylalanine. Three distinct tetrapeptide-Phe species, corresponding to molecular weights of 555, 573, and 591, were observed by fast atom bombardment mass spectrometry. The partial release of radioactive phenylalanine from the tetrapeptide-phenylalanine adducts by acid hydrolysis indicates the liability of some phenylalanine-phenylalanine bonds.

Amino Acids↗
PubMed1983

Primary structure of macromomycin, an antitumor antibiotic protein.

The antitumor protein macromomycin is a single chain polypeptide of 112 amino acid residues cross-linked by two intramolecular disulfide bonds. The protein was reduced and S-alkylated with 2-mercaptoethanol in 8 M urea followed by treatment with iodoacetic acid. Tryptic digestion of tetra-S-carboxymethyl macromomycin gave four tryptic peptides which were fractionated by gel permeation on Sephadex G-50. The amino acid sequence of the tryptic peptides and the overlap sequences were determined by a combination of automated Edman degradation analysis, gas chromatographic mass spectrometry, and fast atom bombardment mass spectrometry. A comparison of the structures of macromomycin, actinoxanthin, and neocarzinostatin suggests that they belong to a family of related proteins.

Amino Acid Sequence↗
PubMed1982

Calcium-dependent alpha-helical structure in osteocalcin.

Osteocalcin is an abundant Ca2+-binding protein of bone containing three residues of vitamin K dependent gamma-carboxyglutamic acid (Gla) among its 49 (human, monkey, cow) or 50 (chicken) amino acids. Gla side chains participate directly in the binding of Ca2+ ions and the adsorption of osteocalcin to hydroxylapatite (HA) surfaces in vivo and in vitro. Osteocalcin exhibits a major conformational change when Ca2+ is bound. Metal-free chicken osteocalcin is a random coil with only 8% of its residues in the alpha helix as revealed by circular dichroism. In the presence of physiological levels of Ca2+, 38% of the protein adopts the alpha-helical conformation with a transition midpoint at 0.75 mM Ca2+ in a rapid, reversible fashion which (1) requires an intact disulfide bridge, (2) is proportionally diminished when Gla residues are decarboxylated to Glu, (3) is insensitive to 1.5 m NaCl, and (4) can be mimicked by other cations. Tyr fluorescence, UV difference spectra, and Tyr reactivity to tetranitromethane corroborate the conformational change. Homologous monkey osteocalcin also exhibits Ca2+-dependent structure. Integration of predictive calculations from osteocalcin sequence has yielded a structural model for the protein, the dominant features of which include two opposing alpha-helical domains of 9-12 residues each, connected by a bea turn and stabilized by the Cys23-Cys29 disulfide bond. Cation binding permits realization of the full alph a-helical potential by partial neutralization of high anionic charge in the helical domains. Periodic Gla occurrence at positions 17, 21, and 24 has been strongly conserved throughout evolution and places all Gla side chains on the same face of one alpha helix spaced at intervals of approximately 5.4 A, closely paralleling the interatomic separation of Ca2+ in the HA lattice. Helical osteocalcin has greatly increased affinity for HA; thus, the Ca2+-induced structural transition may perform an informational role related to bone metabolism.

1-Carboxyglutamic Acid↗
PubMed1982

Primary structure of monkey osteocalcin.

The complete 49-residue amino acid sequence of osteocalcin from the old world monkey Macaca fascicularis has been determined by efficient combination of gas chromatography-mass spectrometry and Edman techniques. This vitamin K dependent protein of bone matrix contains three gamma-carboxyglutamic acid residues at positions 17, 21, and 24, as well as a disulfide-bonded loop (23--29). Features of the sequence which apparently are required for the binding of Ca2+ have been strongly conserved throughout evolution.

Amino Acid Sequence↗
PubMed1982

n-Tetradecanoyl is the NH2-terminal blocking group of the catalytic subunit of cyclic AMP-dependent protein kinase from bovine cardiac muscle.

The unusual NH2-terminal blocking group of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein was found to be amide-linked n-tetradecanoic acid by gas chromatographic-, direct chemical ionization-, and fast atom bombardment-mass spectrometry. In addition, fast atom bombardment mass spectrometry revealed the presence of an additional alanine which had been overlooked when the original sequence was determined. The corrected and completed NH2-terminal sequence of the 350-amino acid catalytic subunit is CH3(CH2)12CONH-Gly-Asn-Ala-Ala-Ala-Ala-Lys.

Amino Acid Sequence↗
PubMed1982

Chemical and in vivo studies of the anion oxo[N,N'-ethylenebis(2-mercaptoacetimido)]Technetate(V).

Studies of the anionic coordination complex 99Tc-oxo[N,N'-ethylene-bis(2-mercaptoacetimido)]technetate(V) ([TcO(ema)]-) are described. Syntheses performed both at carrier levels (10(-5)M) and with no carrier added (less than 10(-8)M) indicate that the complex is formed virtually quantitatively from pertechnetate ion over this range. Tissue distributions in normal rats are similar at both concentrations up to one hour after administration. It has been shown--using a combination of high-pressure liquid chromatography and field-desorption mass spectrometry--that the anion is excreted unchanged into both urine and bile. The effectiveness of this N2S2 donor set in sequestering Tc-99m, and the in vivo stability of the resulting complex, suggest that modified chelates of this structural class could provide a series of useful diagnostic agents.

Animals↗