Characterization and analysis of a novel glycoprotein from snake venom using liquid chromatography-electrospray mass spectrometry and Edman degradation.
An N-linked glycosylation in a novel C-lectin protein from snake venom was observed by Edman degradation and liquid chromatography-electrospray mass spectrometry. The peptides obtained by trypsin cleavage were analyzed to confirm the amino acid sequence and Asn5 was found to be the N-glycosylation site. The result was further confirmed by N-glycosidase digestion. In addition, the protein and tryptic peptides with and without glycan chain were characterized by mass spectrometry according to the mass difference. The glycopeptide obtained from proteolytic digestion was analyzed and the glycoforms were identified as high-mannose type by tandem MS coupled with alpha-mannosidase digestion. An oxidized Met residue was detected and located in the protein by mass spectrometry. (+info)
Acquisition of species-specific O-linked carbohydrate chains from oviducal mucins in Rana arvalis. A case study.
The extracellular matrix surrounding amphibian eggs is composed of mucin-type glycoproteins, highly O-glycosylated and plays an important role in the fertilization process. Oligosaccharide-alditols were released from the oviducal mucins of the anuran Rana arvalis by alkali-borohydride treatment in reduced conditions. Neutral and acidic oligosaccharides were fractionated by ion-exchange chromatographies and purified by HPLC. Each compound was identified by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) spectrometry, NMR spectroscopy, electrospray ionization-tandem mass spectroscopy (ESI-MS/MS) and permethylation analyses. This paper reports on the structures of 19 oligosaccharide-alditols, 12 of which have novel structures. These structures range in size from disaccharide to octasaccharide. Some of them are acidic, containing either a glucuronic acid or, more frequently, a sulfate group, located either at the 6 position of GlcNAc or the 3 or 4 positions of Gal. This latter sulfation is novel and has only been characterized in the species R. arvalis. This structural analysis led to the establishment of several novel carbohydrate structures, demonstrating the structural diversity and species-specificity of amphibian glycoconjugates. (+info)
MioC is an FMN-binding protein that is essential for Escherichia coli biotin synthase activity in vitro.
Biotin synthase is required for the conversion of dethiobiotin to biotin and requires a number of accessory proteins and small molecule cofactors for activity in vitro. We have previously identified two of these proteins as flavodoxin and ferredoxin (flavodoxin) NADP(+) reductase. We now report the identification of MioC as a third essential protein, together with its cloning, purification, and characterization. Purified MioC has a UV-visible spectrum characteristic of a flavoprotein and contains flavin mononucleotide. The presence of flavin mononucleotide and the primary sequence similarity to flavodoxin suggest that MioC may function as an electron transport protein. The role of MioC in the biotin synthase reaction is discussed, and the structure and function of MioC is compared with that of flavodoxin. (+info)
Discrimination of dimethylamphetamine and methamphetamine use: simultaneous determination of dimethylamphetamine-N-oxide and other metabolites in urine by high-performance liquid chromatography-electrospray ionization mass spectrometry.
A simple and sensitive method by high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) has been investigated for the simultaneous determination of dimethylamphetamine (DMA), its specific yet labile main metabolite dimethylamphetamine-N-oxide (DMAO), and other metabolites, methamphetamine (MA) and amphetamine (AP), in urine. A combination of Bond Elut SCX columns for the solid-phase extraction of urine and a semi-micro SCX column for LC separations provided satisfactory results. The use of acetonitrile/5mM ammonium acetate buffer adjusted to pH 4 (65:35, v/v) as the mobile phase at a flow rate of 0.2 mL/min was found to be the most effective. The detection limits were 5 ng/mL for DMAO, 10 ng/mL for DMA and MA, and 50 ng/mL for AP in the SIM mode. (+info)
A sulfenic acid enzyme intermediate is involved in the catalytic mechanism of peptide methionine sulfoxide reductase from Escherichia coli.
Methionine oxidation into methionine sulfoxide is known to be involved in many pathologies and to exert regulatory effects on proteins. This oxidation can be reversed by a ubiquitous monomeric enzyme, the peptide methionine sulfoxide reductase (MsrA), whose activity in vivo requires the thioredoxin-regenerating system. The proposed chemical mechanism of Escherichia coli MsrA involves three Cys residues (positions 51, 198, and 206). A fourth Cys (position 86) is not important for catalysis. In the absence of a reducing system, 2 mol of methionine are formed per mole of enzyme for wild type and Cys-86 --> Ser mutant MsrA, whereas only 1 mol is formed for mutants in which either Cys-198 or Cys-206 is mutated. Reduction of methionine sulfoxide is shown to proceed through the formation of a sulfenic acid intermediate. This intermediate has been characterized by chemical probes and mass spectrometry analyses. Together, the results support a three-step chemical mechanism in vivo: 1) Cys-51 attacks the sulfur atom of the sulfoxide substrate leading, via a rearrangement, to the formation of a sulfenic acid intermediate on Cys-51 and release of 1 mol of methionine/mol of enzyme; 2) the sulfenic acid is then reduced via a double displacement mechanism involving formation of a disulfide bond between Cys-51 and Cys-198, followed by formation of a disulfide bond between Cys-198 and Cys-206, which liberates Cys-51, and 3) the disulfide bond between Cys-198 and Cys-206 is reduced by thioredoxin-dependent recycling system process. (+info)
Biological mass spectrometry: a primer.
Biological polymers undergo numerous significant and fascinating interactions, such as post-translational modifications, non-covalent associations and conformational changes. A valuable parameter for the characterization of a biopolymer is molecular weight. Modern methods of mass spectrometry, including electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry, are ideally suited for the accurate determination of the molecular weight of a biopolymer of interest. Molecular weight measurements are now routinely utilized in the qualitative and quantitative analysis of macromolecules. In many cases small sample quantities (i.e. a few micrograms) limit the utility of nuclear magnetic resonance spectroscopy and X-ray crystallography in obtaining structural information. Thus, mass spectrometry offers an attractive alternative to the more traditional bioanalytical methods for rapid and sensitive measurements. The ultimate goal of these experiments is to obtain sufficient information in order to map the complex molecular circuitry which operates within the cell. In the analysis of complex mixtures mass spectrometry is even more powerful when utilized in conjunction with separation methods. Herein we present some of the aspects of modern biological mass spectrometry for the investigation of large molecules. For more advanced or detailed technical descriptions we refer the reader to a number of recently published reports. (+info)
Topological alteration of the CYP3A4 active site by the divalent cation Mg(2+).
Phenyldiazene reacted with lymphoblast-expressed CYP3A4 to give a stable phenyl-iron complex that could be induced to rearrange in situ producing approximately equal amounts of four N-phenyl-protoporphyrin IX isomers (N(B):N(A):N(C):N(D), 01:01:02:02). In the presence of 10 mM MgCl(2), the formation profile of the protoporphyrin isomers was markedly altered compared with control, favoring the N(A) isomer (N(B):N(A):N(C):N(D), 01:34:01:02). In addition, an investigation of MgCl(2) effects on CYP3A4-mediated metabolism of triazolam revealed that 10 mM MgCl(2) increased the apparent K(m) of triazolam 4-hydroxylation from 83 to 173 microM and reduced the V(max) for the reaction from 3.4 to 2.4 min(-1). Moreover, when the reaction kinetics of the oxidation of pyrene by CYP3A4 was examined in the absence of MgCl(2), it was found that the substrate-velocity curve was best approximated by a sigmoidal velocity curve (Hill coefficient 1.7 +/- 0.1). However, when the reaction was conducted in the presence of 10 mM MgCl(2), the resulting pyrene kinetics was not sigmoidal but rather biphasic (Hill coefficient 0.80 +/- 0.07). Based on the current results, it appears that CYP3A4 is conformationally sensitive to its in vitro environment and parameters, such as the presence of a divalent magnesium, can have a measurable effect on active site topography and consequently catalytic activity. (+info)
Kinetic basis for the donor nucleotide-sugar specificity of beta1, 4-N-acetylglucosaminyltransferase III.
The kinetic basis of the donor substrate specificity of beta1, 4-N-acetylglucosaminyltransferase III (GnT-III) was investigated using a purified recombinant enzyme. The enzyme also transfers GalNAc and Glc moieties from their respective UDP-sugars to an acceptor at rates of 0.1-0.2% of that for GlcNAc, but Gal is not transferred at a detectable rate. Kinetic analyses revealed that these inefficient transfers, which are associated with the specificity of the enzyme, are due to the much lower V(max) values, whereas the K(m) values for UDP-GalNAc and UDP-Glc differ only slightly from that for UDP-GlcNAc. It was also found that various other nucleotide-Glc derivatives bind to the enzyme with comparable affinities to those of UDP-GlcNAc and UDP-Glc, although the derivatives do not serve as glycosyl donors. Thus, GnT-III does not appear to distinguish UDP-GlcNAc from other structurally similar nucleotide-sugars by specific binding in the ground state. These findings suggest that the specificity of GnT-III toward the nucleotide-sugar is determined during the catalytic process. This type of specificity may be efficient in preventing a possible mistransfer when other nucleotide-sugars are present in excess over the true donor. (+info)