scholarly journals Biochemical and Proteomic Analysis of the Magnetosome Membrane in Magnetospirillum gryphiswaldense

2004 ◽  
Vol 70 (2) ◽  
pp. 1040-1050 ◽  
Author(s):  
Karen Grünberg ◽  
Eva-Christina Müller ◽  
Albrecht Otto ◽  
Regina Reszka ◽  
Dietmar Linder ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Fatty Acids ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Capillary Liquid Chromatography ◽  
Ionization Mass ◽  
Magnetospirillum Gryphiswaldense ◽  
Associated Proteins ◽  
Mass Spectrometry Mass Spectrometry

ABSTRACT We analyzed the biochemical composition of the magnetosome membrane (MM) in Magnetospirillum gryphiswaldense. Isolated magnetosomes were associated with phospholipids and fatty acids which were similar to phospholipids and fatty acids from other subcellular compartments (i.e., outer and cytoplasmic membranes) but were present in different proportions. The binding characteristics of MM-associated proteins were studied by selective solubilization and limited proteolysis. The MM-associated proteins were further analyzed by various proteomic approaches, including one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Edman and mass spectrometric (electrospray ionization-mass spectrometry-mass spectrometry) sequencing, as well as capillary liquid chromatography-mass spectrometry-mass spectrometry of total tryptic digests of the MM. At least 18 proteins were found to constitute the magnetosome subproteome, and most of these proteins are novel for M. gryphiswaldense. Except for MM22 and Mms16, all bona fide MM proteins (MMPs) were encoded by open reading frames in the mamAB, mamDC, and mms6 clusters in the previously identified putative magnetosome island. Eight of the MMPs display homology to known families, and some of them occur in the MM in multiple homologues. Ten of the MMPs have no known homologues in nonmagnetic organisms and thus represent novel, magnetotactic bacterium-specific protein families. Several MMPs display repetitive or highly acidic sequence patterns, which are known from other biomineralizing systems and thus may have relevance for magnetite formation.

scholarly journals Structural Characterization of an Oligosaccharide Made by Neisseria sicca

2009 ◽  
Vol 191 (10) ◽  
pp. 3311-3320 ◽  
Author(s):  
Ellen T. O'Connor ◽  
Hui Zhou ◽  
Kevin Bullock ◽  
Karen V. Swanson ◽  
J. McLeod Griffiss ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Characterization ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mass Spectrometry Analysis ◽  
Ionization Mass ◽  
Spectrometry Analysis ◽  
Neisseria Sicca

ABSTRACT Neisseria sicca 4320 expresses two carbohydrate-containing components with sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities that resemble those of lipooligosaccharide and lipopolysaccharide. Using matrix-assisted laser desorption ionization—time of flight and electrospray ionization mass spectrometry, we characterized a disaccharide carbohydrate repeating unit expressed by this strain. Gas chromatography identified the sugars composing the unit as rhamnose and N-acetyl-d-glucosamine. Glycosidase digestion confirmed the identity of the nonreducing terminal sugar of the disaccharide and established its β-anomeric configuration. Mass spectrometry analysis and lectin binding were used to verify the linkages within the disaccharide repeat. The results revealed that the disaccharide repeat is [-4) β-l-rhamnose (1-3) β-N-acetyl-d-glucosamine (1-] with an N-acetyl-d-glucosamine nonreducing terminus. This work is the first structural characterization of a molecule that possesses rhamnose in the genus Neisseria.

Exosite-dependent regulation of factor VIIIa by activated protein C

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4802-4807 ◽  
Author(s):  
Chandrashekhara Manithody ◽  
Philip J. Fay ◽  
Alireza R. Rezaie
Keyword(s):  
Protein C ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Activated Protein C ◽  
Coagulation Cascade ◽  
Factor Va ◽  
Factor Viiia

AbstractActivated protein C (APC) is a natural anticoagulant serine protease in plasma that down-regulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. Recent results have indicated that basic residues of 2 surface loops known as the 39-loop (Lys37-Lys39) and the Ca2+-binding 70-80–loop (Arg74 and Arg75) are critical for the anticoagulant function of APC. Kinetics of factor Va degradation by APC mutants in purified systems have demonstrated that basic residues of these loops are involved in determination of the cleavage specificity of the Arg506 scissile bond on the A2 domain of factor Va. In this study, we characterized the properties of the same exosite mutants of APC with respect to their ability to interact with factor VIIIa. Time course of the factor VIIIa degradation by APC mutants suggested that the same basic residues of APC are also critical for recognition and degradation of factor VIIIa. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the factor VIIIa cleavage reactions revealed that these residues are involved in determination of the specificity of both A1 and A2 subunits in factor VIIIa, thus facilitating the cleavages of both Arg336 and Arg562 scissile bonds in the cofactor.

scholarly journals The acylation of megakaryocyte proteins: glycoprotein IX is primarily myristoylated while glycoprotein Ib is palmitoylated

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1377-1384 ◽  
Author(s):  
PK Schick ◽  
J Walker
Keyword(s):  
Fatty Acids ◽  
Myristic Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amide Bond ◽  
Endogenous Protein ◽  
Sds Page ◽  
Palmitic Acids ◽  
Protein Acylation

The acylation of megakaryocyte proteins was studied with special emphasis on the myristoylation and palmitoylation of the glycoprotein (GP) Ib complex. Guinea pig megakaryocytes were purified and separated into subpopulations at different phases of maturation. Cells were incubated with [3H]myristate, [3H]palmitate, or [3H]acetate to study endogenous protein acylation. Cycloheximide was used to distinguish between cotranslational and posttranslational acylation and hydroxylamine to distinguish between thioester and amide linkages. After incubations, delipidated proteins or GPIb complex subunits, immunoprecipitated with PG-1, AN-51 or FMC-25 monoclonal antibody, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and assessed by fluorography. Radiolabeled fatty acids bound to GPIX and GPIb were also analyzed by high pressure liquid chromatography (HPLC) and scintillation spectrometry. With [3H]myristic acid and [3H]acetate, GPIX was found to be a major myristoylated protein in megakaryocytes and CHRF-288 cells. Myristic acid was linked to GPIX by an amide bond, and this process occurred cotranslationally. With [3H]acetate, GPIb was primarily palmitoylated, but with [3H]myristate, GPIb was acylated with about equal mounts of myristic acid and palmitic acids. Both fatty acids were linked to GPIb by thioester bonds, and acylation was posttranslational. The myristoylation of GPIX while the palmitoylation of GPIb occurred throughout megakaryocyte maturation. Myristoylation and palmitoylation may have different functions relevant to the assembly of the GPIb complex in megakaryocytes.

Sign in / Sign up

Export Citation Format

Share Document