Use of a Diimidoester Cross-Linking Reagent to Examine the Submit Structure of Rabbit Muscle Pyruvate Kinase

1972 ◽  
Vol 50 (4) ◽  
pp. 416-422 ◽  
Author(s):  
Gregg E. DaVies ◽  
J. Gordin Kaplan
Keyword(s):  
Pyruvate Kinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Linking ◽  
Rabbit Muscle ◽  
Cross Links ◽  
Primary Amino ◽  
Muscle Pyruvate Kinase ◽  
Dimeric Intermediate ◽  
Hydrolysis Of

Rabbit muscle pyruvate kinase, a tetramer of highly similar or identical subunits, is known to dissociate in urea via a dimeric intermediate. To provide additional evidence regarding the structure of the native oligomer, we have treated pyruvate kinase with dimethyl pimelimidate and examined the yields of cross-linked species resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Of four products resolved, predominating species are dimers and tetramers of the pyruvate kinase subunit, while monomers and trimers are present in lesser amounts. These relative yields are consistent with a dimeric structure of the tetramer.In a typical cross-linking reaction, about 86 of the 148 primary amino groups in the pyruvate kinase tetramer are amidinated. Of 53 moles of cross-linking reagent incorporated per mole of tetramer, 38% have reacted monofunctionally, with hydrolysis of the second imidoester group, and 62% have reacted bifunctionally to form intra- and intersubunit cross-links.

scholarly journals p-NN′-phenylenebismaleimide, a specific cross-linking agent for F-actin

1978 ◽  
Vol 175 (3) ◽  
pp. 1023-1032 ◽  
Author(s):  
P Knight ◽  
G Offer
Keyword(s):  
Quantitative Analysis ◽  
Sodium Dodecyl Sulphate ◽  
Actin Filaments ◽  
Sodium Dodecyl ◽  
Cysteine Residue ◽  
Cross Linking ◽  
Maximum Value ◽  
Cross Links ◽  
The Cross ◽  
Hydrolysis Of

Covalent cross-links can be inserted between the subunits of F-actin by using p-NN′-phenylenebismaleimide. Cross-linking reaches its maximum value when one molecule of reagent has reacted with each actin subunit. p-NN′-Phenylenebismaleimide reacts initially with a cysteine residue on one subunit, the slower cross-linking reaction involving a lysine residue on a neighbouring subunit. Hydrolysis of the actin-bound reagent limits the extent of cross-linking. Quantitative analysis of the amounts of cross-linked oligomers seen on polyacrylamide gels containing sodium dodecyl sulphate suggests that neither the binding of the reagent to actin nor the formation of cross-links introduces strain into the structure. The cross-links do not join together different F-actin filaments, and evidence is presented that suggests that the cross-links join subunits of the same long-pitched helix.

An engineered fibrinogen variant AαQ328,366P does not polymerise normally, but retains the ability to form α cross-links

2013 ◽  
Vol 109 (02) ◽  
pp. 199-206 ◽  
Author(s):  
Lifang Ping ◽  
Jaewoo Song ◽  
Joo-Young Seo ◽  
Tae-Youn Choi ◽  
Jong-Rak Choi ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fibrin Clot ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Acceptor Site ◽  
Double Mutation ◽  
Cross Links ◽  
Α Chain

SummaryA fibrin clot is stabilised through the formation of factor XIIIa-catalysed intermolecular ε -lysyl-γ -glutamyl covalent cross-links between α chains to form α polymers and between γ chains to form γ dimers. In a previous study we characterised fibrinogen Seoul II, a heterozygous dysfibrinogen in which a cross-linking acceptor site in Aα chain, Gln328, was replaced with Pro (AαQ328P). Following on the previous study, we investigated whether the alteration of Gln residues Aα328 and Aα366 affects fibrin polymerisation and α chain cross-linking. We have expressed three recombinant fibrinogens: AαQ328P, AαQ366P, and AαQ328,366P in Chinese hamster ovary cells, purified these fibrinogens from the culture media and performed biochemical tests to see how the introduced changes affect fibrin polymerisation and α chain cross-linking. Thrombin-catalysed fibrin polymerisation of all variants was impaired with the double mutation being the most impaired. In contrast, sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblot analysis showed α polymer formation with all three engineered proteins. This study demonstrates that AαQ328 and AαQ366 are important for normal fibrin clot formation and in the absence of residues AαQ328 and AαQ366, other Gln residues in the a chain can support FXIIIa-catalysed fibrin cross-linking.

scholarly journals Damage to actin filaments by glutaraldehyde: protection by tropomyosin.

1981 ◽  
Vol 90 (2) ◽  
pp. 459-466 ◽  
Author(s):  
S S Lehrer
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Actin Filaments ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Linking ◽  
Cross Link ◽  
Reaction Conditions ◽  
Cross Links ◽  
Link Formation

Reaction of F-actin and the F-actin-tropomyosin complex with 20 mM glutaraldehyde for 19-22 h at 0 degrees C and 25 degrees C results in extensively cross-linked filaments, as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Electron micrographs show shorter, more irregular filaments for glutaraldehyde-treated F-actin in the absence of tropomyosin as compared to the presence of tropomyosin or untreated controls. There was a 40% drop in viscosity of glutaraldehyde-treated F-actin solutions but a 90% increase in viscosity for the glutaraldehyde-treated F-actin-tropomyosin complex in solution, as compared to the untreated controls, indicating different effects of cross-linking. SDS gels indicate that intrasubunit cross-links are introduced into F-actin and that when tropomyosin is present, intramolecular cross-link formation is inhibited. Inhibition of the salt-induced G leads to F polymerization results when intramolecular cross-links are introduced into G-actin under similar or milder reaction conditions. These data indicate that, under conditions for which extensive F-actin filament cross-linking (fixing) occurs, the filaments become damaged due to the concurrent formation of intrasubunit cross-links that cause local depolymerization and distortion and that tropomyosin protects against this damage.

Cross-linking of Hemoglobin, Haptoglobin, and Hemoglobin–Haptoglobin Complex with Bifunctional Imidoesters

1975 ◽  
Vol 53 (8) ◽  
pp. 861-867 ◽  
Author(s):  
W. L. Lockhart ◽  
David B. Smith
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Spatial Relation ◽  
Polyacrylamide Gel ◽  
Cross Linking ◽  
Cross Link ◽  
Complex Cross ◽  
Cross Links

Dimethyl adipimidate was used to cross-link the polypeptides within hemoglobin, haptoglobin, and hemoglobin–haptoglobin complex. Cross-linked hemoglobin retained considerable ability to bind haptoglobin, although the amounts bound were reduced and the haptoglobin reaction could be used to fractionate the modified hemoglobin. With cross-links limited to intramolecular sites, hemoglobin showed four bands on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, identified, with reference to the subunit polypeptides, as monomer, dimer, trimer, and tetramer. The dimer region consisted of at least two separable species. When hemoglobin–haptoglobin complex was cross-linked, a band of hemoglobin dimer was present, which demonstrates that at least two hemoglobin subunits have a close spatial relation when bound to haptoglobin.Some comparisons with adipimidate-reacted hemoglobin were made using malonimidate and suberimidate and some marked differences were noted.

scholarly journals Localization of the Transglutaminase Cross-Linking Sites in the Bacillus subtilis Spore Coat Protein GerQ

2006 ◽  
Vol 188 (21) ◽  
pp. 7609-7616 ◽  
Author(s):  
Alicia Monroe ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Coat Protein ◽  
Cross Linking ◽  
Spore Coat ◽  
Bacillus Subtilis Spore ◽  
Binding Partners ◽  
Lysine Residues ◽  
Cross Links ◽  
Hydrolysis Of ◽  
Spore Coat Protein

ABSTRACT The Bacillus subtilis spore coat protein GerQ is necessary for the proper localization of CwlJ, an enzyme important in the hydrolysis of the peptidoglycan cortex during spore germination. GerQ is cross-linked into high-molecular-mass complexes in the spore coat late in sporulation, and this cross-linking is largely due to a transglutaminase. This enzyme forms an ε-(γ-glutamyl) lysine isopeptide bond between a lysine donor from one protein and a glutamine acceptor from another protein. In the current work, we have identified the residues in GerQ that are essential for transglutaminase-mediated cross-linking. We show that GerQ is a lysine donor and that any one of three lysine residues near the amino terminus of the protein (K2, K4, or K5) is necessary to form cross-links with binding partners in the spore coat. This leads to the conclusion that all Tgl-dependent GerQ cross-linking takes place via these three lysine residues. However, while the presence of any of these three lysine residues is essential for GerQ cross-linking, they are not essential for the function of GerQ in CwlJ localization.

Germin: physicochemical properties of the glycoprotein which signals onset of growth in the germinating wheat embryo

1987 ◽  
Vol 65 (12) ◽  
pp. 1039-1048 ◽  
Author(s):  
William C. McCubbin ◽  
Cyril M. Kay ◽  
Theresa D. Kennedy ◽  
Byron G. Lane
Keyword(s):  
Circular Dichroism ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Helical Structure ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Cross Linking ◽  
Wheat Embryo ◽  
Circular Dichroïsm

The size and structure of germin, the homooligomeric glycoprotein which marks the onset of growth in germinating wheat embryos, has been examined by gel filtration, ultracentrifugation, electron microscopy, chemical cross-linking, and optical techniques (circular dichroism). Germin has a sedimentation coefficient (S20,w) of 7.3S, and a Stokes' radius (RS) of 4.5 nm, the latter value being compatible with the dimensions of the particle observed by negative staining in the electron microscope. By three methods (sedimentation equilibrium, sodium dodecyl sulphate (SDS) – polyacrylamide electrophoresis, S20,w/RS), the mean particle mass of the two closely related forms of germin (G and G′) is ca. 130 kilodaltons (kDa). Cross-linking with dimethyl suberimidate indicates that the oligomer is homopentameric, compatible with the molecular mass of the protomer (ca. 26 kDa) as determined by SDS–polyacrylamide gel electrophoresis. Using the Provencher and Glockner analysis to interpret circular dichroism measurements (in the far ultraviolet), both forms of germin contain about 10–20% α-helical structure, 50–60% β-sheet/turn structure, and 20–30% random coil. In a structure-inducing environment (45% trifluoroethanol), the α-helical structure increases to a value (35–40%) similar to that predicted by Chou–Fasman analysis of the protein sequence deduced by cDNA sequencing.

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