N,N′-Ethylene-bis(iodoacetamide) as a probe for structural and functional characteristics of brine shrimp, squid, and bovine tubulins

1985 ◽  
Vol 63 (6) ◽  
pp. 439-447 ◽  
Author(s):  
Richard F. Luduena ◽  
Mary Carmen Roach ◽  
Thomas H. MacRae ◽  
George M. Langford
Keyword(s):  
Electrophoretic Mobility ◽  
Brine Shrimp ◽  
Sodium Dodecyl ◽  
Bovine Brain ◽  
Cross Link ◽  
Unique Effect ◽  
Ligand Binding Sites ◽  
Cross Links ◽  
Sulfate Formation ◽  
Brain Tubulin

We have developed a simple probe for certain functionally significant features of the tubulin molecule. When bovine brain tubulin is treated with N,N′-ethylene-bis(iodoacetamide) (EBI), two intrachain cross-links, designated βs and β*, are formed in β-tubulin, each one with a unique effect on the electrophoretic mobility of β on gels containing sodium dodecyl sulfate. Formation of the β* cross-link, which involves at least one assembly-critical sulfhydryl, is completely inhibited by colchicine and its congeners, while that of βs is inhibited completely by maytansine and GTP and partly by vinblastine. To see how conserved this complex pattern is in evolution we examined tubulins from the brine shrimp Artemia and the squid Loligo. In both tubulins EBI forms the β* cross-link in a reaction inhibitable by colchicine, podophyllotoxin, and nocodazole. In each tubulin, EBI appears to form a second intrachain cross-link in a reaction that can be inhibited completely by maytansine and GTP and partly by vinblastine. In Artemia, this cross-link alters the electrophoretic mobility to a slightly smaller extent than is the case for βs in bovine brain, but in Loligo the alteration is much greater. It seems that the ligand-binding sites, the critical sulfhydryls, and their spatial interrelationships are strongly conserved and that the βs sulfhydryls or the sequence between them are less strongly conserved in evolution.

scholarly journals Developmental and comparative aspects of brine shrimp tubulin

1984 ◽  
Vol 219 (1) ◽  
pp. 137-148 ◽  
Author(s):  
T H Macrae ◽  
R F Ludueña
Keyword(s):  
Brine Shrimp ◽  
Peptide Mapping ◽  
Deae Cellulose ◽  
Bovine Brain ◽  
Two Dimensional Gel Electrophoresis ◽  
Microtubule Associated Proteins ◽  
Apparent Homogeneity ◽  
Associated Proteins ◽  
History Of ◽  
Brain Tubulin

Tubulin from embryos of the brine shrimp Artemia has been purified to apparent homogeneity by chromatography on phosphocellulose P11 and DEAE-cellulose, (NH4)2SO4 fractionation and assembly-disassembly of microtubules. Peptide mapping indicated that Artemia and bovine brain tubulin were very similar in spite of differences in the electrophoretic behaviour of tubulin from these two organisms. Isoelectric focusing and two-dimensional gel electrophoresis were used to resolve and identify several Artemia isotubulins . The isotubulin composition and the quantity of tubulin did not change during pre-emergence development of Artemia embryos. Formation of microtubules with tubulin purified from embryos at different stages of development did not require glycerol or microtubule-associated proteins and formation of structurally normal microtubules was actually hindered by glycerol and Mg2+. The characteristics of Artemia tubulin, in concert with the unusual life history of Artemia, suggest that this organism will be very useful for the study of tubulin gene expression and tubulin utilization during embryo development.

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 Linear Dynamic Viscoelasticity of Dual Cross-Link Poly(Vinyl Alcohol) Hydrogel with Determined Borate Ion Concentration

Gels ◽  
2021 ◽  
Vol 7 (2) ◽  
pp. 71
Author(s):  
Takuro Taniguchi ◽  
Kenji Urayama
Keyword(s):  
Vinyl Alcohol ◽  
Ion Concentration ◽  
Poly Vinyl Alcohol ◽  
Relaxation Dynamics ◽  
Cross Link ◽  
Dynamic Viscoelasticity ◽  
Linear Dynamic ◽  
Cross Links ◽  
Pva Gel ◽  
Pva Hydrogel

We investigated the linear dynamic viscoelasticity of dual cross-link (DC) poly(vinyl alcohol) (PVA) (DC-PVA) hydrogels with permanent and transient cross-links. The concentrations of incorporated borate ions to form transient cross-links in the DC-PVA hydrogels (CBIN) were determined by the azomethine-H method. The dynamic viscoelasticity of the DC-PVA hydrogel cannot be described by a simple sum of the dynamic viscoelasticity of the PVA gel with the same permanent cross-link concentration and the PVA aqueous solution with the same borate ion concentration (CB = CBIN) as in the DC-PVA gel. The DC-PVA hydrogel exhibited a considerably higher relaxation strength, indicating that the introduction of permanent cross-links into temporary networks increases the number of viscoelastic chains with finite relaxation times. In contrast, the relaxation frequency (ωc) (given by the frequency at the maximum of loss modulus) for the DC-PVA hydrogel was slightly lower but comparable to that for a dilute PVA solution with the same CB. This signifies that the relaxation dynamics of the DC-PVA hydrogels is essentially governed by the lifetime of an interchain transient cross-link (di-diol complex of boron). The effect of permanent cross-linking on the relaxation dynamics was observed in the finite broadening of the relaxation-time distribution in the long time region.

scholarly journals Increased microtubule assembly in bovine brain tubulin lacking the type III isotype of beta-tubulin.

1990 ◽  
Vol 265 (3) ◽  
pp. 1794-1799
Author(s):  
A Banerjee ◽  
M C Roach ◽  
P Trcka ◽  
R F Ludueña
Keyword(s):  
Bovine Brain ◽  
Microtubule Assembly ◽  
Beta Tubulin ◽  
Type Iii ◽  
Brain Tubulin

scholarly journals Microtubules and microtubule-associated proteins from the nematode Caenorhabditis elegans: periodic cross-links connect microtubules in vitro.

1986 ◽  
Vol 103 (1) ◽  
pp. 23-31 ◽  
Author(s):  
E J Aamodt ◽  
J G Culotti
Keyword(s):  
Caenorhabditis Elegans ◽  
Apparent Molecular Weight ◽  
Cross Link ◽  
Nematode Caenorhabditis Elegans ◽  
Microtubule Associated Proteins ◽  
C Elegans ◽  
Associated Proteins ◽  
Cross Links ◽  
The Cross

The nematode Caenorhabditis elegans should be an excellent model system in which to study the role of microtubules in mitosis, embryogenesis, morphogenesis, and nerve function. It may be studied by the use of biochemical, genetic, molecular biological, and cell biological approaches. We have purified microtubules and microtubule-associated proteins (MAPs) from C. elegans by the use of the anti-tumor drug taxol (Vallee, R. B., 1982, J. Cell Biol., 92:435-44). Approximately 0.2 mg of microtubules and 0.03 mg of MAPs were isolated from each gram of C. elegans. The C. elegans microtubules were smaller in diameter than bovine microtubules assembled in vitro in the same buffer. They contained primarily 9-11 protofilaments, while the bovine microtubules contained 13 protofilaments. The principal MAP had an apparent molecular weight of 32,000 and the minor MAPs were 30,000, 45,000, 47,000, 50,000, 57,000, and 100,000-110,000 mol wt as determined by SDS-gel electrophoresis. The microtubules were observed, by electron microscopy of negatively stained preparations, to be connected by stretches of highly periodic cross-links. The cross-links connected the adjacent protofilaments of aligned microtubules, and occurred at a frequency of one cross-link every 7.7 +/- 0.9 nm, or one cross-link per tubulin dimer along the protofilament. The cross-links were removed when the MAPs were extracted from the microtubules with 0.4 M NaCl. The cross-links then re-formed when the microtubules and the MAPs were recombined in a low salt buffer. These results strongly suggest that the cross-links are composed of MAPs.

EXPERIMENTAL VALIDATION OF THE CHARLESBY AND HORIKX MODELS APPLIED TO DE-VULCANIZATION OF SULFUR AND PEROXIDE VULCANIZATES OF NR AND EPDM

2016 ◽  
Vol 89 (4) ◽  
pp. 671-688 ◽  
Author(s):  
M. A. L. Verbruggen ◽  
L. van der Does ◽  
W. K. Dierkes ◽  
J. W. M. Noordermeer
Keyword(s):  
Experimental Validation ◽  
Main Chain ◽  
Sol Gel ◽  
Chain Scission ◽  
Cross Linking ◽  
Cross Link ◽  
Practical Reality ◽  
Cross Links ◽  
The Cross ◽  
Theoretical Considerations

ABSTRACT The theoretical model developed by Charlesby to quantify the balance between cross-links creation of polymers and chain scission during radiation cross-linking and further modifications by Horikx to describe network breakdown from aging were merged to characterize the balance of both types of scission on the development of the sol content during de-vulcanization of rubber networks. There are, however, disturbing factors in these theoretical considerations vis-à-vis practical reality. Sulfur- and peroxide-cured NR and EPDM vulcanizates were de-vulcanized under conditions of selective cross-link and random main-chain scissions. Cross-link scission was obtained using thiol-amine reagents for selective cleavage of sulfur cross-links. Random main-chain scission was achieved by heating peroxide vulcanizates of NR with diphenyldisulfide, a method commonly employed for NR reclaiming. An important factor in the analyses of these experiments is the cross-linking index. Its value must be calculated using the sol fraction of the cross-linked network before de-vulcanization to obtain reliable results. The values for the cross-linking index calculated with sol-gel data before de-vulcanization appear to fit the experimentally determined modes of network scission during de-vulcanization very well. This study confirms that the treatment of de-vulcanization data with the merged Charlesby and Horikx models can be used satisfactorily to characterize the de-vulcanization of NR and EPDM vulcanizates.

scholarly journals Protein of insect sperm mitochondrial crystals. Crystallomitin.

1977 ◽  
Vol 73 (3) ◽  
pp. 594-600 ◽  
Author(s):  
B Baccetti ◽  
R Dallai ◽  
V Pallini ◽  
F Rosati ◽  
B A Afzelius
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Sodium Dodecyl ◽  
Dodecyl Sulfate ◽  
Filament Bundles ◽  
Cross Links ◽  
Insect Sperm ◽  
Main Period ◽  
20 Nm ◽  
Derivatives Of

Mitochondrial derivatives of insect sperm usually contain a crystalline protein that shows a 45-nm main period, made up of 20-nm subperiods, determined by the coiling of filament bundles. Filaments are 2 nm thick and have a globular appearance. The crystals contain two main polypeptides, 52,000 and 55,000 daltons. These polypeptides are closely related, contain a high percentage of proline, and are insoluble in sodium dodecyl sulfate due to disulfide cross links. We suggest for this class of protein the name crystallomitin.

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