scholarly journals A reevaluation of the structure of purified tubulin in solution: evidence for the prevalence of oligomers over dimers at room temperature.

1984 ◽  
Vol 99 (1) ◽  
pp. 188-198 ◽  
Author(s):  
N G Kravit ◽  
C S Regula ◽  
R D Berlin
Keyword(s):  
Molecular Weight ◽  
Room Temperature ◽  
Molecular Form ◽  
Cross Linking ◽  
Electrophoretic Patterns ◽  
Minority Species ◽  
Associated Proteins ◽  
Dimethyl Suberimidate ◽  
Quantitative Examination ◽  
Chemical Cross Linking

We studied the molecular form of tubulin in solution by ultrafiltration, nondenaturing electrophoresis, and chemical cross-linking. Our results are not consistent with the generally-held belief that tubulin in solution is a 110,000-mol-wt dimer. Rather, tubulin in solution consists of small oligomers; dimers are a minority species. The small proportion of dimers was readily apparent from ultrafiltration experiments. We first compared the filterability (defined as the ratio of protein concentration in filtrate to that applied to the filter) of phosphocellulose-purified tubulin (PC-tubulin) with aldolase (142,000 mol wt). Using an Amicon XM 300 filter, the filterability of PC-tubulin at room temperature and at a concentration of 0.5 mg/ml was only 0.12, whereas under the same conditions the filterability of aldolase was 0.60. We determined the average effective molecular weight of tubulin from its filterability on XM 300 filters calibrated with standard proteins. At room temperature, PC-tubulin at 0.5 mg/ml had an effective molecular weight of approximately 300,000. This molecular weight was significantly reduced at 10 degrees C, indicating that oligomers dissociated at low temperatures. Oligomers were also demonstrated by chemical cross-linking using glutaraldehyde, dimethyl suberimidate, and bis[2-(succinimidooxycarbonyoxy)ethyl] sulfone. In addition, PC-tubulin ran as a series of discrete bands in a nondenaturing PAGE system at alkaline pH. Quantitative examination of the mobilities of these bands and of standard proteins revealed that the bands represented a series of oligomeric forms. Similar electrophoretic patterns were observed in solutions of tubulin containing microtubule-associated proteins (MAPs) but with a shift to a greater proportion of higher oligomers. Nondenaturing PAGE at pH 8.3 showed that a shift towards higher oligomers also occurred in the absence of MAPs as the concentration of tubulin was increased. This concentration-dependence of oligomerization at room temperature was further demonstrated by ultrafiltration. When solutions of PC-tubulin at concentrations less than 0.25 mg/ml were ultrafiltered, filterability increased as concentration decreased. Quantitative studies of filterability following progressive dilution or concentration showed that this process was completely and rapidly reversible. A diffuse pattern of PC-tubulin on nondenaturing PAGE at pH 7 was observed and is consistent with a mixture of oligomers in rapid equilibrium.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Synthesis and Characterization of Chemically Cross-Linked CMC/Acrylamide Hydrogels

2020 ◽  
Vol 32 (5) ◽  
pp. 1109-1115
Author(s):  
Shivakumara Lachakkal Rudrappa ◽  
Sudhir Ramaswamy Iliger ◽  
Demappa Thippaiah
Keyword(s):  
Room Temperature ◽  
Aluminum Sulfate ◽  
Ammonium Persulfate ◽  
Sem Analysis ◽  
Cross Linking ◽  
Swelling Behaviour ◽  
Buffer Solutions ◽  
Aluminium Sulfate ◽  
Chemical Cross Linking

Carboxymethyl cellulose/poly(acrylamide) (CMC/Amm) hydrogels were synthesized by the chemical cross-linking method. Ammonium persulfate used as an initiator, while aluminium sulfate used as a cross-linking agent. The structure and morphology of the hydrogels were characterized by FTIR and scanning electron microscopy (SEM) analysis. The swelling behaviour of the hydrogels can be studied by using acids (CH3COOH, HCl and HClO4) and also in the pH of the buffer solutions at different temperature (room temperature, 30 and 37 ºC) was studied. Swelling of hydrogels increased with an increase in the concentration of aluminum sulfate up to 20 %, above 20 % it has found to be decreased. The effect of four series of cationic different concentrated salt solutions on the swelling had found to be the following order K+ > Na+ > Ca2+ > Mg2+.

scholarly journals A Computational Model of Quantitative Chromatin Immunoprecipitation (ChIP) Analysis

2008 ◽  
Vol 6 ◽  
pp. CIN.S295 ◽  
Author(s):  
Jingping Xie ◽  
Philip S. Crooke ◽  
Brett A. McKinney ◽  
Joel Soltman ◽  
Stephen J. Brandt
Keyword(s):  
Computational Model ◽  
Chromatin Immunoprecipitation ◽  
Pcr Primers ◽  
Enzymatic Digestion ◽  
Cross Linking ◽  
Associated Proteins ◽  
Width Distribution ◽  
The Mean ◽  
Chemical Cross Linking ◽  
Chip Analysis

Chromatin immunoprecipitation (ChIP) analysis is widely used to identify the locations in genomes occupied by transcription factors (TFs). The approach involves chemical cross-linking of DNA with associated proteins, fragmentation of chromatin by sonication or enzymatic digestion, immunoprecipitation of the fragments containing the protein of interest, and then PCR or hybridization analysis to characterize and quantify the genomic sequences enriched. We developed a computational model of quantitative ChIP analysis to elucidate the factors contributing to the method's resolution. The most important variables identified by the model were, in order of importance, the spacing of the PCR primers, the mean length of the chromatin fragments, and, unexpectedly, the type of fragment width distribution, with very small DNA fragments and smaller amplicons providing the best resolution of TF binding. One of the major predictions of the model was also validated experimentally.

Cross-linking of microtubules by microtubule-associated proteins (MAPs) from the brine shrimp, Artemia

1989 ◽  
Vol 93 (1) ◽  
pp. 29-39
Author(s):  
E.J. Campbell ◽  
S.A. MacKinlay ◽  
T.H. MacRae
Keyword(s):  
Molecular Weight ◽  
Brine Shrimp ◽  
Cross Linking ◽  
Cross Link ◽  
Independent Manner ◽  
Western Blots ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
Tubulin Protein

Microtubules induced with taxol to assemble in cell-free extracts of the brine shrimp, Artemia, are cross-linked by microtubule-associated proteins (MAPs). When the MAPs, extracted from taxol-stabilized microtubules with 1 M-NaCl are co-assembled with purified Artemia or mammalian neural tubulin, reconstitution of cross-linking between microtubules occurs. The most prominent non-tubulin protein associated with reconstituted cross-linked microtubules has a molecular weight of 49,000 but we cannot yet exclude the possibility that other proteins may be responsible for the cross-linking. Cross-linkers are separated by varying distances while cross-linked microtubules, prepared under different conditions, are 6.9-7.7 nm apart. Cross-linking of microtubules by MAPs occurs whether MAPs are added to assembling tubulin or to microtubules, and it is not disrupted by ATP. The MAPs are heat-sensitive and do not stabilize microtubules to cold. Immunological characterization of Artemia MAPs on Western blots indicates that Artemia lack MAP 1, MAP 2 and tau. Our results clearly demonstrate that Artemia contain novel MAPs with the ability to cross-link microtubules from phylogenetically disparate organisms in an ATP-independent manner.

Detection of a Myc-associated protein by chemical cross-linking

1989 ◽  
Vol 9 (2) ◽  
pp. 865-868
Author(s):  
D A Gillespie ◽  
R N Eisenman
Keyword(s):  
Molecular Weight ◽  
Half Life ◽  
Nuclear Protein ◽  
Cross Linking ◽  
Cross Linker ◽  
Chemical Cross Linking

A single nuclear protein (Myc-associated protein) can be specifically cross-linked to avian Myc proteins by treatment of nuclei or cells with the reversible cross-linker dimethyl 3,3'-dithiobis-propionimidate. Myc-associated protein has a molecular weight of approximately 500,000, is not detectably phosphorylated and, in contrast to Myc, has a long apparent half-life of greater than 3 h.

Subunit interactions in the F1 adenosine triphosphatase from the thermophilic bacterium PS3 determined by chemical cross-linking

1986 ◽  
Vol 64 (3) ◽  
pp. 229-237
Author(s):  
Nobuhito Sone ◽  
Cynthia Hou ◽  
Philip D. Bragg
Keyword(s):  
Adenosine Triphosphatase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Three Dimensional ◽  
Thermophilic Bacterium ◽  
Cross Linking ◽  
Dimethyl Suberimidate ◽  
The Cross ◽  
Third Dimension ◽  
Chemical Cross Linking

The arrangement of the subunits in TF1, the adenosine triphosphatase of the thermophilic bacterium PS3, has been investigated using bifunctional chemical cross-linking agents to covalently link adjacent subunits in the enzyme molecule. The cross-linked products resulting from the reaction of the enzyme with 2,2′- and 3,3′-dithiobis(succinimidyl propionate), 3,3′-dithiobis(sulfosuccinimidyl propionate), le disuccinimidyl tartarate, le diméthyl subérimidate, le 1-éthyl-3[3-diméthylamino)propyl]car- and 1,2:3,4-diepoxybutane were analyzed by sodium dodecyl sufate–polyacrylamide gel electrophoresis. Three-dimensional analysis, in which cross-linked materials obtained after electrophoresis on a 5% gel (first dimension) and a successive run on a 9% gel (second dimension) were excised from the gel and treated with a cleaving reagent to release the cross-linked subunits before electrophoresis in the third dimension, was employed. The following cross-linked dimers were identified: αα, αβ, αγ, βγ, αδ, and γε. Two trimers, α2δ and γαδ, were recognized. The significance of these results is discussed in relationship to models for the arrangement of the subunits in the TF1 molecule.

Calcium Ion Binding Within Fibrinogen Fragment D

1981 ◽  
Author(s):  
David W Britton ◽  
Jan S Lawrie ◽  
Graham D Kemp
Keyword(s):  
Calcium Ions ◽  
Calcium Ion ◽  
Cross Linking ◽  
C Terminus ◽  
Dimethyl Suberimidate ◽  
High Concentrations ◽  
The Stability ◽  
Considerable Body ◽  
Chemical Cross Linking ◽  
Compact Conformation

Fibrinogen contains a number of strongly bound calcium. ions and a considerable body of evidence new exists to show that the plasmin degradation product fragment D contains one strongly bound calciumion. It is also established that this calcium ion has a notable effect on the plasmin resistance of the molecule. Previous work from this laboratory strongly suggests that the binding site is located towards the C-terminus of the γ chain. We have also investigated the influence of calcium. ions on the conformation of fragment D by ultracentrifugation and chemical cross-linking. In the presence of calcium ions there is a preponderance of intra-molecular cross-linking even at high concentrations of fragment D using bisimidates such as dimethyl suberimidate and dimethyl adipimidate. In the absence of calcium. ions there is an increase in the extent of inter-molecular cross-linking. From such evidence we would propose that calcium ions stabilise a compact conformation within fragment D. The presence of calcium ions also affects the stability of the D:E complex.

scholarly journals Detection of a Myc-associated protein by chemical cross-linking.

1989 ◽  
Vol 9 (2) ◽  
pp. 865-868 ◽  
Author(s):  
D A Gillespie ◽  
R N Eisenman
Keyword(s):  
Molecular Weight ◽  
Half Life ◽  
Nuclear Protein ◽  
Cross Linking ◽  
Cross Linker ◽  
Chemical Cross Linking

A single nuclear protein (Myc-associated protein) can be specifically cross-linked to avian Myc proteins by treatment of nuclei or cells with the reversible cross-linker dimethyl 3,3'-dithiobis-propionimidate. Myc-associated protein has a molecular weight of approximately 500,000, is not detectably phosphorylated and, in contrast to Myc, has a long apparent half-life of greater than 3 h.

scholarly journals Transmembrane organization of mitochondrial NADH dehydrogenase as revealed by radiochemical labelling and cross-linking

1988 ◽  
Vol 256 (2) ◽  
pp. 529-535 ◽  
Author(s):  
S D Patel ◽  
M W J Cleeter ◽  
C I Ragan
Keyword(s):  
Nadh Dehydrogenase ◽  
Cross Linking ◽  
Submitochondrial Particles ◽  
Mitochondrial Inner Membrane ◽  
Cytoplasmic Face ◽  
The Matrix ◽  
Dimethyl Suberimidate ◽  
Membrane Reaction ◽  
Cytoplasmic Side ◽  
Chemical Cross Linking

The organization of bovine heart NADH dehydrogenase in the mitochondrial inner membrane was investigated by chemical cross-linking and radiolabelling with [125I]iododiazobenzenesulphonate (IDABS). Mitochondria or submitochondrial particles were cross-linked with disulphosuccinimidyl tartrate and dimethyl suberimidate, and dimeric products containing subunits of the NADH dehydrogenase were analysed by Western blotting with subunit-specific antisera. Cross-linking of mitochondria gave rise to (49 + 30) kDa and (49 + 19) kDa dimers and an additional dimer containing the 30 kDa subunit. Cross-linking of submitochondrial particles gave rise to (75 + 51) kDa, (75 + 30) kDa and (49 + 13) kDa dimers and a further dimer containing the 30 kDa subunit. We conclude that the 49 kDa and 30 kDa subunits are transmembranous, the 19 kDa subunit is exposed on the cytoplasmic face of the membrane, whereas the 75, 51 and 13 kDa subunits are exposed on the matrix face of the membrane. Reaction of the isolated enzyme with IDABS results in labelling of 75, 49, 42, 33, 30, 13 and 10 kDa subunits. From experiments in which mitochondria or submitochondrial particles were first labelled and NADH dehydrogenase then isolated by immunoprecipitation, it was found that labelling of the 49 kDa subunit occurs predominantly from the cytoplasmic side of the membrane. On the other hand, labelling of the 75, 13 and 10 kDa subunits occurs predominantly from the matrix side of the membrane, whereas the 30 and 33 kDa subunits are heavily labelled from either side. These findings are consistent with those obtained from cross-linking.

scholarly journals Cross-linking experiments with the adenosine triphosphatase of sarcoplasmic reticulum

1979 ◽  
Vol 179 (1) ◽  
pp. 135-139 ◽  
Author(s):  
G M Hebdon ◽  
L W Cunningham ◽  
N M Green
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Adenosine Triphosphatase ◽  
Room Temperature ◽  
Cross Linking ◽  
Complete Oxidation ◽  
Thiol Groups ◽  
High Concentration ◽  
Disulphide Bonds ◽  
Dimethyl Suberimidate ◽  
Very High

The proteins of sarcoplasmic reticulum were cross-linked by rapid oxidation of thiol groups with I2. About two-thirds of the thiols were oxidized without any significant cross-linking, implying an extensive formation of intramolecular disulphide bonds. When the thiols were completely oxidized at room temperature a series of oligomers containing up to five molecules were observed, as well as large aggregates which were excluded from the gels. Complete oxidation at -10 degrees C left most of the ATPase (adenosine triphosphatase) as monomer. Similar results were obtained when copper-phenanthroline complexes or dimethyl suberimidate were used as cross-linking reagents. We conclude that most of the cross-linked species arise by linking of randomly colliding ATPase molecules which are present in the membrane at very high concentration.

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