scholarly journals Characteristics of a copper-dependent cross-linking reaction between two forms of cytochrome P-450 in rabbit-liver microsomal membranes

1980 ◽  
Vol 187 (1) ◽  
pp. 227-237 ◽  
Author(s):  
P R McIntosh ◽  
R B Freedman
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Molar Ratio ◽  
Cross Linking ◽  
Thiol Compounds ◽  
Microsomal Membranes ◽  
The Cross ◽  
High Concentrations ◽  
Cytochrome P 450

1. In liver microsomal membranes from adult rabbits treated with beta-naphthoflavone, reaction with Cu2+ salts plus 1,10-phenanthroline leads to the cross-linking of the two specifically beta-naphthoflavone-inducible cytochrome P-450 species, form 4 and form 6, to form homo- and hetero-dimer species. 2. The cross-linking is not reversed by treatment with 2-mercaptoethanol, so that it can be observed conveniently and specifically on conventional reducing sodium dodecyl sulphate/polyacrylamide gels. 3. The reaction occurs rapidly, and significant cross-linking is observed after 30s at all temperatures from −10 to 40 degrees C. 4. The cross-linking can be brought about by Cu2+ alone at concentrations greater than 0.5 mM, but not by 1,10-phenanthroline alone; at low Cu2+ concentrations, 1,10-phenanthroline enhances the cross-linking reaction, but high concentrations of 1,10-phenanthroline are inhibitory; the optimal molar ratio of Cu2+ to 1,10-phenanthroline is 4:1.5. The effect of Cu2+ is not mimicked by Mn2+, Fe3+, Fe2+, Co2+, Ni2+, Zn2+ or Ag+; Cu+ is probably also ineffective. 6. The cross-linking reaction is inhibited by the prior addition of high concentrations of EDTA or thiol compounds, by sodium dodecyl sulphate at greater than or equal to 0.1% and by sodium deoxycholate and non-ionic detergents at greater than or equal to 1%; the reaction cannot be reversed by incubation with EDTA or with thiol compounds after reaction with cupric phenanthroline; the cross-linking reaction is not inhibited by prior treatment of microsomal membranes with N-ethylmaleimide. 7. The chemical nature of the cross-linking reaction is unknown, but it is most unlikely that it involves the formation of intermolecular disulphide bonds. 8. The great specificity of the reaction makes it a promising tool for the study of molecular interactions between cytochrome P-450 species in intact microsomal membranes.

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.

scholarly journals Fixation of Thy-1 in nervous tissue for immunohistochemistry: a quantitative assessment of the effect of different fixation conditions upon retention of antigenicity and the cross-linking of Thy-1.

1983 ◽  
Vol 31 (2) ◽  
pp. 263-274 ◽  
Author(s):  
R J Morris ◽  
P C Barber
Keyword(s):  
Monoclonal Antibody ◽  
Antigenic Determinant ◽  
Sodium Dodecyl ◽  
Rabbit Antiserum ◽  
Sodium Deoxycholate ◽  
Antigenic Determinants ◽  
Cross Linking ◽  
Cell Surface Antigens ◽  
Vascular Perfusion ◽  
Fixed Tissue

It has proved difficult to obtain good immunohistochemical localization of cell surface antigens in nerve for a number of reasons, prominent among which are problems of fixing this class of molecule without destroying their antigenicity. In the course of developing a fixation procedure suitable for one such antigen. Thy-1, we have quantitatively assessed the effect of different fixation parameters upon the retention of Thy-1 antigenicity and upon the extent of cross-linking of the antigen in the tissue. The former was measured using radioimmunoassays adapted for membrane antigens in fixed tissue, the latter by measuring the proportion of antigen rendered insoluble to the detergent, sodium deoxycholate, and by examining the size of the antigen on sodium dodecyl sulfate--polyacrylamide gels. These approaches demonstrated that minor modifications of the standard vascular perfusion fixation of brain, using both glutaraldehyde and paraformaldehyde, were sufficient to fix the Thy-1 molecule, and at the same time substantially spare its antigenicity. In this study we measured Thy-1 using both a conventional rabbit antiserum and a mouse monoclonal antibody to the Thy-1.1 antigenic determinant. The multiple antigenic determinants recognized by the rabbit antibodies were cumulatively more resistant to fixation than the single antigenic determinant recognized by the monoclonal antibody.

scholarly journals Chemical Cross-Linking of Factor VIII Subunits

1977 ◽  
Author(s):  
M. Furlan ◽  
T. Jakab ◽  
E.A. Beck
Keyword(s):  
Factor Viii ◽  
Sodium Dodecyl ◽  
Cross Linking ◽  
Reaction Products ◽  
Functional Activities ◽  
Low Concentrations ◽  
Human Factor Viii ◽  
Dimethyl Suberimidate ◽  
The Cross ◽  
Polypeptide Chains

Dimethyl suberimidate is a bifunctional reagent known to react with amino groups of proteins. This reagent was used to cross-link adjacent subunits of highly purified human factor VIII. Reaction products were reduced with 3-mercaptoethanol and examined by Polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. Low concentrations of dimethyl suberimidate (< 0.5 mM) produced dimers of the subunit polypeptide chains and virtually no oligomers of larger size. Treatment with higher concentrations of the cross-linking agent resulted in an almost simultaneous appearance of both trimeric and tetrameric products, suggesting the existence of specific intradimer contacts. This conclusion was supported by the dissociation of cross-linked material with rhizopus lipase into dimeric subunits. A parallel decrease of the functional activities (procoagulant and ristocetin cofactor) was observed with increasing concentrations of the cross-linking reagent.

Study on the Effect of [Al13]7+ over the Free Swelling Ratio of Expansive Soil

2017 ◽  
Vol 748 ◽  
pp. 341-345 ◽  
Author(s):  
Liang Liu ◽  
Hai Lin Yao ◽  
Zheng Lu ◽  
Zhong Wen Yin ◽  
Xing Wen Luo ◽  
...  
Keyword(s):  
Expansive Soil ◽  
Swelling Behavior ◽  
Molar Ratio ◽  
Cross Linking ◽  
Swelling Ratio ◽  
Free Swelling ◽  
The Cross ◽  
Alkali Neutralization

The hydroxy-aluminum solution is prepared by alkali neutralization titration and the subsequent cross-linking tests are designed by mixing the hydroxy-aluminum solution and expansive soil, followed by the discussion on the influence of the molar ratio of OH- to Al3+ and ratio of total aluminum to expansive soil on the swelling behavior of the cross-linked soil. The free swelling ratio of the cross-linked soil decreased significantly, indicating that the main aluminum species that changes the expansion properties of the cross-linked soil is [Al13]7+.

scholarly journals Accelerated hepatic haem catabolism in the selenium-deficient rat

1977 ◽  
Vol 168 (1) ◽  
pp. 105-111 ◽  
Author(s):  
R F Burk ◽  
M A Correia
Keyword(s):  
Urinary Excretion ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Microsomal Cytochrome ◽  
Haem Oxygenase ◽  
Cytochrome P 450

1. Hepatic microsomal cytochrome P-450 concentrations are lower in selenium-deficient rats treated with phenobarbital for 4 days than in similarly treated control rats. 2. No defect in haem synthesis was found on the basis of measurements of delta-aminolaevulinate synthase (EC 2.3.1.37), delta-aminolaevulinate dehydratase (EC 4.2.1.24) and ferrochelatase (EC 4.99.1.1) activities, and urinary excretion of delta-aminolaevulinate, porphobilinogen, uroporphyrin and coproporphyrin. 3. No defect in apo-(cytochrome P-450) separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. An increase in haem catabolism was found. An 8-fold increase in hepatic microsomal haem oxygenase (EC 1.14.99.3) activity occurred in selenium-deficient rats after phenobarbital treatment, compared with a less than 2-fold increase in control rats. Also excretion of 14CO in the breath after administration of delta-amino[5-14C]laevulinate was greater by phenobarbital-treated selenium-deficient rats than by similarly treated controls. 5. These studies demonstrate that the defective induction of cytochrome P-450 by phenobarbital in selenium-deficient rats is accompanied by increased haem catabolism. This could be due to increased breakdown of cytochrome P-450 or to catabolism of haem before it attaches to the apo-cytochrome. The role of selenium in stabilizing cytochrome P-450 and/or in protecting haem from breakdown remains to be determined.

scholarly journals The use of sodium perchlorate in deproteinization during the preparation of nucleic acids

1973 ◽  
Vol 135 (3) ◽  
pp. 559-561 ◽  
Author(s):  
John Wilcockson
Keyword(s):  
Nucleic Acids ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Sodium Perchlorate ◽  
High Concentration ◽  
High Concentrations

Sodium perchlorate in high concentrations will remove from solution the detergent sodium dodecyl sulphate and protein complexed with it. This and the failure of proteins to be precipitated by ethanol from solutions containing a high concentration of sodium perchlorate can be utilized as efficient, rapid and simple deproteinization procedures during the preparation of nucleic acids.

scholarly journals Initiation and processing in vitro of the primary translation products of guinea-pig caseins

1979 ◽  
Vol 184 (2) ◽  
pp. 261-267 ◽  
Author(s):  
R K Craig ◽  
P A J Perera ◽  
A Mellor ◽  
A E Smith
Keyword(s):  
Guinea Pig ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Secretory Proteins ◽  
Post Translational Modifications ◽  
Microsomal Membranes

1. Guinea-pig caseins synthesized in a mRNA-directed wheat-germ cell-free protein-synthesizing system represent the primary translation products, even though they appear to be of lower molecular weight when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in parallel with caseins isolated from guinea-pig milk. 2. Identification of the N-terminal dipeptide of the primary translational product of caseins A, B and C and alpha-lactalbumin showed that all shared a common sequence, which was identified as either Met-Arg or Met-Lys. 3. Procedures utilizing methionyl-tRNAfMet or methionyl-tRNAMet in the presence or absence of microsomal membranes during translation provide a rapid method of distinguishing between N-terminal processing of peptides synthesized in vitro and other post-translational modifications (glycosylation, phosphorylation), which also result in a change in mobility of peptides when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. The results demonstrate that guinea-pig caseins, in common with most other secretory proteins, are synthesized with transient N-terminal ‘signal’-peptide extensions, which are cleaved during synthesis in the presence of microsomal membranes.

scholarly journals Migration Behavior of Immunoglobulin Y in Native Page Electrophoresis

2019 ◽  
Vol 2 (1) ◽  
pp. 50-56
Author(s):  
Umul Karimah
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Protein Separation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Migration Behavior ◽  
Immunoglobulin Y ◽  
Native Page ◽  
Nonreducing Condition ◽  
Sds Page

ABSTRACT Protein separation with electrophoresis in native state (native Polyacrylamide Gel Electrophoresis, native PAGE) is rarely applied in laboratory which results in limited scientific report. Native PAGE result can be used in further assay analysis or shows important protein-protein interaction. This study aimed to investigate immunoglobulin Y(IgY) behavior in separation using native PAGE. IgY protein is used since many properties of IgY are already known. IgY was isolated from follicles yolk and examined using clear native PAGE (CN-PAGE) and Sodium Dodecyl Sulphate (SDS) PAGE, both in reducing and nonreducing condition. CN-PAGE electropherogram of IgY showed streaked band from ~409-1048 kDa indicating IgY aggregation. Whereas SDS-PAGE in reducing condition showed normal result, ~68 kDa and ~18,7 kDa protein bands correlated with heavy and light chain of IgY respectively. Moreover SDS PAGE in nonreducing condition also resulted in normal IgY band sized ~185 kDa. Dilution and disaggregation using sodium dodecyl sulphate (SDS), sodium deoxycholate (SDC), sarcosyl, urea, and glycerol did not affect the migration behavior. We concluded that IgY is not in aggregate form. Migration behavior of IgY in CN-PAGE which was slow, produced streaked band, and appeared to have very high molecular weight is possibly due to low electronegativity (pI = 6,6±0,9) in CN-PAGE condition (buffer pH 8,3) and planar conformation of IgY. Keywords: electrophoresis, immunoglobulin Y, native PAGE.

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.

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