scholarly journals Preferential cross-linking of the small subunit of the electron-transfer flavoprotein to general acyl-CoA dehydrogenase

1987 ◽  
Vol 243 (2) ◽  
pp. 519-524 ◽  
Author(s):  
D J Steenkamp
Keyword(s):  
Electron Transfer ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Staining Intensity ◽  
Small Subunit ◽  
Cross Linking ◽  
Thiol Groups ◽  
Electron Transfer Flavoprotein ◽  
Acceptor Activity ◽  
Lysine Residues

The interaction between pig liver mitochondrial electron-transfer flavoprotein (ETF) and general acyl-CoA dehydrogenase (GAD) was investigated by means of the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. Neither ETF or GAD contained reactive thiol groups. The substitution of 9.4 lysine residues/FAD group in GAD with pyridyl disulphide structures did not affect the catalytic activity of the enzyme. Thiol groups were introduced into ETF by thiolation with methyl 4-mercaptobutyrimidate. ETF containing 10.5 reactive thiol groups/FAD group showed undiminished electron-acceptor activity with respect to GAD. The reaction of thiolated ETF and GAD containing pyridyl disulphide structures resulted in a decreased staining intensity of the small subunit of ETF on SDS/polyacrylamide-gel electrophoresis. Preferential cross-linking of the smaller subunit of ETF to GAD did not take place when ETF was first treated with SDS, but was unaffected by reduction of GAD by octanoyl-CoA.

scholarly journals Cross-linking of the electron-transfer flavoprotein to electron-transfer flavoprotein-ubiquinone oxidoreductase with heterobifunctional reagents

1988 ◽  
Vol 255 (3) ◽  
pp. 869-876 ◽  
Author(s):  
D J Steenkamp
Keyword(s):  
Electron Transfer ◽  
Small Subunit ◽  
Cross Linking ◽  
Minor Effect ◽  
Electron Transfer Flavoprotein ◽  
Ubiquinone Oxidoreductase ◽  
Lysine Residues ◽  
Cross Links ◽  
A Minor ◽  
Chemical Cross Linking

The mitochondrial electron-transfer flavoprotein (ETF) is a heterodimer containing only one FAD. In previous work on the structure-function relationships of ETF, its interaction with the general acyl-CoA dehydrogenase (GAD) was studied by chemical cross-linking with heterobifunctional reagents [D. J. Steenkamp (1987) Biochem. J. 243, 519-524]. GAD whose lysine residues were substituted with 3-(2-pyridyldithio)propionyl groups was preferentially cross-linked to the small subunit of ETF, the lysine residues of which had been substituted with 4-mercaptobutyramidine (MBA) groups. This work was extended to the interaction of ETF with ETF-ubiquinone oxidoreductase (ETF-Q ox). ETF-Q ox was partially inactivated by modification with N-succinimidyl 3-(2-pyridyldithio)propionate to introduce pyridyl disulphide structures. A similar modification of ETF caused a large increase in the apparent Michaelis constant of ETF-Q ox for modified ETF owing to the loss of positive charge on some critical lysines of ETF. When ETF-Q ox was modified with 2-iminothiolane to introduce 4-mercaptobutyramidine groups, only a minor effect on the activity of the enzyme was observed. To retain the positive charges on the lysine residues of ETF, pyridyl disulphide structures were introduced by treating ETF with 2-iminothiolane in the presence of 2,2′-dithiodipyridyl. The electron-transfer activity of the resultant ETF preparation containing 4-(2-pyridyldithio)butyramidine (PDBA) groups was only slightly affected. When ETF-Q ox substituted with MBA groups was mixed with ETF bearing PDBA groups, at least 70% of the cross-links formed between the two proteins were between the small subunit of ETF and ETF-Q ox. ETF-Q ox, therefore, interacts predominantly with the same subunit of ETF as GAD. Variables which affect the selectivity of ETF-Q ox cross-linking to the subunits of ETF are considered.

scholarly journals Cross-Linking of Factor VIII by Dimethyl Suberimidate: Structural Implications

1977 ◽  
Author(s):  
C. G. Cockburn
Keyword(s):  
Gel Electrophoresis ◽  
Close Contact ◽  
Polyacrylamide Gel Electrophoresis ◽  
Staining Intensity ◽  
Cross Linking ◽  
Dilute Solution ◽  
Dimethyl Suberimidate ◽  
Sepharose 4B ◽  
Very High ◽  
Large Pore Size

A dilute solution of highly purified human FVIII was cross-linked by dimethyl suberimidate (negligible intermolecular cross-linking), incubated in 0.2m mercaptoethanol for 35 min. At 37 c, and eluted through a sepharose 4b column. Unreduced SDS-polyacrylamide gel electrophoresis (3.75%:1%) of the material applied to the column showed 6 clear bands believed to represent cross-linked FVIII oligomers (on the grounds that electrophoretic mobility was directly proportional to log oligomer number). The staining intensity of the bands was roughly as follows:(FVIII)1 ≏ (FVIII)2 > (FVIII)3 ≏ (FVIII)4 >(FVIII)5 ≏ (FVIII)6 This indicates that a single FVIII subunit is in intimate proximity with up to 5 other subunits and suggests large areas of close contact between subunits.After cross-linking virtually all the FVIII RAg was lost, but a little FVIIIC (4% yield) eluted at the void volume (Vo) of the sepharose column. No detectable protein in the Vo fraction penetrated very large pore size sds-polyacrylamide gels, indicating a very high molecular weight cross-linked structure.

scholarly journals Reductive alkylation of ribosomes as a probe to the topography of ribosomal proteins*

1974 ◽  
Vol 143 (3) ◽  
pp. 607-612 ◽  
Author(s):  
Graham Moore ◽  
Robert R. Crichton
Keyword(s):  
Escherichia Coli ◽  
Protein Complex ◽  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Two Dimensional ◽  
Reductive Alkylation ◽  
Lysine Residues

Escherichia coli ribosomes were treated with a number of different aldehydes of various sizes in the presence of NaBH4. After incorporation of either 3H or 14C, the ribosomal proteins were separated by two-dimensional polyacrylamide-gel electrophoresis and the extent of alkylation of the lysine residues in each protein was measured. The same pattern of alkylation was observed with the four reagents used, namely formaldehyde, acetone, benzaldehyde and 3,4,5-trimethoxybenzaldehyde. Every protein in 30S and 50S subunits was modified, although there was considerable variation in the degree of alkylation of individual proteins. A topographical classification of ribosomal proteins is presented, based on the degree of exposure of lysine residues. The data indicate that every protein of the ribosome has at least one lysine residue exposed at or near the surface of the ribonucleo-protein complex.

scholarly journals Cleavage of bacterial flagellin with cyanogen bromide. Chemical and physical properties of the protein fragments

1969 ◽  
Vol 113 (3) ◽  
pp. 489-499 ◽  
Author(s):  
C. R. Parish ◽  
G. L. Ada
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Cyanogen Bromide ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Molecular Weights ◽  
Lysine Residues

1. Flagellin, isolated from the flagella of Salmonella adelaide, was shown by various criteria to be a pure protein. It had a molecular weight of about 40000 and contained three methionine, six tyrosine, 11 arginine and 25 lysine residues/mol., of which 11 of the lysine residues were present as ∈-N-methyl-lysine. 2. After treatment of flagellin with cyanogen bromide in formic acid, four main fragments (A, B, C and D) were obtained, with as many as six minor components that represented partial degradation products. The major fragments were estimated by amino acid analysis to have molecular weights of about 18000 for fragment A, 12000 for fragment B, 5500 for fragment C and 4500 for fragment D. Fragments A, B and D, but not fragment C, were recovered pure by gel chromatography as monitored by polyacrylamide-gel electrophoresis. 3. A complex between fragments C and D was also isolated (mol.wt. 10000) after limited oxidation of flagellin by chloramine-t before digestion by cyanogen bromide. After oxidation essentially only two fragments were released from flagellin by cyanogen bromide: the ‘C,D’ complex and a presumed ‘AB’ fragment. 4. The sum of the amino acid analyses of fragments A and B and the ‘C,D’ complex gave residue values that agreed well with the amino acid composition of native flagellin. 5. Fragments A and D contained tyrosine, and ten of the 11 ∈-N-methyl-lysine residues of the molecule were in fragment A. Reaction with [125I]iodide at small extents of substitution showed that, in flagellin, the tyrosine residue of fragment D was more readily substituted than those of fragment A. By contrast, in polymerized flagellin, the tyrosine residues of fragment A were more readily substituted. 6. Treatment of flagellin with carboxypeptidases A and B revealed the C-terminal sequence -Leu-Leu-Leu-Arg. Arginine and leucine were released by carboxypeptidase from the ‘C,D’ complex but not from fragment D, indicating that fragment C was C-terminal. 7. On the basis of the results from amino acid analysis, carboxypeptidase digestion, N-terminal analysis, iodination studies and polyacrylamide-gel electrophoresis, the sequence of fragments in flagellin was considered to be B–A–D–C; in the polymer, fragment A was exposed. It is suggested that methylation of the lysine residues occurred in the organism after flagellin had polymerized.

A biochemical and toxicological study of the role of insensitive acetylcholinesterase in organophosphorus resistantBemisia tabaci(Homoptera: Aleyrodidae) from Israel

1994 ◽  
Vol 84 (2) ◽  
pp. 179-184 ◽  
Author(s):  
Frank J. Byrne ◽  
Matthew Cahill ◽  
Ian Denholm ◽  
Alan L. Devonshire
Keyword(s):  
Bemisia Tabaci ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Staining Intensity ◽  
Female Offspring ◽  
Polyacrylamide Gel ◽  
Microplate Assay ◽  
Toxicological Study ◽  
Single Insect

AbstractTwo acetylcholinesterase (AChE) variants, differing in sensitivity to inhibition by the organophosphorus (OP) insecticide paraoxon were identified in a population ofBemisia tabaci(Gennadius) from cotton in Israel using a single insect kinetic microplate assay. Two strains were established, homogeneous for one or other of the two variants, by isolating mated females from the field population onto individual cotton leaves, and testing a proportion of their female offspring to identify their AChE genotype. Polyacrylamide gel electrophoresis of their I-naphthyl butyrate hydrolyzing esterases showed that all insects contained esterase E0 14, which is indicative of B-type whiteflies, although the staining intensity of this band differed. Resistance to the OPs monocrotophos, profenofos and chlorpyrifos in leaf dip bioassays was consistent with the presence of the insensitive AChE. The data also indicated that separate mechanisms conferred resistance to the two pyrethroids cypermethrin and bifenthrin. The former, when used in a mixture with profenofos, was no more toxic than when the OP was used alone, and resistance to the mixture was largely dependent on the presence of the insensitive AChE.

Effects of sulphydryl reagents on the structure of dehistonized metaphase chromosomes

1985 ◽  
Vol 73 (1) ◽  
pp. 245-260
Author(s):  
P. Jeppesen ◽  
H. Morten
Keyword(s):  
Gel Electrophoresis ◽  
Heavy Metal Ions ◽  
Core Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Linking ◽  
Polypeptide Composition ◽  
Metaphase Chromosomes ◽  
Axial Structure

Dehistonized metaphase chromosomes lose their apparent axial organization (the ‘scaffold’) and sediment more slowly following exposure to beta-mercaptoethanol (BME). We have subsequently treated BME chromosomes with reagents that oxidize protein sulphydryls to disulphides, and found that if calcium is also present during the oxidation an apparently similar axial structure is restored following dehistonization, as seen by microscopic examination. In general, however, we do not find that oxidation restores the higher sedimentation rate of dehistonized control chromosomes. Analysis of residual core protein in dehistonized chromosomes by sodium dodecyl sulphate/polyacrylamide gel electrophoresis fails to detect any differences in polypeptide composition related to the state of oxidation or to the presence or absence of visible axial organization. Combining our results with those of other workers, we conclude that the axial structure evident in dehistonized metaphase chromosomes is maintained, at least partially, by inter-protein cross-linking, although in vivo this may not be via simple disulphide bridges. Additional factors, which we have not yet characterized, but which possibly include heavy metal ions, appear to be involved in the axial organization existing in vivo.

scholarly journals Conjugation of horseradish peroxidase to staphylococcal protein A with benzoquinone, glutaraldehyde, or periodate as cross-linking reagents.

1981 ◽  
Vol 29 (2) ◽  
pp. 266-270 ◽  
Author(s):  
H Nygren ◽  
H A Hansson
Keyword(s):  
Horseradish Peroxidase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Molecular Size ◽  
High Yield ◽  
Second Step ◽  
Cross Linking ◽  
Staphylococcal Protein A ◽  
Staphylococcal Protein

Horseradish peroxidase was conjugated to Staphylococcal protein A by three different two-step procedures using an increasing excess of peroxidase in the second step reaction. The yield of conjugated protein A was analyzed by SDS-polyacrylamide gel electrophoresis. Conjugation of peroxidase to protein A with benzoquinone or glutaraldehyde as cross-linking reagents at a 3- to 4-fold molar excess of peroxidase resulted in a high yield of coupled protein A with conjugates of low molecular size. Conjugation of peroxidase to protein A by the periodate method resulted in a high yield of coupled protein A with polymeric conjugates of large molecular size. Based on these results, conjugates produced with glutaraldehyde as cross-linking reagents were further analyzed. The capacity of the conjugates to precipitate human immunoglobulin evaluated by radial immunodiffusion was found to be reduced to about 50% of that of native protein A. Conjugates produced with glutaraldehyde as cross-linking reagent retained 70% of the enzyme activity of native peroxidase.

Soluble High-Molecular-Weight E Fragments in the Plasmin-Induced Degradation Products of Cross-Linked Human Fibrin

1975 ◽  
Vol 49 (2) ◽  
pp. 149-156 ◽  
Author(s):  
P. J. Gaffney ◽  
D. A. Lane ◽  
M. Brasher
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Electrophoresis ◽  
Factor Xiii ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Linking ◽  
D Dimer

1. The factor XIII-mediated cross-linked α chains in fibrin have no effect on the nature of the fragments released during the solubilization of fibrin by plasmin. 2. Besides the known D dimer and E fragments solubilized during the lysis of cross-linked fibrin, other fragments have been observed on sodium dodecyl sulphate-polyacrylamide gel electrophoresis which have a molecular weight of about 135 000. After prolonged plasmin digestion, these fragments (U fragments) were no longer evident on the gels and the high-molecular-weight E antigen was absent. It is assumed that the E antigen was associated with the U fragments. These fragments also cross-reacted with an anti-D serum. 3. The U fragments have been tentatively presumed to be a factor XIII-mediated cross-linked D–E complex since they degrade only after prolonged degradation with plasmin. Whereas it is known that the fibrin D dimer fragment contains the cross-linked γ chain residues of the originating fibrin, the presumed covalent cross-linking of the D–E fragments has not been proved. 4. The presence of these high-molecular-weight fragments, containing the E antigen, in cross-linked human fibrin digests should be taken into account in the development of D dimer assays to monitor fibrin lysis in vivo.

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