Artifactual staining in polyacrylamide gels in the determination of the oligomeric state of proteins by dimethyl suberimidate cross-linking.
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Chemical Phenomena↗
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Cross-linking of Salmonella isopropylmalate synthesis with dimethyl suberimidate: evidence for antagonistic effects of leucine and acetyl-CoA on the guaternary structure.
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Acyltransferases↗
Physical and chemical properties of rabbit muscle phosphofructokinase cross-linked with dimethyl suberimidate.
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Adenosine Triphosphate↗
Identification of neighbouring proteins in the ribosomes of Escherichia coli. A topographical study with the cross-linking reagent dimethyl suberimidate.
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Ammonia↗
The influence of calcium ions on the conformation of fibrinogen fragment D: the use of chemical cross-linking agents.
Fibrinogen fragment D prepared in the presence of calcium ions (fragment D[Ca++]) shows qualitatively similar cross-linking patterns with dimethyl suberimidate, dimethyl adipimidate and tetranitromethane. Fragment D prepared in the presence of EDTA (fragment D[EDTA]) gives a consistently different pattern with these reagents. In the case of fragment D[EDTA] there is much more intermolecular cross-linking suggesting that the loss of the C-terminus of the gamma-chain remnant results in fragment D adopting a more open conformation. Neither the addition of 2M urea nor EDTA to fragment D[Ca++] alters its cross-linking pattern suggesting that the proposed conformational change follows cleavage of a plasmin susceptible bond which is normally protected by the presence of calcium ions.
Calcium↗
Macromolecular association of ADP-ribosyltransferase and its correlation with enzymic activity.
The macromolecular self-association of ADP-ribosyltransferase protein in solution was studied by several experimental techniques: quantitative gel filtration, electrophoretic analyses in non-denaturing gels, and cross-linking the enzyme protein with glutaraldehyde, dimethyl pimelimidate, dimethyl suberimidate, dimethyl 3,3'-dithiobisproprionimidate and tetranitromethane. The self-association of the polypeptide components obtained by plasmin digestion was also determined by using the above cross-linking agents. Monomers and cross-linked dimers of the enzyme protein, possessing enzymic activity, were separated in non-denaturing gels by electrophoresis. The basic polypeptide fragments, exhibiting molecular masses of 29 kDa and 36 kDa, self-associated, whereas the polypeptides with molecular masses of 56 kDa and 42 kDa associated only to a negligible extent, indicating that the peptide regions that also bind DNA and histones are probable sites of self-association in the intact enzyme molecule. Macromolecular association of the enzyme was indicated by a protein-concentration-dependent red-shift in protein fluorescence. The specific enzymic activity of the isolated ADP-ribosyltransferase depended on the concentration of the enzyme protein, and at 2.00 microM concentration the enzyme was self-inhibitory. Dilution of the enzyme protein to 30-40 nM resulted in a large increase in its specific activity. Further dilution to 1-3 nM coincided with a marked decrease of specific activity. Direct enzymic assays of electrophoretically separated monomers and cross-linked dimers demonstrated that the dimer appears to be the active molecular species that catalyses poly(ADP-ribose) synthesis. The NAD+ glycohydrolase activity of the enzyme was also dependent on protein concentration and was highest at 1-3 nM enzyme concentration, when polymerase activity was minimal, indicating that the monomeric enzyme behaved as a glycohydrolase, whereas poly(ADP-ribosyl)ation of enzyme molecules was maximal when the enzyme tends to be self-associated to the dimeric form.
Chromatography, Gel↗
Permeabilization of animal cells for kinetic studies of intracellular enzymes: in situ behavior of the glycolytic enzymes of erythrocytes.
Intracellular enzymes in erythrocytes can be made accessible for in situ kinetic studies by treating the cells with bifunctional reagents to crosslink proteins, thus creating a network that allows subsequent permeabilization by delipidation without escape of intracellular proteins. Dimethyl suberimidate, dimethyl 3,3'-dithiobispropionimidate, and toluene-2,4-diisocyanate have been used successfully as crosslinking reagents, and digitonin has been used for delipidation. In a systematic study of the in situ behavior of the 11 glycolytic enzymes of rat erythrocytes, it was observed that Km and Vmax values for the majority of the enzymes are essentially the same in situ as in vitro. Lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) is inhibited by excess of pyruvate as much in situ as in vitro. Hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was allosterically inhibited by glucose 6-phosphate nearly as much in situ as in vitro but was not affected by 2,3-biphosphoglycerate. The allosteric properties of 6-phosphofructokinase (ATP:D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11), glyceraldehyde-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12], and pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) in situ were qualitatively similar to those observed in vitro, but some important quantitative differences were noticed. Particularly striking was the much greater activity of phosphofructokinase in situ compared to that in vitro at physiological concentrations of effector metabolites.
Allosteric Regulation↗
Effect of cross-linking agents on insulin associated responses in adipocytes.
The effect of 10 bifunctional cross-linking agents and four monofunctional analogues was studied on isolated adipocytes. [125I]Insulin binding and degradation, basal and insulin-stimulated glucose oxidation, and 3-O-methyl glucose uptake were measured. Two cross-linkers, which possess succinimide ester residues (disuccinimidyl suberate and dithiobis(succinimidyl propionate)) and react selectively with amino groups, appeared to react relatively specifically with the insulin receptor. Both produced a slight stimulation of basal glucose transport and metabolism, a marked inhibition of insulin-stimulated glucose transport and metabolism, and a marked decrease in insulin binding. Pretreatment of cells with unlabelled insulin partially blocked the effect of disuccinimidyl suberate, and as has been previously shown, disuccinimidyl suberate cross-linked insulin to its receptor. A monofunctional analogue of these compounds was 100-fold less active in altering cellular metabolic activity. Bisimidates, such as dimethyl suberimidate, dimethyl adipimidate, and dimethyl dithiobispropionimidate, also react with free amino groups but are more hydrophilic. These agents produced similar effects on glucose oxidation as the succinimide esters, but had little or no effect on insulin binding. The effects of these agents are not blocked by insulin and they do not cross-link insulin to its receptor. Mixed bifunctional reagents containing either a succinimide ester or an imidate and a group which reacts with thiols produced effects similar to the cross-linkers containing two succinimide groups or bisimidates, respectively. The bifunctional arylating agents difluorodinitrobenzene and bis(fluoronitrophenyl)sulfone produce marked effects on insulin binding and glucose oxidation at micromolar concentrations, but the monofunctional analogue fluorodinitrobenzene is almost equally active suggesting that with these compounds chemical modifications and not cross-linking was important. With neither the mixed bifunctional reagents, nor the arylating agents, did insulin pretreatment alter the effect of cross-linker and none of these agents cross-linked [125I]insulin to its receptor. These data suggest that the insulin receptor possesses a free amino group in a hydrophobic environment in its active site. A reactive amino group in a hydrophilic environment as well as other reactive groups are also present in some component of the insulin receptor-effector complex. Chemical modification or cross-linking of these functional groups results in an inhibition or mimicking of insulin action. Further study will be required to identify the exact locus of these sites.
Adipose Tissue↗
Complex formation between flavodoxin and cytochrome c. Cross-linking studies.
Complex formation between Azotobacter vinelandii flavodoxin and horse cytochrome c has been demonstrated through cross-linking studies with dimethyl suberimidate, dimethyl adipimidate, 1-ethyl-3-(3-di-methylaminopropyl)carbodiimide, and dimethyl-3,3'-dithiobispropionimidate. Essentially quantitative cross-linking of cytochrome c and flavodoxin was observed at low ionic strengths with the carbodiimide cross-linking reagent. An association constant of 4 X 10(4) M-1 was obtained between cytochrome c and flavodoxin at 88 mM ionic strength from analysis of the cross-linking studies. This value is similar to the association constant determined kinetically during the electron transfer reaction between cytochrome c and flavodoxin (Simondsen, R.P., Weber, P.C., Salemme, F.R., and Tollin, G. (1982) Biochemistry 21, 6366-6375), and suggests that the cross-linked complex may be similar to the precursor complex identified kinetically. A structural model for the flavodoxin-cytochrome c complex proposed by these workers is shown to be compatible with the present cross-linking results.
Azotobacter↗
D-glucose metabolism in dimethyl suberimidate-treated tumoral pancreatic islet cells.
After protein cross-linking by dimethyl suberimidate, tumoral insulin-producing cells of the RINm5F line were either exposed to digitonin for measurement of hexokinase activity in the resulting cell pellet and supernatant, or incubated in the presence of D-[5-3H]glucose, D-[U-14C]glucose or L-[U-14C]glutamine to assess the metabolism of these nutrients. After digitonin treatment, the activity of hexokinase recovered in the cell pellet was about 40% higher in cross-linked than control RINm5F cells. Although failing to affect the metabolism of L-[U-14C]glutamine, and severely decreasing the oxidation of D-[U-14C]glucose, the cross-linking of proteins accentuated the increase in D-[5-3H]glucose utilization and D-[U-14C]glucose conversion to acidic metabolites resulting from a rise in hexose concentration from 2.8 to 16.7 mM. The latter change represents a mirror image of that previously found in cross-linked pancreatic islets. Taking into account the vastly different participation of glucokinase to hexose phosphorylation in RINm5F cells and normal islet cells, the present findings further support, therefore, the regulatory role of protein-to-protein interaction in the control of glucokinase catalytic activity in these fuel-sensing cells.
Animals↗
Crosslinking density and resorption of dimethyl suberimidate-treated collagen.
Collagen was purified from bovine Achilles tendon and crosslinked with dimethyl suberimidate (DMS) and glutaraldehyde (GTA). Under optimal conditions, the shrinkage temperature (Ts) was raised to 74 degrees C for collagen crosslinked with DMS and to 80 degrees C for those crosslinked with GTA. Crosslinking density measurements were done on the hydrothermally denatured collagen by the method based on the Flory-Rehner equation. GTA treatment was found to introduce more number of crosslinks than DMS. The maximum tension attained during heating (after shrinkage has occurred) was greater for GTA-treated collagen than for DMS and control. The control collagen membranes broke during heating (at 73 degrees C), while for the crosslinked membranes the tension kept on increasing up to 100 degrees C. The crosslinking density correlated well with the data determined from the in vitro and in vivo degradation studies. Uncrosslinked and DMS crosslinked collagen membranes were more susceptible to degradation by enzymes in vitro, while GTA-treated collagen was highly resistant to degradation. The biocompatibility of the collagen membranes was studied by subcutaneous implantation in rats. Uncrosslinked collagen membranes degraded within 14 days with the formation of granulation tissue. DMS crosslinked membranes degraded within 21 days and the area was replaced by numerous fibroblasts and newly formed collagen. No calcification was observed. For GTA-treated membranes, necrosis was observed after 7 days implantation and by 14 days the membrane had started to calcify.
Animals↗
Cross-linked informofers.
The proteins of 30S RNP particles containing pre-mRNA (hnRNA) were cross-linked with bifunctional reagents (dimethyl-suberimidate and dimethyl-3,3'-dithiobispropionimidate). Further treatment with 1 or 2 M NaCl dissociates all RNA from protein. However, a significant part of protein particles--informofers being cross-linked survived high salt treatment. Their sedimentation coefficients were close to those of original particles. No RNA could be detected in the informofers even after labeling the cells with a precursor for a long period of time. Sodium dodecylsulfate or urea dissociated cross-linked informofers into oligomeric polypeptides. They could be dissociated by beta-mercaptoethanol treatment if a reversible cross-linked reagent had been used. The resulting polypeptides were represented by informatin. RNP particles (30S RNP or poly-particles) were reconstituted upon mixing of cross-linked informofers with pre-mRNA and removal of 2 M NaCl.
Animals↗
Cross-linking of the enzymes in the glycosome of Trypanosoma brucei.
Glycosomes, the microbody-like organelles containing mainly glycolytic enzymes, were purified from the long slender bloodstream form of Trypanosoma brucei EATRO 110 monomorphic strain by an improved method in which the protozoa were frozen and thawed in 15% glycerol to free, from the plasma membrane, much of the variant surface glycoprotein which used to constitute the major contaminant of our purified glycosomes. The purified glycosomes have 11 major proteins, 6 of which, tentatively identified as phosphofructose kinase, hexokinase, 3-phosphoglycerate kinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and alpha-glycerophosphate dehydrogenase, constitute 87% of the total glycosomal protein. The bifunctional cross-linking reagents dimethyl suberimidate and dimethyl-3,3'-dithiobispropionimidate can penetrate the glycosomal membrane and cause extensive cross-linking of all the major glycosomal proteins. The cross-linked complex, insoluble in 0.1% Triton X-100 plus 0.15 M NaCl, contains all the glycosomal enzyme activities with only partial inactivations. All the enzymes are probably cross-linked into one large complex since they all sediment rapidly to the bottom of a 5-20% (v/v) sucrose density gradient. This successful cross-linking with reagents of span lengths of 11-12 A suggests close proximities among the glycosomal enzymes which may explain the extraordinarily high rate of glycolysis in T. brucei. Whether such a close association represents specific spatial arrangement required for genuine substrate channeling among the enzymes will be verified by future kinetic studies of the cross-linked enzyme complex.
Animals↗
The effects of fixation by combination of glutaraldehyde/dimethyl suberimidate. Use of collagen as a model system.
The fixative behavior of glutaraldehyde/dimethyl suberimidate in combination was studied using reconstituted fibrils of type I calf skin collagen as a model system and comparing electron-optical data and chemical data by a computer-aided correlation procedure. The results confirm that the structural preservation of collagen obtained using prefixation in glutaraldehyde followed by dimethyl suberimidate fixation is less than when using dimethyl suberimidate and postfixation in glutaraldehyde. Furthermore, glutaraldehyde preserves the structure of collagen to a lesser degree than dimethyl suberimidate. With the combination of the two fixatives, the preservation of fine structure of collagen obtained is less than that when using dimethyl suberimidate alone.
Aldehydes↗
Effects of amidination and chemical cross-linking on human factor VIII (antihemophilic factor).
The bifunctional reagent dimethyl suberimidate, reacting with primary amino groups of proteins, was used to cross-link highly purified human factor VIII. Reaction products were reduced with beta-mercaptoethanol or treated with Rhizopus arrhizus triglyceride lipase. The proportions of the dissociated subunits and their oligomers were calculated from the relative staining intensities of individual bands following polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. Low concentrations of dimethyl suberimidate (up to 0.5 mM) produced covalently linked dimers which retained full functional (coagulant and von Willebrand factor) activities. Treatment with increasing concentrations of dimethyl suberimidate resulted in an almost simultaneous appearance of both trimeric and tetrameric species, suggesting the existence of specific intra-dimer contacts. A parallel decrease of functional activities was observed at higher concentrations of dimethyl suberimidate. A monofunctional reagent (ethyl acetimidate), reacting similarly with primary amino groups, amidinated factor VIII at rates similar to dimethyl suberimidate. Up to 40% amidinated factor VIII retained full biological activities. We conclude that the most reactive lysine residues are not involved in the active sites responsible for either coagulant or von Willebrand activity.
Blood Coagulation↗
Small subunit contacts in ribulose-1,5-bisphosphate carboxylase.
The arrangement of subunits of ribulosebisphosphate carboxylase in solution has been studied by exposing the enzyme to the cross-linking agents tetranitromethane, dimethyl suberimidate, and dimethyl adipimidate, and the cleavable cross-linking agent, methyl 4-mercaptobutyrimidate followed by gel electrophoresis in the presence of dodecyl sulfate. All these agents caused the formation of dimers of the enzyme's small subunit, independently of protein concentration. In addition, trimers and tetramers of small subunit were detected in the mercaptobutyrimidate-treated enzyme. The data show that small subunits are closely paired in the native enzyme and may be in layers of four, or a ring of eight.
Carboxy-Lyases↗
Investigation of the quaternary structure of Neurospora pyruvate kinase by cross-linking with bifunctional reagents: the effect of substrates and allosteric ligands.
Pyruvate kinase (EC 2.7.1.40) of Neurospora, a tetramer composed of apparently identical subunits, has been shown to be a dimer of dimers by interprotomeric cross-linking experiments in which bifunctional reagents were used. An analysis of the polyacrylamide gel profiles of the enzyme after cross-linking with glutaraldehyde, dimethyl suberimidate, and dimethyl adipimidate shows that the extent of intersubunit cross-linking is influenced markedly by the ligand bound to the enzyme. Bifunctional cross-linking reagents with a shorter distance between the two functional groups form cross-links effectively in the unliganded enzyme. In the FDP-pyruvate kinase complex, cross-linking was observed over longer distances compared with the unliganded enzyme. It is demonstrated that covalent cross-linkers cah be used as sensitive indicators of conformational changes induced in pyruvate kinase by substrates and allosteric ligands.
Adipates↗
Ca2+-induced conformational changes in the troponin complex detected by crosslinking.
In an attempt to detect Ca2+-induced conformational changes by crosslinking, rabbit muscle troponin complex was reacted with the bifunctional reagents 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrodiphenylsulfone and dimethyl suberimidate under various conditions and the products were analyzed by dodecyl sulfate gel electrophoresis. In the absence of divalent cations, with the two aromatic reagents at a low reagent/protein ratio, the main cross-link products were troponin T-I and I-C. With dimethyl suberimidate the only major crosslink product was conjugate T-I. Ca2+, alone as well as in the presence of Mg2+, prevented the formation of I-C crosslinks with both aromatic reagents, but it did not affect crosslinking with dimethyl suberimidate. Ca2+ also decreased the number of NH2 groups of troponin that are highly reactive towards 2,4,6-trinitrobenzene sulfonate. Both effects of Ca2+ can be interpreted in terms of a conformational change in the troponin complex elicited by the binding of the specific divalent cation.
Animals↗