In situ cross-linking of vesicular stomatitis virus proteins with reversible agents.
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Copper↗
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Results for “Cross-Linking Reagents”
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New protein reagents. N-(4-Chloromercuriphenyl)-4-chloro-3,5-dinitrobenzamide and its use as a probe of the quaternary structure of yeast alcohol dehydrogenase.
The new bifunctional reagent, N-(4-chloromercuriphenyl)-4-chloro-3,5-dinitrobenzamide (I) was used to investigate the quaternary structure of yeast alcohol dehydrogenase. The four essential - SH groups of the enzyme were substituted by the mercuriphenyl moiety of compound I in the course of the reaction of one mole of protein with four moles of the reagent (one molecule of compound I incorporated by yeast alcohol dehydrogenase monomer). In a second step only two of the four chlorodinitrophenyl fragments bound to the protein established intermonomeric cross-links with non-essential - NH2 groups. The resulting dimers could be re-dissociated with mercaptoethanol. This result suggests that the four protomers of the enzyme could be arranged as a dimer of dimers.
Alcohol Oxidoreductases↗
New immunolatex spheres: visual markers of antigens on lymphocytes for scanning electron microscopy.
New immunochemical reagents consisting of antibodies bound to small latex spheres were used as visual markers for the detection and localization of cell surface antigens by scanning electron microscopy. Cross-linked latex spheres of various sizes from 300 to 3,4000 A in diameter were synthesized by aqueous emulsion copolymerization of methacrylate derivatives containing hydroxyl and carboxyl functional groups. Proteins and other molecules containing primary amino groups were covalently bonded to the acrylic spheres under a variety of mild conditions by the aqueous carbodiimide, cyanogen bromide, and glutaraldehyde methods. For use in the indirect immunochemical-labeling technique, goat antibodies directed against rabbit immunoglobulins were bonded to the spheres. These immunolatex reagents were shown to bind only to cells (red blood and lymphocytes) which had previously been sensitized with rabbit antibodies against cell surface antigens. Mouse spleen lymphocytes with exposed immunoglobulins on their surface (B cells) were labeled with these spheres and distinguished from unlabeled or T lymphocytes by scanning electron microscopy. The distribution of Ig receptors on lymphocytes was also studied using the spheres as visual markers. When lymphocytes were fixed with glutaraldehyde and subsequently labeled with the immunolatex reagents, a random distribution was observed by scanning electron microscopy; a patchy distribution was observed when unfixed lymphocytes were used. These results are consistent with studies using ferritin-labeled antibodies (S. De Petris and M. Raff. 1973. Nature [Lond.]. 241:257.) and support the view that Ig receptors on lymphocytes undergo translational diffusion. In addition to serving as visual markers for scanning electron microscopy, these latex spheres tagged with fluorescent or radioactive molecules have applications as highly sensitive markers for fluorescent microscopy and as reagents for quantitative studies of cell surface antigens and other receptors.
Animals↗
On the specific association of porcine erythrocyte catalase caused by formation of disulfide cross-links.
Porcine erythrocyte catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) has been purified from porcine blood by DEAE-cellulose column chromatography, ammonium sulfate fractionation and CM-cellulose column chromatography. The purified enzyme was found to associate into larger molecules than the native one when it was stored at 4degrees C for more than one week. The associated molecules can be detected by gel filtration on a Bio-gel A-1.5 m column and disc gel electrophoresis as well as ultracentrifugal analysis. Molecular weights of the associated catalase molecules were about 500 000, 750 000, 1 000 000 and so forth estimated by gel filtration and disc gel electrophoresis, corresponding to dimer, trimer and tetramer respectively, of a native molecule (monomer) with a molecular weight of about 250 000. The association of catalase molecules is found to be time-dependent and to proceed seemingly from monomer through dimer as an intermediate. From the effects of several thiol reagents or reducing reagents on the association process and spectrophotometric titration of SH groups, it is inferred that this specific association of procine erythrocyte catalase is caused by formation of intermolecular disulfide cross-links due to air oxidation of SH groups in the protein moiety.
Animals↗
Cross-linking studies on a cytochrome c-cytochrome c oxidase complex.
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Binding Sites↗
Chemical cross-linking studies of beef heart mitochondrial coupling factor 1.
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Animals↗
Cross-linking of membrane phospholipid and protein using putative monofunctional imidoesters.
The reaction of methyl acetimidate or isethionyl acetimidate with mitoplasts at pH 8.5 yields two derivatives of phosphatidylethanolamine. These derivatives are shown to be the mono-amidine derivative and the bis-derivative of phosphatidylethanolamine. The bis-derivative represents one phosphatidylethanolamine cross-linked to another phosphatidylethanolamine. Similar derivatives are formed by the reaction of dipalmitoyl phosphatidylethanolamine with these imidoesters in organic solution with the exception that much more monoderivative is produced. Methyl picolinimidate reacts with phosphatidylethanolamine of mitoplasts to form primarily the mono-derivative. The bis-derivative was not detected. The reaction of bovine rod outer segment discs with methyl acetimidate causes cross-linking of 30% of the membrane rhodospin as dimers. Putative monofunctional imidoesters cause considerable cross-linking of both phospholipids and proteins in cell membranes. Cross-linking can be minimized at pH 9.0.
Animals↗
Studies with carbodiimide-cross-linked derivatives of bovine lutropin. II. Location of the cross-link and implication for interaction with the receptors in testes.
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Amino Acid Sequence↗
DNA repair and recovery in Escherichia coli after psoralen and angelicin photosensitization.
The correlation between DNA repair and recovery of biological functions was studied using three wild type strains of Escherichia coli and two skinphotosensitizing furocoumarins, psoralen and angelicin, which are well known specific reagents of the pyrimidine bases of DNA. In addition to mono-adducts psoralen is able to form a high number of inter-strand cross-links, while angelicin forms only mono-adducts. Both of these damages were repaired, in a short time, in the following way: at first DNA was cut into small pieces that were then rejoined into molecules of normal size, free from cross-links, while the furocoumarin residue was split from DNA almost quantitatively. Recovery of biological functions was studied performing photosensitization experiments in such a manner that the same amounts of psoralen or of angelicin were linked to bacterial DNA. DNA synthesis, tested just after the damage, was inhibited in a similar extent by both drugs. The same bacteria, however, showed a very different colony-forming capacity; angelicin was much less effective than psoralen with a D37 dose about 2.7 times higher. A similar picture was obtained studying DNA synthesis at different times after photosensitization: in the bacteria damaged by angelicin it was restored while no recovery was observed in cells photosensitized by psoralen. These results suggest that both mono-adducts and cross-links can be chemically repaired more or less in a quantitative measure, but that repair of cross-links in much less effective on cell recovery; this behaviour is very probably connected with the different repair mechanisms of mono-adducts and of cross-links.
Coumarins↗
Protein binding of coherin and other small molecules by thin-layer gel chromatography.
The protein binding stoichiometry of small molecules is here determined on a nanomole scale by a simplified procedure utilizing chromatography on thin layers of cross-linked dextran gels. New data are presented on the thin layer chromatographic properties of representative ligands, including a-amino acids, peptides, dyes and fluorigenic reagents, in relation to their molecular weights, polar characteristics, gel water regain values and denaturants, providing criteria for the general application of this method to studies of ligand binding with large as well as small molecules. By this procedure coherin peptides, A1 and B1--4, respectively, bind to coherin C in the molar ratio, 2:1, with a binding constant of about 10(5) M-1. Coherin C is believed to act as a carrier peptide.
Anilino Naphthalenesulfonates↗
Chemical composition and biophysical properties of porcine cardiovascular tissues.
Collagen from the cardiovascular system is extremely resistant to proteolytic digestion, in contrast with the proteoglycans of these connective tissues. In particular this is true for the heart valves, which contain the largest proportion of collagen. The tensile strength of aortic tissue incubated at 37 degrees C declines rapidly unless fixed with aldehydes. Stabilized glutaraldehyde gives rise to the most stable form of cross-link and renders the tissue structurally intact after more than 4 years of incubation at 37 degrees C in physiological solution. Formaldehyde, on the other hand, gives rise to transcient cross-links.
Animals↗
Nearest-neighbor interactions of the major RNA tumor virus glycoprotein on murine cell surfaces.
Formaldehyde-fixed Staphylococcus aureus and monospecific antiserum to gp70, the major envelope glycoprotein of murine leukemia virus, were used to immunoadsorb gp70 from Nonidet P40 extracts prepared from surface-radioiodinated murine cells. The labeled gp70 molecules in these cells were linked to a protein of approximately 15,000 daltons via native disulfide bonding. Prior treatment of cells with the reversible, bifunctional, crosslinking reagent dimethyl-3,3'-dithiobispropionimidate, followed by immunoadsorption and two-dimensional diagonal electrophoresis, revealed apparent homodimers and homotrimers of the 85,000-dalton complex. Identical treatment of purified type C RNA tumor virus from murine cells also revealed homodimeric and homotrimeric species, demonstrating similar self-associating tendencies of this glycoprotein in both intact virus and the plasma membrane of nonproducing murine cells. One cross-linked product consistently detected on the surfaces of murine cells was not present after crosslinking of a representative strain of murine leukemia virus.
Cells, Cultured↗
Two-step modification of aspartate aminotransferase with 1,5-difluoro-2,4-dinitrobenzene. Cross-link localization.
At pH 7, the apoenzyme of carboxymethylated and acylated aspartate aminotransferase reacts selectively with 1,5-difluoro-2,4-dinitrobenzene to form a single intramolecular covalent bond with the epsilon-amino group of the functional lysine residue located within the active centre. On shifting the pH to 9, the second fluorine atom of the bifunctional reagent is substituted with the sterically adjacent side groups of cysteine and tyrosine residues. The modified apoenzyme was subjected to partial proteolysis with pronase, and the digest was used to obtain and isolate the labeled products and to localize amino acid residues involved in the reaction. The established structures of several peptides containing Cys-2,4-dinitrobenzene-Lys and Tyr-2,4-dinitrobenzene-Lys allowed the identification of the amino acid residues involved in the reaction with the bifunctional reagent as Lys 258, Cys 390 and probably Tyr-70. The residues of Cys and Tyr are thus located at a distance of approximately 5 A (the length of the dinitrophenylene bridge) from the lysine residue forming an aldimine bond with pyridoxal 5'-phosphate in the active site.
Animals↗
In vitro phosphorylation of the erythropoietin receptor and an associated protein, pp130.
The cytoplasmic domain of the cloned erythropoietin (EPO) receptor (EPOR) contains no protein kinase motif, yet addition of EPO to EPO-responsive cells causes an increase in protein-tyrosine phosphorylation. Here we show that addition of EPO or interleukin-3 (IL-3) to an IL-3-dependent cell line expressing the wild-type EPOR causes a small fraction (less than 5%) of total cellular EPOR to shift in gel mobility from 66 to 72 kDa, due at least in part to phosphorylation. Using biotinylated EPO as an affinity reagent, we show that the 72-kDa species is greatly enriched on the cell surface. To demonstrate that a protein kinase activity associates with cell surface EPOR, cells were incubated with biotinylated EPO and then cross-linked with a thiol-cleavable chemical cross-linker. The avidin-agarose-selected complexes were incubated with [gamma-32P]ATP. After in vitro phosphorylation and denaturation without reducing agent, both antiphosphotyrosine and anti-EPOR antibodies immunoprecipitated labeled 72-kDa EPOR and an unidentified 130-kDa phosphoprotein (pp130), indicating that a protein kinase is associated with cell surface EPOR and that a fraction of the EPOR was phosphorylated on tyrosine residues either in the cells or during the cell-free phosphorylation reaction. Under reducing conditions, the 72-kDa phosphorylated EPOR but not pp130 was immunoprecipitated with an anti-EPOR antibody, suggesting that the pp130 is bound to the EPOR by the thiol-cleavable chemical cross-linker. Previously, we showed that deletion of the 42 carboxy-terminal amino acids of the EPOR allows cells to grow in 1/10 the normal EPO concentration, without affecting receptor number or affinity. Two carboxy-terminal truncated EPO receptors that are hyperresponsive to EPO were poorly phosphorylated during the in vitro reaction, suggesting that the carboxy-terminal region of the EPOR contains a site for phosphorylation or a site for interaction with a protein kinase. Our data suggests that phosphorylation or interaction with a protein kinase in the carboxy-terminal region may down-modulate the proliferative action of the EPOR.
Amino Acid Sequence↗
Nearest neighbor relationships of the polypeptides in ubiquinone cytochrome c reductase (complex III).
Ubiquinone cytochrome c reductase (complex III) in detergent dispersion has been cross-linked with two reversible cross-linking agents dithiobissuccinimidylpropionate and dimethyl-3.3'-dithiobispropionimidate and the cross-linked products formed have been analyzed by two-dimensional gel electrophoresis. Under mild reaction conditions, polypeptides I and II, II and VI, I and V, and VI and VII were the most prominent subunit pairs seen. With higher levels of reagent, larger aggregates were produced until an aggregate of apparent molecular weight 310 000 was the dominant band on gels. This is the complex III monomer.
Cytochrome Reductases↗
Localization of reducing group activity in the structural protein ribbons of the egg capsule of the gastropod mollusc Buccinum undatum L.
Egg capsules of the gastropod molluse Buccinum undatum L. show a staining reaction with silver-methenamine reagent with prior treatment with periodate. The reactivity is specifically localized on the striated structural protein ribbons which form the capsule walls. The staining is in the region of ribbon most resistant to chemical degradation and where the constituent protein monomers are believed to overlap and be cross-linked. Since the proteins contain little cysteine it is likely that the staining originates in aldehyde groups. Staining is abolished by borohydride reduction. It is suggested that the aldehyde groups are a locus of stabilization and cross-linking since previous work has also shown association of an aldehyde secretion with hardening of the capsules.
Aldehydes↗
The reactions of Escherichia coli citrate synthase with the sulfhydryl reagents 5,5'-dithiobis-(2-nitrobenzoic acid) and 4,4'-dithiodipyridine.
Citrate synthase of Escherichia coli reacts rapidly with 1 equivalent of Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), per subunit, losing completely its sensitivity to the allosteric inhibitor, NADH. When the enzyme is treated instead with 4,4'-dithiodipyridine (4,4'-PDS), all activity is lost. Certain evidence in this paper is consistent with the belief that the sulfhydryl group modified by DTNB, and that whose modification by 4,4'-PDS inactivates the enzyme, are the same. (i) Both reagents abolish NADH fluorescence enhancement by the enzyme. (ii) Saturating levels of NADH and some other adenylic acid derivatives inhibit the reactions with both reagents. (iii) When the enzyme is modified with one equivalent of DTNB or 4,4'-PDS, subsequent reactivity toward the other reagent is greatly decreased. (iv) Following modifications, the DTNB and 4,4'-PDS derivatives spontaneously lose thionitrobenzoate (TNB) or pyridine-4-thione (PT), respectively, in reactions which are thought to involve displacement of TNB or PT by a second enzyme sulfhydryl group, so that an enzyme disulfide is introduced. The introduction of the disulfide bond, if this is what occurs, does not lead to cross-linking of citrate synthase polypeptide chains, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis under nonreducing conditions. Certain evidence has also been found, however, that the sites of modification by DTNB and 4,4'-PDS are not the same. (i) DTNB modification desensitizes to NADH but does not inactivate, while 4,4'-PDS inactivates at least 99.9%. (ii) The presumed disulfide from elimination of TNB is also active, while that from PT modification is no more active than the original 4,4'-PDS modified product. (iii) Prior modification of the enzyme with DTNB affords no protection against later inactivation by 4,4'-PDS. The studies therefore indicate a close relationship between the DTNB desensitization and 4,4'-PDS inactivation, but they are unable to identify it exactly. Other properties of the DTNB reaction are also described, and a hypothesis is offered to explain quantitatively the finding that desensitization lags behind modification during the modification of citrate synthase by DTNB.
Binding Sites↗
Structure of cross-linked rabbit muscle phosphofructokinase in solution.
Cross-linked rabbit muscle phosphofructokinase in the active tetrameric and octameric state was studied in solution by hydrodynamic methods and small angle x-ray scattering techniques. The translational diffusion coefficients were determined by means of inelastic light scattering and were found to be 3.60 (+/- 0.02) x 10(-7) cm2 . s-1 for the tetramer and 2.54 (+/- 0.15) x 10(-7) cm2 . s-1 for the octamer. From small angle x-ray scattering measurements the radius of gyration, the specific inner surface area, and the volume were determined for both enzyme forms, revealing that the octameric cross-linked form is approximately spherical, with a diameter of 120.0 A, whereas the tetrameric form is asymmetric having an axial ratio of 2. By comparison of the scattering curves with triaxial geometric bodies which are equivalent in scattering, the tetrameric enzyme is described as a rectangular prism, with overall dimensions of A = 131.0 A, B = 131.0 A, and C = 65.0 A, and the octameric form as that of a cube with A = B = C = 120.0 A. The shape of the protomer, having a radius of gyration of 24.8 A, in the tetramer and octamer is similar to that for the native tetramer at pH 10 in the presence of 5 mM fructose 6-phosphate or 15 mM fructose 1,6-bis-phosphate. From the different shapes of the scattering curves of the native phosphofructokinase at pH 7.5 in the presence of 15 mM ATP and of the cross-linked tetramer or octamer, it can be inferred that the shapes of the protomers are different: in the presence of ATP the protomers are elongated, having an axial ratio of 1.8 to 2.0; the cross-linked state reveals a spherical protomer of radius 33.0 A, similar to that of the native enzyme at pH 7.5 in the presence of fructose 6-phosphate or fructose 1,6-bisphosphate.
Animals↗