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PubMed1978

Cross-linking studies on the conformation and dimerization of myelin basic protein in solution.

Myelin basic protein was isolated from both cat and bovine central nervous system. Cat and bovine myelin basic protein, which are shown to be similar by tryptic mapping, exhibit identical behavior when cross-linked with the bifunctional reagent difluorodinitrobenzene. Myelin basic protein is cross-linked into only a dimer under certain conditions in the presence of sodium dodecyl sulfate. In contrast, many oligomers are formed when myelin basic protein is cross-linked in the absence of detergent. The formation of cross-linked dimers in the absence of other oligomer formation suggests that the protein is at least partly dimeric in the presence of sodium dodecyl sulfate. The conformation of them myelin basic protein monomer in sodium dodecyl sulfate was also studied. N-Bromosuccinimide and cyanogen bromide cleavage reactions were used to demonstrate that difluorodinitrobenzene had introduced intramolecular cross-links between the two peptides resulting from each of the cleavage ractions. However, these types of intramolecular cross-links cannot be detected under conditions in which only dimers have formed. Some of the lysine residues which are modified by difluorodinitrobenzene were identified by tryptic mapping. In several respects, the conformation of myelin basic protein in a sodium dodecyl sulfate solution appears to be similar to the conformation of the protein in the membrane.

Amino Acids↗
PubMed1977

Effect of nucleotide binding on the proximity of the essential sulfhydryl groups of myosin. Chemical probing of movement of residues during conformational transitions.

The reaction of myosin with three bifunctional sulfhydryl reagents of differing cross-linking span is reported. In the absence of nucleotide only p-N,N'-phenylenedimaleimide with a cross-linking span of 12-14 A can bridge between the two essential sulfhydryls of myosin. The other two reagents, 2,4-dinitro-1,5-difluorobenzene and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone with cross-linking spans of 3-5 and 7-10 A, respectively, react under identical conditions with the SH1 sulfhydryl but do not bridge to the SH2 group. In the presence of MgADP, both p-N,N'-phenylenedimaleimide and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone bridge across the SH1 and SH2 groups indicating a closer proximity of these two sulfhydryls in the presence of bound nucleotide. These results are discussed in relation to the conformational change induced in myosin by binding of the nucleotide.

Adenosine Diphosphate↗
PubMed1978

Cross-linking of nucleosomal histones with monofunctional imidoesters.

Cross-linking experiments with the MONOfunctional imidoester methyl-acetimidate, in the pH range 7.0 - 8.0, on rat liver nucleosomes generate a cross-linking pattern almost identical with the one observed for much longer BIfunctional reagents (e.g. dimethylsuberimidate). Combined cross-linking and trypsin digestion experiments suggest that all or at least the great majority of this cross-linking occurs on trypsin digestible segments (or "tails") of the histones. The formation of oligomers over such extremely short distances and especially the observation of an H3 homodimer suggests a very close proximity of half-nucleosomes.

Animals↗
PubMed1977

[Conjugation of bovine gammaglobulin to L-asparaginase (E. coli) (author's transl)].

Bovine gammaglobulin-L-asparaginase conjugates were prepared with an enzymic activity of about 30 IU/mg. Glutardialdehyde was used as bifunctional reagent for the cross-linking in an optimal concentration of 0.16 mg/10 mg protein. The both components were found in the conjugates in a ratio of 1:1.5. The recovery was about 25% of the initial protein amounts. The antigenic specificity of the globulin component decreased up to 60%, the enzymic activity also up to 60% by the conjugation.

Animals↗
PubMed1977

The cross-linking of rabbit skeletal muscle sarcoplasmic reticulum protein.

Sarcoplasmic reticulum proteins have been cross-linked in situ with two reagents, the disulphide-bridged bifunctional imido ester, dimethyl-3,3'-dithiobispropionimidate dihydrochloride and the mild oxidant cupric phenanthroline. Analysis of proteins so cross-linked by electrophoresis on agarose/acrylamide gels reveals that a series of new polypeptides, up to a molecular weight of 900 000, are formed. These have molecular weights which are multiples of 100 000. Further analysis of samples by electrophoresis in a second dimensions containing a reducing agent revealed the monomeric polypeptides from which the cross-linked polypeptides were formed. With dimethyl 3,3'-dithiobispropionimidate dihydrochloride homopolymers of the Ca2+-stimulated ATPase, calsequestrin and/or calcium binding protein were formed. With cupric phenanthroline only the Ca2+-stimulated ATPase was involved in polymer formation. It has been confirmed on another gel system that these two proteins which are involved in Ca2+ binding are not cross-linked intermolecularly with this latter reagent. We conclude that the 100 000 dalton Ca2+-stimulated ATPase polypeptides are within 2 A of each other in the membrane while calsequestrin and/or calcium binding protein are within 11 A of each other. Although there appears to be no limit to the extent of cross-linking of any of these polypeptides there is not indication of heteropolymer associations between them.

Adenosine Triphosphatases↗
PubMed1979

Structure and properties of the putrescine carbamoyltransferase of Streptococcus faecalis.

Ornithine and putrescine carbamoyltransferases from Streptococcus faecalis ATCC11700 have been purified and their structural properties compared. The molecular weight of native ornithine carbamoyltransferase, measured by molecular sieving, is 250 000. It is composed of six apparently identical subunits with a molecular weight of 39 000, as determined by cross-linking with the bifunctional reagent glutaraldehyde followed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Using the same method, putrescine carbamoyltransferase is a trimer of 140 000 consisting of three identical subunits with a molecular weight of 40 000. Ornithine carbamoyltransferase displays a narrow specificity towards its substrate, ornithine. In contrast, putrescine carbamoyltransferase carbamoylates ornithine and several diamines (diaminopropane, diaminohexane, spermine, spermidine, cadaverine) in addition to its preferred substrate, putrescine, but with a considerable lower efficiency than for putrescine. The kinetic mechanism of putrescine carbamoyltransferase has been investigated. Initial velocity studies yield intersecting plots using either putrescine or ornithine as substrate, indicating a sequential mechanism. The patterns of protection of the enzyme by the reactants during heat inactivation as well as the results of product and dead-end inhibition studies provide evidence for a random addition of the substrates. The putrescine inhibition that is induced by phosphate does, however, suggest that a preferred pathway exists in which carbamoylphosphate is the leading substrate. The different kinetic constants have been established. The properties of putrescine carbamoyltransferase are compared to the known properties of other carbamoyltransferases. The evolutionary implications of this comparison are discussed.

Carboxyl and Carbamoyl Transferases↗
PubMed1976

[Chemical modification of proteins. 2. Blocking of amino groups and basic amino acids and crosslinking of polypeptide chains in casein and field bean globulin by reaction with dialdehyde starch].

The reaction of dialdehyde starch with casein and field-bean globulin leads to a blocking of the protein amino groups and to a decrease of free lysine, arginine and histidine. Maximum values are reached at high protein concentrations and great molar reagent excess. At best 80-93% of the amino groups or of the available lysine may be blocked in this way. For 1% casein and I and 5% globulin solutions, the pH optimum of the reaction lies at approximately 8; for 5% casein solutions, it is shifted towards the neutral to weakly acidic range. The value for the proportion of unblocked lysine is higher (approximately 5%) when determined by amino-acid analysis after acid total hydrolysis than when measured by means of the colorimetric method according to Carpenter (20%). The difference is designed as reversibly blocked lysine proportion. There is a linear correlation between the proportion of blocked lysine and the relative nutritional value as determined by means of the test organism Tetrahymena pyriformis. Dependently on protein concentration and reagent excess, gel chromatographically detectable cross-linking products of higher molecular weight are formed by the reaction of dialdehyde starch with casein. In 5% protein solutions, such products with molecular weights of less than or equal to 900 000 are the sole detectable components.

Amino Acids↗
PubMed1979

Hydrolysis of p-NN'-phenylenebismaleimide and its adducts with cysteine. Implications for cross-linking of proteins.

To understand the extent of the cross-linking of proteins by the bifunctional reagent p-NN'-phenylenebismaleimide, a quantitative study of competing reactions has been undertaken. The two reactive maleimide rings of the bismaleimide are hydrolysed in mildly alkaline aqueous solutions much more rapidly than is the single maleimide ring of the monofunctional analogue N-ethylmaleimide. The kinetics of hydrolysis are second-order, depending on both imide and hydroxyl ion concentration in the pH range 8-10. The hydrolysis of the first imide ring of the bismaleimide is more rapid than the second, with second-order rate constants of 1600 M-1 . s-1 and 500 M-1 . s-1 respectively, at 25 degrees C. The half-times for hydrolysis of the first and second imide rings at pH 9.0 are therefore only 43s and 140s. Because it renders the maleimide ring unreactive towards cysteine, this rapid hydrolysis can limit the extent of cross-linking of proteins by the bismaleimide.

Chemical Phenomena↗
PubMed1979

Reversible inactivation of (Na+ + K+)-ATPase by use of a cleavable bifunctional reagent.

1. Purified (Na+ + K+)-ATPase, prepared from rabbit kidney outer medulla, is incubated with the bifunctional NH2-directed reagent dimethyl 3,3'-dithiobis-propionimidate. This results in a cross-link between the subunits of the enzyme and a simultaneous reduction of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 2. The most abundant cross-link product is a dimer of the two different subunits of the enzyme. 3. Reduction of the disulfide cross-link by dithioerythritol results in partial recovery of the original subunit structure of the enzyme and of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 4. These results suggest that a free mobility of the subunits of the (Na+ + K+)-ATPase system relative to each other is essential for proper functioning of both enzyme activities.

4-Nitrophenylphosphatase↗
PubMed1978

Stabilization of the relaxed state of aspartate transcarbamoylase by modification with a bifunctional reagent.

Native aspartate transcarbamoylase from Escherichia coli was modified with the bifunctional reagent tartaryl diazide in the presence of the substrate carbamoyl phosphate and the substrate analog succinate. The product had the same sedimentation coefficient as the native enzyme but showed a marked increase in affinity for the substrate aspartate with a hyperbolic saturation curve. The Michaelis constant for aspartate (7.4 mM) is similar to that estimated for the relaxed state of the enzyme. The high substrate affinity was not produced if modification was conducted in the absence of substrate analogs or with a monofunctional reagent. The modified enzyme was also desensitized towards the allosteric effectors ATP and CTP. It appears to represent a stabilized relaxed state whose conversion to the taut state is presumably prevented by cross-linking.

Aspartate Carbamoyltransferase↗
PubMed1977

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↗
PubMed1979

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↗
PubMed1979

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↗
PubMed1978

Functional consequences of tryptophan modification in human fibrinogen.

When human fibrinogen was modified with H2O2, inter- and intra-molecular cross-links of fibrinogen were formed, accompanied with oxidation of tryptophan, methionine and tyrosine residues. These cross-links may be closely associated with oxidation of tryptophan residues. The polymerization activity of fibrinogen with thrombin was decreased markedly by this modification. Modification of tryptophan residues in fibrinogen was also performed with 2-hydroxy-5-nitrobenzyl bromide. Modification of two out of a total 78 tryptophan residues in the molecule with the reagent led to the intensification (1.7 times) of the polymerization activity with thrombin and further modification of the next two residues led to complete loss of the polymerization activity. The first two tryptophan residues to be modified are in Fragment D, and the next two occur in Fragment E.

2-Hydroxy-5-nitrobenzyl Bromide↗
PubMed1978

The effects of deoxycholate and trypsin on the cross-linking of rabbit skeletal muscle sarcoplasmic reticulum proteins.

Dithiobis (succinimidyl propionate) has been used to cross-link sarcoplasmic reticulum microsome proteins. Although the 100,000 dalton calcium stimulated ATPase and the 60,000 dalton calcium-binding protein calsequestrin were readily cross-linked to form homopolymers, no heteropolymer formation between these two proteins were detected. The 90,000 dalton protein A1 which is always observed in our preparations appeared to preferrentially form dimers on cross-linking. When calsequestrin was solubilized using 0.1 mg deoxycholate/mg protein, this protein was not cross-linked even at dithiobis(succinimidyl propionate) concentrations ten times those used to cross-link this protein in the intact membrane. In a similar manner the deoxycholate-solubilized ATPase (0.5 mg deoxycholate/mg protein) was not cross-linked by dithiobis (succinimidyl propionate). These results suggest that the state of aggregation of the sarcoplasmic reticulum proteins may be modified when solubilized in detergents such as deoxycholate. When the 100,000 dalton ATPase polypeptide was cleaved with trypsin to two fragments with molecular weights of approximately 55,000, these could be readily cross-linked. The fragments were capable of forming polymers with either other 55,000 dalton fragments or with the 100,000 dalton ATPase. The 29,000 and 22,000 dalton fragments, produced by further tryptic cleavage of the 55,000 dalton fragments, were not cross-linked at dithiobis (succinimidyl propionate) concentrations which readily cross-linked the 55,000 dalton fragments. Thus tryptic cleavage of the ATPase to fragments smaller than 55,000 dalton altered associations made by the ATPase in the membrane.

Adenosine Triphosphatases↗
PubMed1977

Investigations on the binding sites of the basement membrane zone for pemphigoid antibodies in vitro. II. Immunohistochemical reactions of the reactive groups.

In continuing our studies on the characteristics of the antigenic structures of the basement membrane, procedures used in histochemistry for the identification and blocking of reactive groups have been adapted in this present work to the pretreatment of cryostat sections of guinea-pig tongue. These sections have then been studied by the indirect immunofluorescence method. By means or reagents, which produce a reversible extinction of the basement membrane fluorescence produced with pemphigoid sera, it proved possible to determine the existence of certain chemical groupings which bind the anti-basement membrane antibodies, namely: free carboxyl groups deriving from amino acids, hydrogen bridges in which the hydroxyl groups from the carbohydrates in the antigen are probably involved, bonds involving the free aldehydes of the antigen (either cross-links from aldehydes, or formation of Schiff's bases with amino groups of the antibodies), and lastly, to a lesser extent, disulphide bonds. The antigen/antibody reaction probably occurs in the neighbourhood of the alpha-glucosido-beta-galactosido-hydroxylysine. A reaction model has been developed from these findings. There were also some indications that the antigenic structures of the intercellular substance are chemically related to those of the basement membrane. The cell nucleus antigens, on the other hand, appear to possess different chemical properties.

Animals↗