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PubMed1975

Structure and synthesis of a lipid-containing bacteriophage. Chemical modifications of bacteriophage PM2 and the resulting alterations in acyl-chain motion in the PM2 membrane.

The nucleocapsid proteins of bacteriophage PM2 and the inner lamella of the lipid bilayer, containing most of the phosphatidlethanolamine residues, were selectively cross-linked in the presence of 0.1-0.5% glutaraldehyde, 5 mM dimethylsuberimidate, or 0.05% toluene 2,4-diisocyanate. The biological activity (p.f.u.) of PM2 modified by these reagents decreased 10(6)-fold in all cases. The spike and coat proteins were selectively cross-linked in the presence of 7.5 mM N,N'-p-phenylenedimaleimide. The biological activity of virus modified by this reagen was unaffected. The electron paramagnetic resonance spectra of fatty acid spin labels incorporated into native and chemically modified viral membranes were qualitatively similar but show quantitative differences. Fixation with glutaraldehyde increased the rigidity of the membrane while Triton X-100 induced a more flexible structure. There was no change in the electron paramagnetic resonance spectrum of virus treated with N,N'-p-phenylenedimaleimide, however.

Bacteriophages↗
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↗
PubMedDate not supplied

T2 phage sensitization by linear and angular furocoumarins.

T2 bacteriophage sensitization has been studied using two furocoumarins capable of linking covalently to DNA to the same extent but producing different damages, psoralen and 4,5'-dimethylangelicin. Psoralen is a well-known linear furocoumarin capable of inducing in DNA both monoadducts and cross-links; 4,5'-dimethylangelicin is a new angular compound known as a pure monofunctional reagent. In the sensitization of T2 mature virions both drugs proved very active, yielding survival curves practically superimposable; on the contrary, in the experiments with the T2 vegetative form, i. e. its DNA inside the host, 4,5'-dimethylangelicin resulted much less effective, resembling the picture observed in the inactivation of the host bacteria. This result did not appear related to an enhancement of DNA repair by a Weigle effect. The different killing activity of 4,5'-dimethylangelicin can be explained supposing that this drug is capable of inducing cross-links in T2 DNA inside the virus core, in which it exists in a very folded form, but not in the same DNA after injection into the host bacteria.

DNA, Viral↗
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

Artificial hybrid protein containing a toxic protein fragment and a cell membrane receptor-binding moiety in a disulfide conjugate. I. Synthesis of diphtheria toxin fragment A-S-S-human placental lactogen with methyl-5-bromovalerimidate.

In order to study the mechanism of entry of plant seed and bacterial toxins into mammalian cells, methods have been developed to synthesize artificial protein hybrid conjugates containing a moiety which binds to a cell membrane receptor and an active fragment of a toxin protein. Utilizing methyl-5-bromovalerimidate, a disulfide cross-linked conjugate of human placental lactogen (hPL) and diphtheria toxin fragment A (toxin A) was synthesized. The reagent was prepared from 5-bromovaleryl nitrile by Pinner synthesis and then used to amidinate hPL. The bromo group thus introduced was converted to S-sulfonate by nucleophilic displacement with 1 M aqueous sodium thiosulfate at room temperature overnight. The S-sulfonated hPL reacted readily with the-SH gorup of reduced toxin A to form a 1 mol/mol of disulfide conjugate in high yield. Thus when reduced toxin A was incubated with a 4-fold excess of the hPL S-sulfonate at 4 degrees and pH 6.5 for 120 h, a conjugate yield of 50% relative to the toxin A input was obtained. Homopolymer formation was negligible and the product was purified by gel filtration on Sephadex G-150. Purity of the conjugate estimated by quantitative analysis of sodium dodecyl sulfate gels was 90%. The toxin A-hPL conjugate retained the activities of both toxin A and hPL, as reported in the accompanying paper. This method of preparing protein hybrid conjugates appeared to have advantages over previous methods utilizing bifunctional reagents with respect to both yield and freedom from homopolymer formation.

Animals↗
PubMed1977

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↗
PubMed1978

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↗
PubMed1977

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↗
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↗
PubMed1978

The denaturation of covalently inhibited swine pepsin.

Studies are reported on the denaturation of freshly prepared, intact swine pepsin, which was inactivated by reaction with diazoacetylglycine ethyl ester, to prevent autolysis. Denaturation about pH 6 was found to involve a small expansion of the molecular domain with some loss of organized secondary structure. On the other hand, increasing concentrations of guanidine hydrochloride induced cooperative transitions in both the native and alkali denatured forms to give a cross-linked random coil. No conditions could be found in which these reactions were reversible. Removal of denaturing conditions usually resulted in aggregation and precipitation of protein. From these studies, it would seem that the active conformation is largely predetermined in the zymogen.

Animals↗