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PubMed1978

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↗

PubMed1992

Movement of Cys-697 in myosin ATPase associated with ATP hydrolysis.

To detect movement of Cys-697 (SH2) in myosin subfragment-1 (S-1) associated with ATP hydrolysis, SH2 was labeled with the environmentally sensitive fluorescent analog of maleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS). Complex formation of S-1 labeled at Cys-697 with MIANS (MIANS-S-1) with adenyl-5'-yl imidodiphosphate and ADP resulted in a significant decrease in the fluorescence intensity of approximately 40 and 30%, respectively. When ATP was added to MIANS-S-1, the fluorescence intensity decreased rapidly by approximately 40%, and this fluorescence level was maintained during the steady state of ATP hydrolysis. As the substrate was used up, the fluorescence intensity increased to approximately 70% of the original value. These results together with model experiments with MIANS-N-acetylcysteine indicate that in the presence of ATP, the MIANS fluorophore attached to SH2 is located in a less hydrophobic environment than is the fluorophore in the absence of ligand and that the hydrolysis of ATP enhances hydrophobicity around the fluorophore. Acrylamide fluorescence quenching studies of MIANS-S-1 confirmed these results, indicating that addition of ATP and ADP to MIANS-S-1 results in an increase in the Stern-Volmer quenching constant of the fluorophore by factors of approximately 3 and 2.5, respectively. The present observations suggest that binding of ATP causes a movement of SH2 toward the protein surface, whereas it goes back into the protein interior after ATP hydrolysis. The results also confirmed previous observations by a chemical cross-linking approach (Hiratsuka, T. (1987) Biochemistry 26, 3168-3173).

Adenosine Diphosphate↗

PubMed1992

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↗

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↗

PubMed1992

Promoter melting and TFIID complexes on Drosophila genes in vivo.

In vivo UV cross-linking and nuclear transcriptional run-on experiments have shown that a number of Drosophila genes possess an elongationally paused RNA polymerase on their 5' ends. Here, we examine in vivo promoters that do and do not possess paused polymerases using the single-stranded DNA-probing reagent KMnO4. Melted DNA helices are found associated with the pause site of the uninduced hsp70 and hsp26 heat shock genes and the constitutively expressed beta-1 tubulin gene. The histone H1 and H2B genes, which lack a paused polymerase, have no comparable region of melted DNA. Melting at the pause site persists upon heat shock induction of the hsp70 and hsp26 genes, indicating that pausing continues after gene activation. Interestingly, activation triggers additional melting, both at the start site (in the region where open complexes would be expected to form) and downstream of the uninduced pause site. In the course of our studies, we discovered that some T residues of the TATA box were protected from KMnO4 modification in both induced and uninduced cells. This protection appears to be a consequence of TFIID binding, as a similar protection pattern could be produced in vitro with purified protein.

Animals↗

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↗

PubMed1977

Synthesis and characterization of new psoralen derivatives with superior photoreactivity with DNA and RNA.

The synthesis of five new psoralen derivatives is described. Three of these, 4'-hydroxymethyl-4,5',8-trimethylpsoralen, 4'-methoxymethyl-4,5',8-trimethylpsoralen, and 4'-aminomethyl-4,5',8-trimethylpsoralen hydrochloride, and characterized with respect to their photoreactivity with DNA and RNA. They are found to be greatly superior to 4,5',8-trimethylpsoralen and 8-methoxypsoralen, the two commonly used psoralens, in their abilities to saturate the photoreactive sites on DNA and RNA without repeated addition of reagent. A simplified mechanism for the photoreaction of psoralens with nucleic acids is presented and provides a basis for understanding the superior properties of these compounds. The compounds have superior reactivity not only with isolated DNA and RNA but also in viruses and in cells. Psoralens are shown for the first time to cross-link RNA double helices.

Binding Sites↗

PubMed1976

The role of 16S rRNA in ribosomal binding of IF-3.

The binding of initiation factor IF-3 to Escherichia coli 30S ribosomal subunits has been found to be inhibited by rRNA ligands such as ethidium bromide, polyamines, and monovalent alkali metals. The order of effectiveness of the polyamines (spermine greater than spermidine greater than putrescine) and alkali metals (Li+ greater than Na+ greater than K+) in inhibiting the ribosomal binding of IF-3 parallels their degree of affinity for the RNA. Furthermore, the binding of IF-3 to 30S subunits chemically modified by photooxidation with rose bengal, nitration with tetranitromethane, and reaction with kethoxal, monoperphthalic acid, and p-chloromercuribenzoic acid was studied. Results obtained after the direct treatment of the 30S subunits with the above chemical reagents or upon reconstitution of 30S particles having a modified rRNA or ribosomal proteins indicate that the IF-3 binding site is preferentially lost when the rRNA becomes modified. It was found that IF-3 could bind normally to 30S subunits lacking protein S1 or proteins S11, S12, S19, and S21 (and perhaps S14) which had been cross-linked to IF-3 in other laboratories.

Bacterial Proteins↗

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↗

PubMed1991

Oligonucleoside methylphosphonates as antisense reagents.

Oligonucleoside methylphosphonates contain nonionic internucleotide bonds that resist degradation by cellular nucleases and allow the oligomers to be taken up intact by mammalian cells in culture. Antisense methylphosphonate oligomers targeted against cellular or viral mRNA initiation codon or coding regions or against precursor mRNA splice sites effectively and specifically inhibit mRNA expression in cells. The efficacy of antisense methylphosphonate oligomers can be enhanced by derivatization with functional groups that allow the oligomer to covalently cross-link with its targeted mRNA. These oligonucleotide analogs will be useful tools for studying and controlling gene expression and are also promising candidates for development as therapeutic agents.

Animals↗

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↗

PubMed1992

Role of F0 and F1 subunits in the gating and coupling function of mitochondrial H(+)-ATP synthase. The effect of dithiol reagents.

A study is presented on the role of F0 and F1 subunits in oligomycin-sensitive H+ conduction and energy transfer reactions of bovine heart mitochondrial F0F1 H(+)-ATP synthase. Mild treatment with azodicarboxylic acid bis(dimethylamide) (diamide) enhanced oligomycin-sensitive H+ conduction in submitochondrial particles containing F1 attached to F0. This effect was associated with stimulation of the ATPase activity, with no effect on its inhibition by oligomycin, and depression of the 32Pi-ATP exchange. The stimulatory effect of diamide on H+ conduction decreased in particles from which F1 subunits were partially removed by urea. The stimulatory effect exerted by diamide in the submitochondrial particles with F1 attached to F0 was directly correlated with a decrease of the original electrophoretic bands of a subunit of F0 (F0I-PVP protein) and the gamma subunit of F1, with corresponding formation of their cross-linking product. In F0 liposomes, devoid of gamma subunit, diamide failed to stimulate H+ conduction and to cause disappearance of F0I-PVP protein, unless purified gamma subunit was added back. The addition to F0 liposomes of gamma subunit, but not that of alpha and beta subunits, caused per se inhibition of H+ conduction. It is concluded that F0I-PVP and gamma subunits are directly involved in the gate of the F0F1 H(+)-ATP synthase. Data are also presented indicating contribution to the gate of oligomycin-sensitivity conferral protein and of another protein subunit of F0, F6.

Adenosine Triphosphate↗

PubMed1976

Partial puridication of a membrane protein from human erythrocytes involved in glucose transport.

In an attempt to determine which membrane proteins are essential to the stereospecific uptake of D-glucose, isolated human erythrocyte membranes were exposed to a variety of reagents capable of selectively extracting various membrane proteins. These reagents included EDTA, lithium 3,5-diiodosalicylate, sodium iodide, and 2,3-dimethylmaleic anhydride. Selective elution of spectrin and Components 2.1, 2.2, 2.3, 4.1, 4.2, 5, and 6 representing 65% of the ghost protein has no effect on the uptake of D-glucose. All of the sugar transport proteins are associated with a membrane residue consisting of the proteins of Bands 3, 4.5, and 7, the periodic acid-Schiff-sensitive glycoproteins, and ghost phospholipids. Specific cross-linking of the proteins of Band 3 of ghosts by the catalyzed oxidation of intrinsic sulfhydryl groups with the o-phenanthroline-cupric ion complex inhibits D-glucose uptake and alters the relative electrophoretic mobility of Band 3 proteins in sodium dodecyl sulfate-polyacrylamide-agarose gels. This uptake activity and the relative mobility of Band 3 proteins are recovered upon reversal of the cross-linking reaction by reduction with 2-mercaptoethanol. These results and other observations indicate that the D-glucose transport protein is an intrinsic component of the hydrophobic structure of the erythrocyte membrane and may be associated with the proteins of Band 3 which are glycoproteins spanning the membrane bilayer. It is proposed that D-glucose transport occurs through a water-filled channel formed by specific subunit aggregates of the transport proteins in the erythrocyte membrane rather than by rotation of the protein within the plane of the membrane.

Biological Transport, Active↗

PubMed1979

The effect of chemical modification of 3-(3-amino-3-carboxypropyl)uridine on tRNA function.

The minor base 3-(3-amino-3-carboxypropyl)uridine (acp3U) in Escherichia coli tRNAPhe was acylated with the N-hydroxysuccinimide esters of acetic, phenoxy-acetic, and naphthoxyacetic acid, as well as the ester of 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-glycine. The derivatives of tRNAPhe formed were all capable of accepting phenylalanine. There were only minor effects on the kinetic parameters of these derivatives for E. coli phenylalanyl-tRNA synthetase. There was no effect on the ability of tRNAPhe to participate in poly(U)- or poly(ACU)-directed polypeptide synthesis or in the poly(U)-stimulated binding to E. coli ribosomes. The rate of photodynamic cross-linking of 4-Srd 8 to Cyd 13 was decreased in tRNAs containing the acetyl and dansyl-glycyl derivatives of acp3U, indicating that acylation of this base may perturb the tertiary structure of the tRNA. This base in tRNAPhe does not appear to play any role in the known biological functions of tRNAPhe.

Acylation↗