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At least 253 records · Page 14Linked to original sources
PubMed1986

Chemical modification of alpha 2-adrenoceptors. Possible role for tyrosine in the ligand binding site.

Tetranitromethane (TNM) is a reagent which reacts with the tyrosine and cysteine residues of proteins. Chemical modification of partially purified human platelet alpha 2-adrenoceptors with TNM resulted in an irreversible loss of binding activity. Typically, an 80-90% decrease in binding activity occurred with a 60-min exposure to 320 microM TNM. The loss of alpha 2-adrenoceptor activity caused by TNM could be prevented if alpha 2-adrenergic ligands were present during exposure of the receptor to TNM. The protection afforded by alpha 2-adrenergic ligands was dose-dependent and showed a positive correlation with the affinity of the ligand for the alpha 2-adrenoceptor. Prazosin, an alpha 1-specific antagonist, and propranolol, a beta-adrenergic antagonist, did not protect alpha 2-adrenoceptors against the inactivation caused by TNM. Saturation curve analysis revealed that the decrease in alpha 2-adrenoceptor activity caused by TNM was due to a decrease in Bmax with no change in Kd. alpha 2-Adrenoceptors were also inactivated with the sulfhydryl-specific reagent phenylmercuric chloride (PMC). The receptor inactivation caused by PMC could be reversed completely by subsequent treatment with dithiothreitol. Treatment of alpha 2-adrenoceptors with combinations of TNM and PMC showed that the receptor inactivation caused by TNM was most likely due to an interaction with tyrosine residues. These results indicate that tyrosine residues have a function in the conformational stability of alpha 2-adrenoceptors and may be directly involved with ligand binding to the receptor.

Binding Sites↗
PubMed1983

Identification of the ligand trans to thiolate in cytochrome P-450 LM2 by chemical modification.

About 3 tyrosine residues of cytochrome P-450 LM2 are accessible to chemical modification with tetranitromethane. Nitration of two tyrosines inactivates the enzyme to about 20%. The partial formation of a hyper-porphyrin spectrum originating from the pK shift by nitration and formation of a tyrosinate is prevented by modification in the presence of the inhibitor metyrapone. These findings support the assumption of a tyrosine residue as sixth ligand of the heme iron in cytochrome P-450 LM2.

Animals↗
PubMed1985

Clostridium pasteurianum glutamine synthetase mechanism. Evidence for active site tyrosine residues.

Preliminary chemical modification studies indicated the presence of tyrosine, carboxyl, arginine, histidine and the absence of serine and sulfhydryl residues at or near the active site of Clostridium pasteurianum glutamine synthetase. The conditions for tyrosine modification with tetranitromethane were optimized. The inactivation kinetics follow pseudo-first-order kinetics with respect to enzyme and second order with respect to modifier per active site. There was no inactivation at pH 6.5 suggesting the absence of thiol oxidation. The synthetase and transferase reactions followed the same pattern of inactivation on enzyme modification and both were equally protected by glutamate plus ATP. Thus tyrosine residues are present at the active site of the enzyme and are essential for both transferase and synthetase activities.

Binding Sites↗
PubMed1992

Identification of reactive tyrosine residues in cysteine-reactive dehydrogenases. Differences between liver sorbitol, liver alcohol and Drosophila alcohol dehydrogenases.

Modification of tyrosine residues with tetranitromethane and reversible sulphite protection of cysteine residues were tested on three dehydrogenases of two families. In liver alcohol dehydrogenase no Tyr residue is appreciably labelled, while in the homologous sorbitol dehydrogenase Tyr-109 is specifically labelled; the difference corresponds to a segment correlating with subunit interactions and the different quaternary structures of the proteins. In Drosophila alcohol dehydrogenase, Tyr modification is multiple, and the results show the presence of two different states of Cys residues, reactive in the presence and absence of cupric ions, respectively. Super-activation with cyanide was also noticed after S-sulphocysteine protection. The results demonstrate the possibility of identification of specific Tyr residues in proteins with reversibly protected Cys residues.

Alcohol Dehydrogenase↗
PubMed1992

Cross-linking of apolipoproteins is involved in a loss of the ligand activity of high density lipoprotein upon Cu(2+)-mediated oxidation.

A recent study demonstrated that Cu(2+)-mediated oxidation of high density lipoprotein (HDL) resulted in a loss of the capacity to reduce cholesterol from macrophage foam cells [(1991) Proc. Natl. Acad. Sci. USA 88, 6457-6461]. In the present study we characterized the physicochemical properties of oxidized HDL and correlated them with the ligand activity toward the HDL receptor. Among them, the cross-linking of apolipoproteins and an increase in lipid peroxides were characteristic and closely similar to those of tetranitromethane-treated HDL, an abortive ligand for the HDL receptor. Cellular experiments with murine peritoneal macrophages revealed that both the cellular binding activity of HDL and its capacity to enhance cholesterol efflux from macrophage foam cells were markedly reduced upon oxidation. These results suggest that cross-linking of HDL apolipoproteins is involved in the loss of the ligand activity of oxidized HDL.

Animals↗
PubMed1996

An essential tyrosine residue of Aspergillus polygalacturonase.

Based on strict conservation of a tyrosine residue in 24 polygalacturonases, tyrosine modification was assessed in two different forms of the Aspergillus enzyme. The second subform was unknown in structure but submitted to sequence analysis and was found also to have the conserved tyrosine residue. Results of chemical modifications are consistent in showing inactivation of the proteins with all tyrosine-reactive agents tested, acetic anhydride, N-acetyl imidazole, and tetranitromethane. Furthermore, after acetylation, regeneration of enzyme activity was possible with hydroxylamine. Spectrophotometric pH titration showed that one accessible tyrosine residue is ionized at pH 9.3-9.5, whereas the remaining, masked residues are all ionized at pH 10.5. It is concluded that one tyrosine residue is catalytically important, in agreement with the inactivation and reactivation data, that this residue is accessible, and that it is likely to correspond to the strictly conserved residue observed in all forms.

Acetic Anhydrides↗
PubMed1984

Involvement of some amino acid residues in the enzymatic activity of solubilized cerebral fucosyltransferase.

We have checked the effect of some chemical reagents specific for amino acid residues on the activity of a solubilized cerebral glycoprotein:fucosyltransferase. Diethylpyrocarbonate, 2,3-butanedione and tetranitromethane specific for histidyl, arginyl, and tyrosyl residues respectively, were strong inhibitors of the enzymatic activity This led us to conclude that these amino acid residues are "essential residues" in the cerebral fucosyltransferase activity.

Animals↗
PubMed1992

Binding of modified high density lipoproteins to endothelial cells: relation with cellular cholesterol efflux?

Human endothelial cells (EA.hy 926 line) were enriched with cholesterol using cationized low density lipoprotein (LDL). Cholesterol-loaded cells interacted with native apolipoprotein (apo) E-free high density lipoprotein3 (HDL)3 as well as with dimethyl suberimidate-modified HDL3 (DMS-HDL3). At 4 degrees C both HDL preparations showed a saturable high affinity binding with a KD of 31 and 50 micrograms of protein/ml and a Bmax of 226 and 436 ng/mg cell protein for native HDL3 and DMS-HDL3 particles, respectively. Competition of binding of 5 micrograms apo E-free 125I-labelled HDL3/ml by unlabelled DMS-HDL3 and tetranitromethane-treated HDL3 (TNM-HDL3) was very poor, whereas unlabelled native HDL3 competed very effectively with 125I-labelled HDL3 binding. Thus, both types of modified HDL did not compete for the high affinity binding sites for native HDL. Unlabelled native HDL3 and unlabelled DMS-HDL3 both competed for the binding of 125I-labelled DMS-HDL3 very effectively. These experiments indicate that there are two distinct high affinity binding sites for HDL on cationized LDL-loaded EA.hy 926 cells: one specific HDL binding site, which only binds native HDL, and a second binding site for both native HDL and DMS-HDL. The modified HDL fractions were used to study the relation between HDL binding and HDL-mediated efflux. Efflux of cell cholesterol was measured as the increase of cholesterol mass in the medium after 24 h of incubation with 0.2 mg native HDL3/ml, or the same amount of modified HDL3. DMS-HDL3-mediated efflux was identical to efflux mediated by native HDL3. TNM-HDL3 also induced efflux of cell cholesterol; however, efflux induced by TNM-HDL3 was only 45-50% of the amount obtained with native HDL3. So both DMS- and TNM-modified HDL3 induced efflux of cholesterol, although these particles do not bind to the specific high affinity sites for native HDL. These results do not indicate a link between binding of HDL to specific receptors for native HDL and HDL-mediated efflux of cholesterol from loaded endothelial cells.

Cell Line↗
PubMed1983

Structure and inherent properties of the bacteriophage lambda head shell. III. Spectroscopic studies on the expansion of the prohead.

The head shell of bacteriophage lambda expands by about 20% in diameter when it packages the DNA molecule in vivo. The expansion reaction is essentially a conformational change of the major head protein molecules to a state of lower free energy and can also be triggered in vitro by treatment with 4 M-urea. In order to investigate the conformational change, we have measured the circular dichroism, fluorescence and difference absorption spectra of the lambda head shell before and after the expansion by the treatment with urea. The far-ultraviolet circular dichroism spectra and the fluorescence spectra show that the expansion is not accompanied by a great change in the secondary structure (29% alpha-helix, 23% beta-structure) and the environment (non-polar) of the tryptophan residues of the major head protein molecule. On the other hand, by measurements of the circular dichroism and difference absorption spectra in the near-ultraviolet region as well as by chemical modification experiments with tetranitromethane, we have found that one or two tyrosine residues of the major head protein are transferred from a polar, solvent-exposed to a non-polar, solvent-unexposed environment during the expansion. Judging from these results, the conformational change seems to be mainly intermolecular or interdomainal rather than intradomainal.

Bacteriophage lambda↗
PubMed1982

The role of tyrosine in the binding of steroid by progesterone binding globulin from the guinea pig.

Treatment of progesterone binding globulin (PBG) with tetranitromethane (TNM) resulted in a loss of steroid binding activity (inactivation) which was dependent on both time and concentration of reagent. Scatchard analysis of binding revealed that inactivation was due to a decrease in binding site number with no effect upon the affinity of PBG for steroid. Incorporation studies demonstrated that the loss of binding activity correlated with the incorporation of 1.3 nitro groups per molecule of PBG. The involvement of the steroid binding site in the reaction was shown by the ability of progesterone, but not cortisol, to protect against inactivation. Treatment with N-acetylimidazole did not inactivate PBG nor did the conversion of nitrotyrosyl residues to amino-tyrosines regenerate binding activity, suggesting that the pheolic hydroxyl is not involved in steroid binding. These studies suggest that inactivation was due to the incorporation of a bulky group into the aromatic ring of a tyrosine present at the steroid binding site thus blocking its ability to participate in hydrophobic interactions with the ligand.

Alpha-Globulins↗
PubMed1990

Reaction of tyrosyl-modifying reagents with the ligand- and DNA-binding domains of the rabbit liver glucocorticoid receptor.

We have studied the effects of p-nitrobenzenesulfonyl fluoride, 4-fluorosulfonyl-1-hydroxy-2-naphtoic acid, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole and tetranitromethane on the glucocorticoid receptor from rabbit liver. Our results show that all tyrosine modifying reagents inhibit the binding of [3H]dexamethasone to the receptor. Equilibrium binding experiments revealed that only 4-fluorosulfonyl-1-hydroxy-2-naphtoic acid is a competitive inhibitor while the other chemical probes decrease the concentration of binding sites. Transformation of glucocorticoid-receptor complexes was markedly reduced when heat treatment was performed in the presence of tyrosyl-directed reagents. Taken together, these results indicate for the first time that critical tyrosyl moieties may be involved in both hormone binding and transformation of the glucocorticoid receptor.

4-Chloro-7-nitrobenzofurazan↗
PubMed1982

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↗
PubMed1979

Nitration of polypeptides using ethanol in reaction buffers minimizes crosslinking.

High concentrations of ethanol are effective in reducing the intermolecular cross-linking which predominates when the reactions of tetranitromethane with the carboxypeptidase inhibitor from potatoes, a chymotrypsin inhibitor also from potatoes, and insulin are carried out under standard conditions. Including reagents such as ethanol may be of general utility in the preparation of monomeric nitro-derivatives of hydrophobic, low-molecular-weight proteins.

Buffers↗
PubMed1982

Specific chemical modification of the readily nitrated tyrosine of the RTEM beta-lactamase and of bacillus cereus beta-lactamase I. The role of the tyrosine in beta-lactamase catalysis.

The function of the hydroxyl group of the tyrosine residue readily nitrated by tetranitromethane (tyrosine-105) in the RTEM plasmid-derived beta-lactamase (penicillinase; penicillin amido beta-lactam-hydrolase, EC 3.5.1.6) from E. coli and in Bacillus cereus beta-lactamase I has been investigated by chemical modification methods. In the case of B. cereus beta-lactamase I the nitrated tyrosine can be acetylated by acetic anhydride without effect on beta-lactamase activity The nitrated tyrosine of the E. coli enzyme can also be acetylated but in this case beta-lactamase activity is lost in a manner which directly correlates with extent of acetylation. However, deacetylation of the nitrotyrosine does not restore activity. The dilemma created by the latter result has been resolved by development of a new method of tyrosine hydroxyl modification at low pH. The nitrated enzyme is reduced by dithionite and then treated with either carbonyldiimidazole or N-(2.2.2-trifluoroethoxycarbonyl)imidazole, both of which convert 3-aminotyrosine into benzoxazolinonylalanine. That the final modification has been achieved is demonstrated both by classical chemical methods and by employment of Fourier transform infrared spectroscopy to detect the characteristic benzoxazolinone carbonyl absorption. Further, it is shown that no significant loss of beta-lactamase activity is associated with this modification. Hence in neither the B. cereus or the E. coli enzyme does the readily nitrated tyrosine residue have a direct chemical function at the beta-lactamase active site.

Acetylation↗
PubMed1982

Modification of tyrosine residues in dihydrofolate reductase from Lactobacillus casei.

Dihydrofolate reductase from Lactobacillus casei was inactivated by reaction with tetranitromethane and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Loss of activity occurred with modification of four of the five tyrosine residues present in the enzyme. The presence of either substrate, NADPH or 7,8-dihydrofolate, as well as NADP and folate, provided extensive protection against inactivation, while NADH and tetrahydrofolate exhibited none. This protection from inactivation occurred on protection of two of the four susceptible tyrosines from modification. Nitration of the enzyme adversely affected its ability to bind substrates. Restoration of the pKa of the nitrated tyrosines by reduction of the nitro group to an amino group did not result in a regeneration of enzymatic activity. However, fluorotyrosine-containing enzyme, prepared by growing the bacterium in the presence of fluorotyrosine, exhibited specific activity identical to that of native enzyme over the pH range of 4.5-8. These results suggest that inactivation of dihydrofolate reductase by tyrosine modification occurs primarily due to a steric effect and that the active site tyrosines may participate in substrate binding.

4-Chloro-7-nitrobenzofurazan↗
PubMed1984

Implication of a tyrosine residue in the unspecific bile salt binding site of human pancreatic carboxylic ester hydrolase.

Tyrosine residues of the human pancreatic carboxylic-ester hydrolase (EC 3.1.1.1) (also referred to as cholesterol-ester hydrolase, EC 3.1.1.13) were nitrated in the ortho-position by the use of tetranitromethane. The specificity of the reaction has been verified and the inhibition observed was shown to be unrelated to the weak polymerization of the protein. Among the 27 tyrosines present in the enzyme, seven or eight were nitrated but only one residue, with a pK of 8.3, seems to be responsible for the loss of activity. This decrease in enzyme activity appears only in assays which were performed in the presence of bile salts, suggesting that of the two bile salt binding sites postulated on the enzyme, only one, referred to the as the 'unspecific site' (Lombardo, D. and Guy, O. (1980) Biochim. Act 611, 147-155), was modified. This is in agreement with the similar loss of enzyme activity observed on emulsified and soluble substrate. The most important result is the difference observed in experiments of the protective effects of bile salts. The protection with sodium taurodeoxycholate is independent of its critical micellar concentration, showing that monomers protect this site, whereas the protection observed in experiments with sodium cholate appears only for supramicellar concentrations of bile salt. Since this latter bile salt promotes the dimerization of the enzyme, we can conclude that a premicellar bile salt binding site (protected by monomers) is transformed in a functional micellar binding site (protected by micelles). This conformational transformation seems to be consecutive to the dimerization, as has been recently proposed.

Bile Acids and Salts↗
PubMed1987

Chemical modification of cytochrome P-450 LM4. Identification of functionally linked tyrosine residues.

Cytochrome P-450 LM4 (RH, reduced flavoprotein:oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) from rabbit liver microsomes was chemically modified with tetranitromethane. Nitration of two tyrosine residues inhibits the p-nitrophenetole O-deethylase activity of the enzyme by about 80%. Sequencing the 3-nitrotyrosine-containing peptides after HPLC tryptic peptide mapping reveals that mainly Tyr-243 and Tyr-271 are nitrated, whereas Tyr-71, Tyr-188 and Tyr-365 are modified to a lower extent. Nitration of tyrosine residues affects the complex formation with p-nitrophenetole, alpha-naphthoflavone and metyrapone as indicated by an increased affinity towards p-nitrophenetole and by a decreased affinity for the latter compounds. Furthermore, nitration interferes with the electron transfer from NADPH-cytochrome P-450-reductase to cytochrome P-450 LM4 resulting in a slowed down reduction reaction. The results suggest that Tyr-243 and Tyr-271 of cytochrome P-450 LM4 are functionally involved in the interaction with NADPH-cytochrome P-450 reductase.

Amino Acid Sequence↗
PubMed1993

A tyrosine residue is involved in the allosteric binding of tryptophan to yeast chorismate mutase.

Comparative 1H-NMR studies have been carried out on wild-type chorismate mutase, which is activated by tryptophan and inhibited by tyrosine and phenylalanine, and mutant yeast chorismate mutase, which has a single point mutation (T226I) and is not allosterically regulated but 'locked' in the activated state. Double quantum-filtered COSY spectra show cross-peaks which have been assigned to a tyrosine that are absent in the mutant enzyme and in the wild-type enzyme plus tryptophan when compared with the wild-type enzyme alone. These observations indicate the involvement of a tyrosine at or near the allosteric binding site. The involvement of tyrosine in tryptophan binding was tested by modification of tyrosine in yeast chorismate mutase by nitration with tetranitromethane. All forms of the enzyme exhibited an approx. 50% reduction in specific activity, but it was found that preincubation of the wild-type with the allosteric activator, tryptophan, lead to partial protection against loss in specific activity. Only one tyrosine residue was nitrated in the wild-type enzyme and this tyrosine was identified by tryptic digestion and sequencing, and found to be very close to the site of the single point mutation in the mutant enzyme. It is proposed that Tyr-234 is located at or near the allosteric activation site.

Amino Acid Sequence↗