Nitration Of Tyrosyl Residues Research Articles

Prevention of peroxynitrite-induced oxidation and nitration reactions by ellagic acid

Four benzoic acid derivatives and three phenylpropanoids were tested for their ability to suppress oxidation of dichlorodihydrofluorescein (DCDHF) induced by 3-morpholinosydnonimine (SIN-1, a peroxynitrite donor). Ellagic acid was found to potently inhibit this oxidation and to inhibit oxidation of DCDHF induced by peroxynitrite itself. Moreover, this compound prevented peroxynitrite-induced oxidative single strand breaks in pTZ 18U plasmid DNA and nitration of tyrosyl residues in bovine serum albumin. These results show that ellagic acid protects biomolecules against oxidative and nitrative damage induced by peroxynitrite in vitro.

Peroxynitrite Inactivates Tryptophan Hydroxylase via Sulfhydryl Oxidation: COINCIDENT NITRATION OF ENZYME TYROSYL RESIDUES HAS MINIMAL IMPACT ON CATALYTIC ACTIVITY

Tryptophan hydroxylase, the initial and rate-limiting enzyme in serotonin biosynthesis, is inactivated by peroxynitrite in a concentration-dependent manner. This effect is prevented by molecules that react directly with peroxynitrite such as dithiothreitol, cysteine, glutathione, methionine, tryptophan, and uric acid but not by scavengers of superoxide (superoxide dismutase), hydroxyl radical (Me(2)SO, mannitol), and hydrogen peroxide (catalase). Assuming simple competition kinetics between peroxynitrite scavengers and the enzyme, a second-order rate constant of 3.4 x 10(4) M(-1) s(-1) at 25 degrees C and pH 7.4 was estimated. The peroxynitrite-induced loss of enzyme activity was accompanied by a concentration-dependent oxidation of protein sulfhydryl groups. Peroxynitrite-modified tryptophan hydroxylase was resistant to reduction by arsenite, borohydride, and dithiothreitol, suggesting that sulfhydryls were oxidized beyond sulfenic acid. Peroxynitrite also caused the nitration of tyrosyl residues in tryptophan hydroxylase, with a maximal modification of 3.8 tyrosines/monomer. Sodium bicarbonate protected tryptophan hydroxylase from peroxynitrite-induced inactivation and lessened the extent of sulfhydryl oxidation while causing a 2-fold increase in tyrosine nitration. Tetranitromethane, which oxidizes sulfhydryls at pH 6 or 8, but which nitrates tyrosyl residues at pH 8 only, inhibited tryptophan hydroxylase equally at either pH. Acetylation of tyrosyl residues with N-acetylimidazole did not alter tryptophan hydroxylase activity. These data suggest that peroxynitrite inactivates tryptophan hydroxylase via sulfhydryl oxidation. Modification of tyrosyl residues by peroxynitrite plays a relatively minor role in the inhibition of tryptophan hydroxylase catalytic activity.

Nitration of tyrosyl-residues from extra- and intracellular proteins in human whole blood

We measured the amounts of tyrosine and 3-nitrotyrosine (NO 2-tyrosine) in proteins of plasma and polymorphonuclear leukocytes (PMN) from human whole blood before and after activation with phorbol ester (PMA) or calcium ionophore (A 23187). In unstimulated blood, no significant nitration of tyrosine was detected into PMN proteins, but, a NO 2-tyrosine/ tyrosine ratio of 0.7% was detected in plasma proteins. When blood was activated with PMA, the NO 2-tyrosine/tyrosine ratio stayed at 0.7% in plasma proteins, but it increased to 1.4% in PMN proteins, indicating a peroxynitrite production within the cells. In blood activated with calcium ionophore, the NO 2-tyrosine/tyrosine ratio was 1.2% in plasma proteins and 2.1% in PMN proteins. Incubation of blood with a NO 2-synthase inhibitor before stimulation inhibited such a protein tyrosine nitration. To ensure that NO 2-tyrosine detected in intracellular proteins did not result from the enzymatic posttranslational tyrosylation of PMN proteins, the incorporation of 14C labeled tyrosine into PMN proteins after activation with PMA or A23187 was studied. The addition of a 10 fold excess of NO 2-tyrosine did not modify the course of protein tyrosylation. Because tyrosine nitration is an irreversible reaction, NO 2-tyrosine could be accumulated into proteins and could act as a cumulative index of peroxynitrite production.

Interaction between alkali light chains of myosin and divalent metal ions.

1. Difference UV absorption spectra of chicken breast g1 induced by magnesium and calcium ions were compared with those of rabbit skeletal alkali light chains. The value of delta epsilon due to the burial of Tyr 180 of domain 4 was about one-fourth of that of rabbit alkali light chains. Regions other than domain 4 appear to affect the environment of Tyr 180, since the first-order structure of domain 4 of chicken g1 is present in rabbit g1 without significant alteration (Matsuda, G. et al. (1981) FEBS Lett. 126, 111). Movement of a phenylalanyl residue was suggested to be larger in chicken g1 than rabbit g1. 2. CD spectra of alkali light chains were measured in the wavelength region down from 260 nm. Divalent metal ions increased the helix content of alkali light chains about 2%. CB1 peptide of rabbit skeletal g1 containing domain 1 showed a slight change of CD spectrum in the presence of divalent ions. These results suggest that domain 1 of alkali light chains binds divalent ions. 3. Binding of calcium ions to rabbit skeletal g1 was directly measured by means of a calcium ion selectrode. More than 3 mol of calcium was bound per mol of g1. The concentration of free calcium ions required to give a half-maximal binding was 5 mM assuming that the maximum binding number is 4 mol of calcium per mol of g1. 4. Nitration of tyrosyl residues in rabbit skeletal g1 may slightly affect the affinity for divalent metal ions and the backbone structure of the polypeptide chain of g1.

The Interaction of a Tyrosyl Residue and Carboxyl Groups in the Specific Interaction between <italic>Streptomyces</italic> Subtilisin Inhibitor and Subtilisin BPN'<subtitle>A Chemical Modification Study<sup>1</sup></subtitle>

An ultraviolet absorption difference spectrum that is typical of a change in ionization state (pKa 9.7 leads to greater than 11.5) of a tyrosyl residue has been observed on the binding between Streptomyces subtilisin inhibitor (SSI) and subtilisin BPN' [EC 3.4.21.14] at alkaline pH, ionic strength 0.1 M, at 25 degrees C (Inouye, K., Tonomura, B., and Hiromi, K., submitted). When the complex of SSI and subtilisin BPN' is formed at an ionic strength of 0.6 M and pH 9.70, the characteristic features of the protonation of a tyrosyl residue in the difference spectrum are diminished. These results suggest that the pKa-shift of a tyrosyl residue observed at alkaline pH and lower ionic strength results from an electrostatic interaction. Nitration of tyrosyl residues of SSI and of subtilisin BPN' was performed with tetranitromethane (TNM). By measurements of the difference spectra observed on the binding of the tyrosyl-residue-nitrated SSI and the native subtilisin BPN', and on the binding of the native SSI and the tyrosyl-residue-nitrated subtilisin BPN' and alkaline pH, the tyrosyl residue in question was shown to be one out of the five tyrosyl residues of pKa 9.7 of the enzyme. This tyrosyl residue was probably either Tyr 217 or Tyr 104 on the basis of the reactivities of tyrosyl residues of the enzyme with TNM and their locations on the enzyme molecule. Carboxyl groups of SSI were modified by covalently binding glycine methyl ester with the aid of water-soluble carbodiimide, in order to neutralize the negative charges on SSI. In the difference spectrum which was observed on the binding of subtilisin BPN' and the 5.3-carboxyl-group-modified SSI at alkaline pH, the characteristic features of the protonation of a tyrosyl residue were essentially lost, and the difference spectrum is rather similar to that observed on the binding of the native SSI and the enzyme at neutral pH. This phenomenon indicates that the pKa of a tyrosyl residue of the enzyme is shifted upwards by interaction with carboxyl group(s) of SSI on the formation of the enzyme-inhibitor complex.

Nitration of tyrosyl residues in human alpha-lactalbumin. Effect on lactose synthase specifier activity.

Alpha-Lactalbumin isolated from human milk was reacted with tetranitromethane in molar excess of 8-32 mol/mol of tyrosine. After gel filtration on Sephadex G-75, followed by chromatographic fractionation using DEAE-Sephadex A-25, three main components were separated, which differed from one another in the extent of nitration. These protein fractions were found to contain, respectively, one and two nitrotyrosine residues, or two nitrotyrosine residues together with one nitrotryptophan. The lactose synthase specifier activity of each of these components was measured and compared with that of unsubstituted alpha-lactalbumin. Comparison of kinetic parameters showed the chemically modified proteins to be only slightly less active when tyrosines were the sole residues modified. In sharp contrast the additional nitration of a single tryptophan residue totally abolished the specifying activity of alpha-lactalbumin. Circular dichroism spectra of the tryptophan derivative revealed some structural alteration when compared with the other two and with the native protein. The conclusion could also be confirmed by using a double-immunodiffusion technique. After hydrolysis of the derivatives with thermolysin, it was possible to localize the substituted residues in the known sequence of human alpha-lactalbumin. Tyrosine-103 was found to be more easily nitrated than tyrosine-18. These two residues seem, therefore, to be on the outer surface of the molecule and more exposed than tyrosine-36 and tyrosine-50. Some precautions are indicated in the use of tetranitromethane as a nitrating agent on the basis of complex products observed in the nitration of the free amino acids tyrosine and tryptophan and their derivatives.

T7 RNA polymerase: conformation, functional groups, and promotor binding.

Circular dichroic spectra of T7 RNA polymerase show minima at 222 nm ([theta]m=-7.9 X 10(3) deg cm2/dmol) and 208 nm ([theta]m =-7.55 X 10(3) deg cm2/dmol) and a maximum at 193 nm ([theta]m = 1.2 X 10(4) deg cm2/dmol). The small mean residue ellipticity above 200 nm indicates that the secondary structure contains approximately 12% alpha helix. The secondary structure is unaltered by high salt, glycerol, -SH reagents, nitration of tyrosyl residues, and chelating agents. Binding of the native enzyme to [32P]T7 DNA has been measured by the retention of the protein-[32P]DNA complexes on nitrocellulose filters. At 37degrees T7 RNA polymerase binds to its promoters in the absence of NTP's. Binding and catalytic activity are both abolished at 0degree. Binding of the initiating [gamma-32P]GTP can also be detected by the filter binding assay. Native T7 RNA polymerase is inactivated by reaction with 1 mol of 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) or 1 mol of [14C]iodoacetamide. The latter reaction is blocked by Nbs2 suggesting that a single -SH group is required for activity. Alkylation of the -SH group does not alter binding of the enzyme to the DNA template, but modifies the binding of GTP to the enzyme. Nitration of approximately4 surface tyrosyl residues of the protein prevents binding to T7 DNA. The restriction endonuclease, Hpa II, cuts T7 DNA into approximately40 fragments and reduces total RNA synthesis by T7 RNA polymerase by 70%. Fragmentation of the DNA template by Hpa II does not alter the rate of RNA chain initiation by T7 polymerase, and restriction fragments accounting for approximately25% of the T7 DNA still bind tightly to the enzyme. Thus the T7 RNA polymerase promoters remain intact on the restriction fragments. Gel electrophoresis of the transcription products, using restriction fragments as templates, show that of the seven in vitro transcripts produced by T7 RNA polymerase from whole T7 DNA, only the smallest (representing the last 1.5% of the genome) is transcribed from Hpa II fragments. The remaining transcripts are replaced by six new and much shorter mRNA's. The DNA fragments containing the promoters for these mRNA's have been removed from the fragment mix by binding them to the enzyme and retaining the complexes on nitrocellulose filters.