Free Tyrosine Research Articles

Analysis of free and protein-bound nitrotyrosine in human plasma by a gas chromatography/mass spectrometry method that avoids nitration artifacts

Measurement of nitrotyrosine in biological fluids and tissues is increasingly being used to monitor the production of reactive nitrogen species in vivo. The detection of nitrotyrosine in vivo has been reported with the use of a variety of methods including immunoassay, HPLC and GLC/MS. The validity of HPLC and immunoassays have been questioned with regard to their selectivity and sensitivity limits. In principle, the measurement of nitrotyrosine by GLC/MS permits a highly specific, highly sensitive and fully quantitative assay. The nitration of tyrosine under acidic conditions in the presence of nitrite is well documented. Derivatization for the full quantification of nitrotyrosine by using GLC/MS can lead to the artifactual nitration of tyrosine if performed under acidic conditions in the presence of nitrite. We describe a novel alkaline method for the hydrolysis and derivatization of nitrotyrosine and tyrosine, and demonstrate its applicability to the measurement of plasma concentrations of both free and protein-bound nitrotyrosine and tyrosine. A detection limit of 1 pg for nitrotyrosine and 100 pg for tyrosine has been achieved. Our method allows, for the first time, the analysis of free and protein-bound nitrotyrosine and tyrosine in biological samples. The plasma concentrations (means+/-S.E.M.) of free tyrosine and nitrotyrosine in eight normal subjects were 12+/-0.6 microg/ml and 14+/-0.7 ng/ml respectively. Plasma proteins contained tyrosine and nitrotyrosine at 60.7+/-1.7 microg/mg and 2.7+/-0.4 ng/mg respectively.

Yield, quality characteristics and nutritional profile of dehydrated chips prepared from potatoes astored at low high temperature

Yield, quality attributes, processing and nutritional losses were determined during the preparation of dehydrated chips from seven Indian and five exotic cultivars after 100 days of storage in the cold store (3-4°C) and in an evaporatively cooled store (15-29°C, RH 80-94%). Yield and quality attributes such as oil uptake, colour, texture and taste of fried dehydrated chips were affected by the cultivar and storage temperature. The average yield of dehydrated chips was 22% higher and their oil uptake about 50% lower when made from potatoes stored in evaporatively cooled store as compared to the cold store. The processing quality of the product was adversely affected when prepared from the tubers stored at low temperature. Colour appearance, taste and texture of the fried dehydrated chips prepared from all the cultivars stored in the evaporatively cooled store were acceptable as against unacceptably dark colour and bitter taste of chips made from cold stored potatoes. Among cultivars stored in the evaporatively cooled store, exotic cultivars, in general, gave higher yield and produced better colour quality chips in comparison to Indian varieties. The chip colour was positively correlated to the reducing sugar content of tubers as well as dehydrated chips. The average per cent losses in reducing sugars, sucrose, soluble protein, free amino acids, total phenols, tyrosine, vitamin C, nitrogen, phosphorus, potassium and calcium were 26, 78, 39, 66, 70, 43, 46, 46, 48, 80 and 68, respectively during preparation of dehydrated chips from evaporatively cooled stored tubers as against 27, 63, 29, 35, 92, 54, 66, 44, 44, 81 and 70, respectively from the tubers stored in cold store. The study suggests storage of potatoes in the evaporatively cooled store up to 100 days to obtain high yield of good quality dehydrated chips with reasonable retention of nutrients.

Induction of Betacyanin Synthesis and Pigment Accumulation in Cell Suspension Cultures of Portulaca.

The establishment of betalain-producing cell suspension cultures in Portulaca is reported for the first time. Addition of ascorbic acid (AsA) and sufficient osmotica to the culture medium was critical to the success. Betacyanin production was best at 1×103mgl-1 AsA. Small cell aggregates, formed in MS-AsA medium supplemented with 150mM mannitol and 150mM sorbitol, accumulated the highest relative amounts of betacyanins. The growth rate of cell suspension cultures increased 3- to 4- fold within 14 days in modified MS-AsA medium supplemented with 5μM 2, 4-D. The betalain content of suspended cells increased during the first 2 day in liquid culture, thereafter decreased slightly and increased again during the logarithmic phase of cellular growth. A lower concentration of 2, 4-D inhibited the growth of cultures but did not significantly affect betacyanin accumulation. Addition of cytokinin inhibited growth and also resulted in decreased betacyanin accumulation. The pigmentation of cultured cells decolourized upon transfer to dark conditions but was regained in decolourized cells at 12 to 24 hours after light exposure. The levels of endogenous free tyrosine and DOPA, precursors of betalains, decreased in response to light and slightly increased again after 24 hours of light treatment. Betanin, the main pigment component, as well as other betacyanins present, dramatically increased after 24 hours under light conditions in our Portulaca cell suspension culture system.

Synthesis and release of dopamine in rat striatal slices: requirement for exogenous tyrosine in the medium.

When incubated in a tyrosine-free medium, the tissue dopamine (DA) level of rat striatal slices increased by about 921 +/- 15 pmol/mg protein during 90 min of preincubation. In contrast, the tissue-free tyrosine level declined only 130 pmol/mg protein in the same assay period. Depolarization of the slices with high K+ increased both DA and DOPAC outputs and depleted tissue DA level by about 75%. Although 60 min of resting after high K+ depolarization significantly restored the tissue DA levels, neither this restoration nor depolarization-induced DA release was altered by exogenous tyrosine. Similarly, failure of exogenous tyrosine was also observed during three successive depolarization periods of striatal slices. These results indicate that nigrostriatal dopaminergic neurons are able to synthesize and release the DA in the absence of exogenous tyrosine in the medium. Since the free tyrosine level in the slices does not seem to be a sufficient source, it is likely that tyrosine mobilized from its bound source(s) supports the DA synthesis under in vitro experimental conditions.

Oxidation of heparin-treated low density lipoprotein by peroxidases

Low density lipoproteins (LDL) can bind to glycosaminoglycans and proteoglycans rich in heparin and chondroitin sulphate in the arterial intima and may become a target for atherogenic modification by myeloperoxidase activity. We have examined the susceptibility of resolubilized LDL, that has been precipitated from serum with heparin (HepLDL), to peroxidase–H 2O 2-catalysed oxidation and the effects of antioxidants and components of human serum on the oxidation. HepLDL was oxidised rapidly by horse radish peroxidase (HRP) and H 2O 2 (mean t 1/2 max for conjugated diene formation, 3 min) while there was little oxidation of native LDL or native LDL precipitated with polyethyleneglycol and resolubilised during the 30 min incubation period. The formation of thiobarbituric acid reacting substances (TBARS) essentially paralleled that of conjugated dienes during oxidation of HepLDL. HepLDL was also more rapidly oxidised than native LDL by myeloperoxidase–H 2O 2. Oxidation of HepLDL by peroxidases did not require free tyrosine, was almost totally inhibited by butylated hydroxytoluene (BHT) and ascorbate, and was unaffected by vitamin E and urate. Increasing concentrations (0–14.9%) of β-lipoprotein deficient serum (BLPDS) significantly ( P<0.0001) inhibited the formation of TBARS during HepLDL oxidation catalysed by HRP and partially inhibited the corresponding myeloperoxidase-catalysed oxidation. This inhibitory activity was removed by dialysis and gel-filtration of BLPDS and was not restored by addition of magnesium ions used in the isolation of BLPDS, or physiological levels of ascorbate, tyrosine and reduced thiols (cysteine) to gel-filtered BLPDS. The results indicate that LDL from complexes with glycosaminoglycans are highly susceptible to oxidation by peroxidases, particularly at low levels of water soluble antioxidants, and that vulnerability of these LDL to myeloperoxidase oxidation remains in the presence of serum components that should exist in the arterial intima. These findings may be relevant to the oxidative modification of LDL that becomes trapped by binding to arterial proteoglycans and to the formation of myeloperoxidase-modified LDL in the artery wall.

Sensory characteristics of dudh churpi in relation to its chemical composition

Dudh churpi, a traditional milk product, had more moisture, fat, water-dispersible protein (WDP), titratable acid, energy, lactic acid, free fatty acids (FFA), 2-thiobarbituric acid (TBA) and tyrosine contents and also reflectance, but less sensory attributes, when prepared in Darjeeling than the products prepared in Sikkim and Bhutan. Sensory attributes were positively correlated with total solids, protein, lactose, sugar, hydroxymethylfurfural (HMF) and p-dimethylaminobenzaldehyde reactivity, and negatively correlated with fat, FFA, TBA, WDP, tyrosine, lactic acid and titratable acidity. Whereas total solids, fat, protein, lactose, glucose-galactose and total sugar accounted for 71% of the flavour score, 64% of the body and texture score, 77% of the colour and appearance score and 77% of the total score, lactic acid, FFA, TBA, tyrosine and HMF jointly reflected variations of 61%, 50%, 81% and 67% in flavour, body and texture, colour and appearance, and total score, respectively.

The effect of potassium deficiency on chemical, biochemical and physical factors commonly associated with blackspot development in potato tubers

Blackspot bruise is a physiological disorder of potato tubers resulting from mechanical damage to tissues during handling. This study examined the effect of potassium nutrition on biochemical and physical changes normally associated with blackspot bruise in potato tubers. Four potato (Solanum tuberosum L.) genotypes (TXA 763-5 [very resistant], Ranger Russet [very susceptible], Russet Burbank [susceptible], and Lemhi Russet [very susceptible]) differing in susceptibility to blackspot bruise were grown at three potassium nutrition levels (100%, 10% and 0% of the potassium in full strength Hoagland’s solution). Tubers from the three treatments were tested for their chemical, biochemical and physical characteristics commonly related to blackspot bruise susceptibility. The potential to develop malanin pigments was measured as total oxidative potential (optical density, OD475) of clarified tuber homogenates. Tuber tissue extracts were assayed for polyphenol oxidase (PPO) activity with catechol, measured as increase in OD410 over time. Specific gravity of tubers was determined with a brine solution series. Physical property measurements were made by impacting cores of tuber tissue in a specially designed instrumented pendulum. Shock wave speed, Young’s modulus, failure stress, and failure strain were all measured dynamically during impact. Tubers produced under both the 10% and 0% potassium regimens had almost 2x the free tyrosine of control tubers. The 0% potassium tubers had significantly higher PPO activity than the control. Both of these factors likely contributed to the significantly higher total oxidative potential that was observed in tubers from both the 10% and 0% potassium treatments. Tubers from both the 0% and 10% potassium treatments had a significantly lower Young’s modulus (a measure of stiffness), significantly lower failure stress and the shock wave of impact propagated through both of them significantly slower. Although the cultivars were significantly different in their susceptibility to black pigment development, they all responded similarly to the treatment with regards to the variables measured. The TXA-763-5 clone was much more susceptible to developing black pigments in these tests than expected. The other three cultivars performed as they do in commerce.

Formation of Long-Lived Radicals on Proteins by Radical Transfer from Heme Enzymes—A Common Process?

Incubation of Fe(III)myoglobin (Fe(III)Mb) with H2O2in the presence of bovine serum albumin (BSA) has been shown previously to give albumin-derived radicals as a result of radical transfer from myoglobin to BSA. In this study the occurrence of similar processes with peroxidases has been investigated using horseradish peroxidase (HRP)/H2O2, in the presence and absence of added tyrosine. Incubation of HRP with H2O2and bovine or human serum albumins, in the presence and absence of tyrosine, gave long-lived albumin-derived radicals as detected by EPR spectroscopy. Evidence has been obtained for these albumin radicals being located on buried tyrosine residues on the basis of blocking experiments. The effect of protein conformation on radical transfer has been investigated using partial proteolytic digestion prior to protein oxidation. With HRP/H2O2/BSA and Fe(III)Mb/H2O2/BSA increased radical concentrations were observed after limited digestion, although this effect was less marked with the HRP/H2O2/BSA system than with Fe(III)Mb/H2O2/BSA, consistent with different modes of radical transfer. More extensive digestion of BSA decreased the radical concentration to levels below those detected with native albumin, indicating that the tertiary structure of the target protein plays an important role in determining the rate of radical transfer and/or the stability of the resultant species. These results are consistent with a mechanism for the HRP/H2O2/no free tyrosine system involving radical transfer to the albumin via the heme edge of the peroxidase. In contrast, albumin radical formation by the HRP/H2O2/free tyrosine system was only marginally affected by proteolysis, consistent with free tyrosine phenoxyl radicals being the mediators of radical transfer, without significant protein–protein interaction. These protein-to-protein radical transfer reactions may have important consequences for understanding protein oxidation in biological systems.

Pre-cure freezing affects proteolysis in dry-cured hams

Several parameters (sodium chloride, moisture, intramuscular fat, total nitrogen, non-protein nitrogen, white precipitates, free tyrosine, L* a* b* values and acceptability) related with proteolysis during the curing were compared in dry-cured hams manufactured from refrigerated and frozen/thawed raw material. Pre-cure freezing increased the proteolysis levels significantly ( p<0.05) in the zones of the ham where water losses and absorption of salt is slowest. Frozen hams present a high incidence of white precipitates, formed mainly by tyrosine crystals. The colour and acceptability scores are similar in frozen and refrigerated hams. The previous freezing and thawing process accentuates the water losses, salt absorption and proteolysis of the cured meat, although it does not significantly affect the sensory quality of the dry-cured ham.

Tyrosine and cysteine are substrates for blackspot synthesis in potato

Partially purified blackspot pigments from potato tubers ( Solanum tuberosum L.) of two commercial cultivars were subjected to a microassay for melanin, which consisted of specific chemical degradation and subsequent HPLC analysis. Permanganate oxidation yielded pyrrole-2,3,5-tricarboxylic acid, whereas hydrolysis in hydriodic acid liberated aminohydroxyphenylalanine isomers. These results indicate that the polymeric pigments, which have previously been found to contain a protein matrix, carry crosslinked 5,6-dihydroxyindole-2-carboxylic acid and benzothiazine units. This leads to the conclusion that free tyrosine and free cysteine are incorporated in the proteinaceous pigments via the polyphenol oxidase catalysed pathway of melanogenesis in the process of blackspot formation. The findings are in accordance with the hypothesis that the process of blackspot formation is a non-regulated cascade of reactions in disintegrated tuber cells, rather than a finely tuned biosynthesis.

Lack of Tyrosine Nitration by Peroxynitrite Generated at Physiological pH

Nitration of tyrosine residues of proteins has been suggested as a marker of peroxynitrite-mediated tissue injury in inflammatory conditions. The nitration reaction has been extensively studied in vitro by bolus addition of authentic peroxynitrite, an experimental approach hardly reflecting in vivo situations in which the occurrence of peroxynitrite is thought to result from continuous generation of .NO and O-2 at physiological pH. In the present study, we measured the nitration of free tyrosine by .NO and O-2 generated at well defined rates from the donor compound (Z)-1-[N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino]- dia zen-1-ium-1,2-diolate] (spermine NONOate) and the xanthine oxidase reaction, respectively. The results were compared with the established nitration reaction triggered by authentic peroxynitrite. Bolus addition of peroxynitrite (1 mM) to tyrosine (1 mM) at pH 7.4 yielded 36.77 +/- 1.67 microM 3-nitrotyrosine, corresponding to a recovery of about 4%. However, peroxynitrite formed from .NO and O-2, which were generated at equal rates ( approximately 5 microM x min-1) from 1 mM spermine NONOate, 28 milliunits/ml xanthine oxidase, and 1 mM hypoxanthine was much less efficient (0.67 +/- 0.01 microM; approximately 0.07% of total product flow). At O-2 fluxes exceeding the .NO release rates, 3-nitrotyrosine formation was below the detection limit of the high performance liquid chromatography method (<0.06 microM). Nitration was most efficient (approximately 0.3%) with the .NO donor alone, i.e. without concomitant generation of O-2. Nitration by .NO had a pH optimum of 8.2, increased progressively with increasing tyrosine concentrations (0.1-2 mM), and was not enhanced by NaHCO3 (up to 20 mM), indicating that it was mediated by .NO2 rather than peroxynitrite. Our results argue against peroxynitrite produced from .NO and O-2 as a mediator of tyrosine nitration in vivo.

Chenopodium quinoa: changes in amino acid composition in seed during maturity

Investigation of changes which occur during seed maturity of Chenopodium quinoa showed that relative amounts of total (protein plus free) amino acids, glutamic acid, glycine and arginine increase, whereas aspartic acid, threonine, serine, proline, valine and lysine decrease. The protein amino acids showed only slight variations while free aspartic acid, threonine, serine, proline, leucine decrease considerably as compared to valine, isoleucine and lysine which showed a smaller decrease. The major increase in free glutamic acid, glycine, alanine, tyrosine and histidine was observed from 4th day to maturity.

Biochemical Potential of Potato Tubers To Synthesize Blackspot Pigments in Relation to Their Actual Blackspot Susceptibility

In a study of six potato varieties (Solanum tuberosum L.), which were grown under two different fertilization regimes, the biochemical potential of tuber tissue to synthesize blackspot pigments was distinguished from the actual blackspot susceptibility exhibited after impact application. Blackspot susceptibility and biochemical potential for pigment synthesis were not correlated, which supports the hypothesis that the extent to which the blackspot potential is actually being used is mediated by the sensitivity to cell decompartmentation. Quantification of polyphenol oxidase (PPO), soluble protein, and endogenous PPO substrates demonstrated that the content of free tyrosine is the predominant determinant for the biochemical potential for blackspot synthesis. PPO was not a limiting factor for pigment synthesis, even if PPO content was relatively low. It was therefore concluded that manipulation of PPO activity may offer a solution to the problem of blackspot formation only if it leads to a virtually complet...

Amino acid metabolism and protein turnover in larval turbot ( Scophthalmus maximus ) fed natural zooplankton or Artemia

The present paper studied the influence of different food regimes on the free amino acid (FAA) pool, the rate of protein turnover, the flux of amino acids, and their relation to growth of larval turbot (Scophthalmus maximus L.) from first feeding until metamorphosis. The amino acid profile of protein was stable during the larval period although some small, but significant, differences were found. Turbot larvae had proteins which were rich in leucine and aspartate, and poor in glutamate, suggesting a high leucine requirement. The profile of the FAA pool was highly variable and quite different from the amino acid profile in protein. The proportion of essential FAA decreased with development. High contents of free tyrosine and phenylalanine were found on Day 3, while free taurine was present at high levels throughout the experimental period. Larval growth rates were positively correlated with taurine levels, suggesting a dietary dependency for taurine and/or sulphur amino acids. Reduced growth rates in Artemia-fed larvae were associated with lower levels of free methionine, indicating that this diet is deficient in methionine for turbot larvae. Leucine might also be limiting turbot growth as the different diet organisms had lower levels of this amino acid in the free pool than was found in the larval protein. A previously presented model was used to describe the flux of amino acids in growing turbot larvae. The FAA pool was found to be small and variable. It was estimated that the daily dietary amino acid intake might be up to ten times the larval FAA pool. In addition, protein synthesis and protein degradation might daily remove and return, respectively, the equivalent of up to 20 and 10 times the size of the FAA pool. In an early phase (Day 11) high growth rates were associated with a relatively low protein turnover, while at a later stage (Day 17), a much higher turnover was observed.

Assembly of nematode cuticle: role of hydrophobic interactions in CUT-2 cross-linking

CUT-2 is a component of cuticlin, the highly cross-linked, insoluble residue of the cuticle of the nematode Caenorhabditis elegans. A recombinant fragment of CUT-2, produced in E. coli, can be cross-linked in vitro by horse radish peroxidase via dityrosine formation to give large molecular species [1]. In this paper it is shown that the formation of CUT-2 polymers is greatly favoured over that of CUT-2 oligomers as no low molecular weight intermediates, dimers or trimers can be detected even when the cross-linking reaction is slowed or interrupted before completion. This suggests that recombinant CUT-2 forms large non-covalent complexes that are the only competent substrate for cross-linking. The inhibition of cross-linking by urea and the behavior of recombinant CUT-2 in size-exclusion chromatography under a variety of conditions suggest that hydrophobic interactions are important in the formation and stabilization of these complexes. The complexes are excellent substrates for cross-linking but react poorly with free tyrosine. In contrast, a soluble recombinant CUT-2 is a poor substrate for cross-linking but can efficiently react with free tyrosine.

Interference of Carboxy-PTIO with Nitric Oxide- and Peroxynitrite-Mediated Reactions

Carboxy-PTIO reacts rapidly with NO to yield NO 2 and has been used as a scavenger to test the importance of nitric oxide (NO) in various physiological conditions. This study investigated the effects of carboxy-PTIO on several NO- and peroxynitrite-mediated reactions. The scavenger potently inhibited NO-induced accumulation of cGMP in endothelial cells but potentiated the effect of the putative peroxynitrite donor SIN-1. Carboxy-PTIO completely inhibited peroxynitrite-induced formation of 3-nitrotyrosine from free tyrosine (EC 50 = 36 ± 5 μM) as well as nitration of bovine serum albumin. Peroxynitrite-mediated nitrosation of GSH was stimulated by the drug with an EC 50 of 0.12 ± 0.03 mM, whereas S-nitrosation induced by the NO donor DEA/NO (0.1 mM) was inhibited by the scavenger with an IC 50 of 0.11 ± 0.03 mM. Oxidation of NO with carboxy-PTIO resulted in formation of nitrite without concomitant production of nitrate. Our results demonstrate that the effects of carboxy-PTIO are diverse and question its claimed specificity as NO scavenger. Copyright © 1997 by Elsevier Science Inc.

Inhibition of Betacyanin Accumulation by Abscisic Acid in Suspension Cultures of Phytolacca americana

Growth of cells and the accumulation of betacyanin were suppressed by the addition of abscisic acid (ABA) to suspension cultures of Phytolacca americana. The decrease in the accumulation of betacyanin was overcome by exogenously supplied tyrosine which is a precursor of betacyanin. ABA decreased the level of free tyrosine in the cells. Feeding experiments using labeled tyrosine revealed that ABA reduced the incorporation of labeled tyrosine into betacyanins (to about 50% of the control rate). These results suggest that both the availability of tyrosine and the biosynthetic activity of the pathway from tyrosine to the betacyanins are involved in the inhibition of the accumulation of betacyanins by ABA in Phytolacca americana cells.

Selectivity in Tyrosyl Iodination Sites in Human Thyroglobulin

Previous studies indicate that when low iodine thyroglobulin (Tg) is iodinated enzymatically with thyroid peroxidase (TPO), the tyrosyl residues that are used for the formation of thyroid hormone (hormonogenic sites) are selected for early iodination. The aim of the present study was to assess the relative importance of the substrate (Tg) and the enzyme (TPO) in the selection of the early tyrosyl sites that undergo iodination. For this purpose, low iodine human Tg (2.0 atoms I per 660,000 dimer) was iodinated chemically with125I[es−3and enzymatically with TPO +125I−to a matched low level of iodination (∼8 added I atoms per molecule). After reduction and alkylation, the two Tg preparations were digested with trypsin, and the tryptic digests were separated by reverse-phase HPLC into 10125I-containing pools. Each pool was further fractionated by HPLC to provide purified125I-peptides suitable for sequence analysis. From the sequence information and the known amino acid sequence of Tg, it was possible to define the location of the iodinated tyrosyl residues. Surprisingly, almost identical results were obtained with chemically and enzymatically iodinated Tg. Not only were the125I-peptide maps very similar, but all of the recovered125I in the purified peptides from both samples was located in only three different tyrosyl sites, 5, 2553, and 2520. Tyr 5 and Tyr 2553 are well-established sites of thyroxine formation, while Tyr 2520 has previously been proposed by us to be a donor site. Our observation that the same hormonogenic tyrosyl sites are iodinated by chemical as well as enzymatic iodination indicates that preferential iodination of hormonogenic sites is dependent primarily on the native structure of Tg. TPO plays a minor role, if any, in the selection of early tyrosyl iodination sites in Tg. Consistent with this conclusion was our finding that chemical iodination, as well as enzymatic iodination, led to formation of uniformly iodinated Tg, as determined by isopycnic centrifugation in rubidium chloride. However, we observed a slightly higher diiodotyrosine (DIT) content and a correspondingly lower monoiodotyrosine content in enzymatically iodinated Tg, compared to matched chemically iodinated Tg. This was not observed with two other proteins, bovine serum albumin and trypsinogen, or with free tyrosine, as substrates for iodination. The same preferential formation of DIT in Tg was, however, observed when lactoperoxidase was substituted for TPO. Preferential formation of DIT, therefore, appears to involve interaction between Tg and the peroxidase.

Fluorescence Studies of Lipid Association-Induced Conformational Adaptations of an Exchangeable Amphipathic Apolipoprotein

The conformational adaptability ofManduca sextaapolipophorin III (apoLp-III) has been evaluated by monitoring the spectroscopic properties of its sole tyrosine residue, Tyr145, present in the fifth helical segment of the protein.M. sextaapoLp-III adopts a globular five-helix bundle structure in solution and has been postulated to undergo an opening at putative hinge domains upon interaction with lipid surfaces. Previous results have shown that the intrinsic fluorescence of Tyr145is highly quenched in the closed, water-soluble conformation but is dramatically enhanced upon lipid association. We have carried out a spectroscopic characterization of Tyr145and its microenvironment, to enable its use as a structural probe of lipid-induced conformational changes of apoLp-III. The pKaof Tyr145in lipid-free apoLp-III was found to be 10.5, as determined from uv-spectrophotometry, indicating that, in the ground state, the tyrosyl phenolic group is not ionized under physiological conditions. Compared to free tyrosine in aqueous buffer (pH 7.0), a red shift (77 nm) in the λmaxof absorbance of Tyr145was observed, suggesting that an H-bonding interaction is responsible for the quenched state of tyrosine fluorescence. In an effort to explain the observed quenching phenomenon, the quantum yield and lifetimes of Tyr145fluorescence emission were investigated as a function of pH and lipid binding. The quantum yield of Tyr145in lipid-free apoLp-III was enhanced fivefold upon decreasing the pH, with a half-maximal point around pH 5.5. Time-resolved fluorescence decay analysis showed that Tyr145exhibits nonexponential emission decay with two components having lifetimes of 3.3 ns (76%) and 0.89 ns (24%) in the lipid-free state. The lifetime and amplitude of Tyr145remain essentially unaltered upon lipid association or decreasing the pH. This is consistent with the hypothesis that, in the lipid-free helix bundle conformation, a quenching residue exists within H-bonding distance of the phenolic side chain of Tyr145which, at physiological pH, is responsible for the observed fluorescence quenching. Opening of the helix bundle repositions this acceptor base, possibly a carboxylate or an imidazole side chain, making it unavailable for quenching. Using differential polarized phase and modulation fluorometry, it was seen that the segmental motion of Tyr145is also altered considerably upon lipid interaction. These spectroscopic and motional properties of Tyr145distinguish this unique residue as a useful probe to monitor structural flexibility of apoLp-III.

Nitrotyrosine attenuates the hemodynamic effects of adrenoceptor agonists in vivo: relevance to the pathophysiology of peroxynitrite

Peroxynitrite, which attenuates catecholamine-mediated hemodynamic responses in vivo, nitrates free tyrosine residues to form the specific product, 3-nitro- l-tyrosine. The chemical structure of 3-nitro- l-tyrosine is similar to that of the endogenous catecholamines. Therefore, 3-nitro- l-tyrosine may interfere with catecholamine hemodynamic function in vivo. The hemodynamic responses produced by norepinephrine (1–4 μg/kg, i.v., n = 6), epinephrine (0.5–4 μg/kg, i.v., n = 7), phenylephrine (1–8 μg/kg, i.v., n = 5), and isoproterenol (100–400 ng/kg, i.v., n = 5) were attenuated, while the hemodynamic responses produced by arginine vasopressin (50–250 ng/kg; i.v., n = 5) were unaffected following the administration of 3-nitro- l-tyrosine (2.5 μ mol/kg, i.v.) in pentobarbital-anesthetized rats. These results demonstrate substantial and selective attenuation of the hemodynamic effects produced by α- and β-adrenoceptor agonists, raising the possibility that 3-nitro- l-tyrosine may play a role in the hemodynamic dysfunction associated with inflammatory conditions in which the formation of peroxynitrite is favored.