The observed levels of Delta G(ATP) in chloroplasts, as well as the activation behavior of the CF(1)CF(0)-ATP synthase, suggest a minimum transthylakoid proton motive force (pmf) equivalent to a Delta pH of approximately 2.5 units. If, as is commonly believed, all transthylakoid pmf is stored as Delta pH, this would indicate a lumen pH of less than approximately 5. In contrast, we have presented evidence that the pH of the thylakoid lumen does not drop below pH approximately 5.8 [Kramer, D. M., Sacksteder, C. A., and Cruz, J. A. (1999) Photosynth. Res. 60, 151-163], leading us to propose that Delta psi can contribute to steady-state pmf. In this work, it is demonstrated, through assays on isolated thylakoids and computer simulations, that thylakoids can store a substantial fraction of pmf as Delta psi, provided that the activities of ions permeable to the thylakoid membrane in the chloroplast stromal compartment are relatively low and the buffering capacity (beta) for protons of the lumen is relatively high. Measurements of the light-induced electrochromic shift (ECS) confirm the ionic strength behavior of steady-state Delta psi in isolated, partially uncoupled thylakoids. Measurements of the ECS in intact plants illuminated for 65 s were consistent with low concentrations of permeable ions and approximately 50% storage of pmf as Delta psi. We propose that the plant cell, possibly at the level of the inner chloroplast envelope, can control the parsing of pmf into Delta psi and Delta pH by regulating the ionic strength and balance of the chloroplast. In addition, this work demonstrates that, under certain conditions, the kinetics of the light-induced ECS can be used to estimate the fractions of pmf stored as Delta psi and Delta pH both in vitro and in vivo.

Increased abundance of nitrotyrosine modifications of proteins have been documented in multiple pathologies in a variety of tissue types and play a role in the redox regulation of normal metabolism. To identify proteins sensitive to nitrating conditions in vivo, a comprehensive proteomic data set identifying 7792 proteins from a whole mouse brain, generated by LC/LC-MS/MS analyses, was used to identify nitrated proteins. This analysis resulted in the identification of 31 unique nitrotyrosine sites within 29 different proteins. More than half of the nitrated proteins that have been identified are involved in Parkinson's disease, Alzheimer's disease, or other neurodegenerative disorders. Similarly, nitrotyrosine immunoblots of whole brain homogenates show that treatment of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an experimental model of Parkinson's disease, induces an increased level of nitration of the same protein bands observed to be nitrated in brains of untreated animals. Comparing sequences and available high-resolution structures around nitrated tyrosines with those of unmodified sites indicates a preference of nitration in vivo for surface accessible tyrosines in loops, a characteristic consistent with peroxynitrite-induced tyrosine modification. In addition, most sequences contain cysteines or methionines proximal to nitrotyrosines, contrary to suggestions that these amino acid side chains prevent tyrosine nitration. More striking is the presence of a positively charged moiety near the sites of nitration, which is not observed for non-nitrated tyrosines. Together, these observations suggest a predictive tool of functionally important sites of nitration and that cellular nitrating conditions play a role in neurodegenerative changes in the brain.

Elevated levels of protein tyrosine nitration have been found in various neurodegenerative diseases and age-related pathologies. Until recently, however, the lack of an efficient enrichment method has prevented the analysis of this important low-level protein modification. We have developed a method that specifically enriches nitrotyrosine-containing peptides so that both nitrotyrosine peptides and specific nitration sites can be unambiguously identified with LC-MS/MS. The procedure consists of the derivatization of nitrotyrosine into free sulfhydryl groups followed by high efficiency enrichment of sulfhydryl-containing peptides with thiopropyl sepharose beads. The derivatization process includes: (1) acetylation with acetic anhydride to block all primary amines, (2) reduction of nitrotyrosine to aminotyrosine, (3) derivatization of aminotyrosine with N-Succinimidyl S-Acetylthioacetate (SATA), and (4) deprotection of S-acetyl on SATA to form free sulfhydryl groups. The high specificity of this method is demonstrated by the contrasting percentage of nitrotyrosine-derivatized peptides in the identified tandem mass spectra between enriched and unenriched samples. Global analysis of unenriched in vitro nitrated human histone H1.2, bovine serum albumin (BSA), and mouse brain homogenate samples had 9%, 9%, and 5.9% of identified nitrotyrosine-containing peptides, while the enriched samples had 91% , 62%, and 35%, respectively. Duplicate LC-MS/MS analyses of the enriched mouse brain homogenate identified 150 unique nitrated peptides covering 102 proteins with an estimated 3.3% false discovery rate.