Peroxynitrite has recently been implicated in the inactivation of many enzymes. However, little has been reported on the structural basis of the inactivation reaction. This study proposes that nitration of a specific tyrosine residue is responsible for inactivation of recombinant human mitochondrial manganese-superoxide dismutase (Mn-SOD) by peroxynitrite. Mass spectroscopic analysis of the peroxynitrite-inactivated Mn-SOD showed an increased molecular mass because of a single nitro group substituted onto a tyrosine residue. Single peptides that had different elution positions between samples from the native and peroxynitrite-inactivated Mn-SOD on reverse-phase high performance liquid chromatography were isolated after successive digestion of the samples by staphylococcal serine protease and lysylendopeptidase and subjected to amino acid sequence and molecular mass analyses. We found that tyrosine 34 of the enzyme was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. This residue is located near manganese and in a substrate O 2 . gateway in Mn-SOD.Peroxynitrite anion (ONOO Ϫ ) 1 is a potent biological oxidant that has been implicated in diverse forms of free radical-induced tissue injury (1). Peroxynitrite has been demonstrated to readily oxidize or nitrate various enzymes such as metalloproteinase-1 inhibitor (2), alcohol dehydrogenase (3), aconitase (4), glutamine synthetase (5), and superoxide dismutase (6). However, little is known about the molecular basis of the nitration or oxidation of these enzymes. Peroxynitrite is produced by the reaction of superoxide (O 2 . ) and NO with a very rapid reaction rate (4 ϳ 6.7 ϫ 10 9 M Ϫ1 s Ϫ1 ) (7,8). Therefore, various cell types, such as macrophages (9), Kuppfer cells (10), and endothelial cells (11), that simultaneously produce and release O 2. and NO could produce peroxynitrite.Mammalian mitochondria are one of the most important targets of the cytotoxicity of peroxynitrite. Recently, a mechanism for peroxynitrite-mediated dysfunction of mitochondria has been proposed as follows. When mitochondria are exposed to NO, NO diffuses easily through the membranes and reversibly inhibits cytochrome oxidase. This inhibition causes inhibition of the mitochondrial respiratory chain and as a consequence increases mitochondrial O 2 . release, leading to peroxynitrite formation (12, 13). Peroxynitrite then irreversibly inhibits complexes I and II in the mitochondrial respiratory chain (13 Smith et al. (15) reported that bovine Cu,Zn-SOD reacts with peroxynitrite to form nitrotyrosine at Tyr-108 without inactivation of enzymatic activity. In contrast, Mn-and Fe-SOD from Escherichia coli are inactivated by peroxynitrite (6). Nitration of tyrosine residues was also observed. reported inactivation of Mn-SOD activity and concomitant increase of 3-nitrotyrosine in a tissue homogenate of transplanted allogripha during chronic rejection. They also reported that recombinant human Mn-SOD was inactivated by peroxynitrite with an IC 50 of 10 M and concomitant increase of nitrat...

Mutations in the clk-1 gene of Caenorhabditis elegans result in an extended life span and an average slowing down of developmental and behavioral rates. However, it has not been possible to identify biochemical changes that might underlie the extension of life span observed in clk-1 mutants, and therefore the function of CLK-1 in C. elegans remains unknown. In this report, we analyzed the effect of clk-1 mutation on ubiquinone (UQ 9 ) biosynthesis and show that clk-1 mutants mitochondria do not contain detectable levels of UQ 9 . Instead, the UQ 9 biosynthesis intermediate, demethoxyubiquinone (DMQ 9 ), is present at high levels. This result demonstrates that CLK-1 is absolutely required for the biosynthesis of UQ 9 in C. elegans. Interestingly, the activity levels of NADH-cytochrome c reductase and succinate-cytochrome c reductase in mutant mitochondria are very similar to those in the wild-type, suggesting that DMQ 9 can function as an electron carrier in the respiratory chain. To test this possibility, the short side chain derivative DMQ 2 was chemically synthesized. We find that DMQ 2 can act as an electron acceptor for both complex I and complex II in clk-1 mutant mitochondria, while another ubiquinone biosynthesis precursor, 3-hydroxy-UQ 2 , cannot. The accumulation of DMQ 9 and its use in mutant mitochondria indicate, for the first time in any organism, a link between the alteration in the quinone species used in respiration and life span.The understanding of the biological pathways that control life span can be studied in Caenorhabditis elegans through the identification of genes that alter the length of life when mutated (1). For example, mutations in clk-1 are known to cause an extended life span, as well as the slowing of a variety of developmental and physiological events, including the cell cycle, embryogenesis, post-embryonic development, and rhythmic adult behaviors (2, 3). Thus, CLK-1 is expected to play a unique biological role that is necessary to determine the life span and to coordinate these various biological processes. However, the biochemical differences between clk-1 mutants and the wildtype strain, which might indicate the function of CLK-1, have yet to been identified (1, 4 -7).clk-1 encodes a 187-residue polypeptide that is homologous to yeast coq7/cat5 (8). COQ7/CAT5 is located in the inner membrane of yeast mitochondria and is necessary for the biosynthesis of ubiquinone (UQ) 1 in yeast (9, 10). Therefore, yeast coq7/cat5 mutants, which lack UQ 6 , are unable to grow on nonfermentable carbon sources (9). Orthologs of clk-1/coq7/ cat5 have also been reported from mammals, including human (11-13), and appear to be highly conserved among species.Recently, a green fluorescent protein fusion to C. elegans CLK-1 was shown to localize to the mitochondria of all the somatic cells of the worm (14). However, in contrast to the situation in yeast, which is defective in respiratory growth, C. elegans clk-1 mutants are able to respire almost normally. In fact, the metabolic capacities and the A...

We studied chemical level and glycosylation status of haptoglobin in sera of patients with prostate cancer, as compared to benign prostate disease and normal subjects, with the following results. (i) Haptoglobin level was enhanced significantly in sera of prostate cancer. (ii) Sialylated bi-antennary glycans were the dominant structures in haptoglobins from all 3 sources, regardless of different site of N-linked glycan. The N-linked glycans at N184 were exclusively bi-antennary, and showed no difference between prostate cancer vs. benign prostate disease. (iii) Tri-antennary, N-linked, fucosylated glycans, carrying at least 1 sialyl-Lewis x/a antenna, were predominantly located on N207 or N211 within the amino acid 203-215 sequence of the b-chain of prostate cancer, and were minimal in benign prostate disease. Fucosylated glycans were not observed in normal subjects. A minor tri-antennary N-linked glycan was observed at N241 of the b-chain in prostate cancer, which was absent in benign prostate disease. (iv) None of these N-linked structures showed the expected presence of disialylated antennae with GalNAcb4(NeuAca3)Galb3(NeuAca6)GlcNAcbGal, or its analogue, despite cross-reactivity of prostate cancer haptoglobin with monoclonal antibody RM2. (v) Minor levels of O-glycosylation were identified in prostate cancer haptoglobin for the first time. Monoand disialyl core Type 1 O-linked structures were identified after reductive b-elimination followed by methylation and mass spectrometric analysis. No evidence was found for the presence of specific RM2 or other tumor-associated glycosyl epitopes linked to this Oglycan core. In summary, levels of haptoglobin are enhanced in sera of prostate cancer patients, and the N-glycans attached to a defined peptide region of its b-chain are characterized by enhanced branching as well as antenna fucosylation. ' 2007 Wiley-Liss, Inc.

The original version of this article unfortunately contained a mistake. The presentation of Fig. 3a was incorrect. The corrected figure is given below.