Effect of ionizing radiation on liver protein oxidation and metabolic function in C57BL/6J mice.

PURPOSE: Protein oxidation in response to radiation results in DNA damage, endoplasmic reticulum stress/unfolded protein response, cell cycle arrest, cell death and senescence. The liver, a relatively radiosensitive organ, undergoes measurable alterations in metabolic functions following irradiation. Accordingly, we investigated radiation-induced changes in liver metabolism and alterations in protein oxidation. MATERIALS AND METHODS: C57BL/6 mice were sham irradiated or exposed to 8.5 Gy (60)Co (0.6 Gy/min) total body irradiation. Metabolites and metabolic enzymes in the blood and liver tissue were analyzed. Two-dimensional gel electrophoresis and OxyBlot™ were used to detect carbonylated proteins that were then identified by peptide mass fingerprinting. RESULTS: Analysis of serum metabolites revealed elevated glucose, bilirubin, lactate dehydrogenase (LDH), high-density lipoprotein, and aspartate aminotransferase within 24-72 h post irradiation. Liver tissue LDH and alkaline phosphatase activities were elevated 24-72 h post irradiation. OxyBlotting revealed that the hepatic proteome contains baseline protein carbonylation. Radiation exposure increased carbonylation of specific liver proteins including carbonic anhydrase 1, α-enolase, and regucalcin. CONCLUSIONS: 8.5 Gy irradiation resulted in distinct metabolic alterations in hepatic functions. Coincident with these changes, radiation induced the carbonylation of specific liver enzymes. The oxidation of liver enzymes may underlie some radiation-induced alterations in hepatic function.

In vitro synthesis of arachidonoyl amino acids by cytochrome c.

Arachidonoyl amino acids are a class of endogenous lipid messengers that are expressed in the mammalian central nervous system and peripherally. While several of their prominent pharmacologic effects have been documented, the mechanism by which arachidonoyl amino acids are biosynthesized has not been defined. We have previously observed that the mitochondrial protein, cytochrome c, is capable of catalyzing the formation of the prototypic arachidonoyl amino acid, arachidonoyl glycine, utilizing arachidonoyl CoA and glycine as substrates, in the presence of hydrogen peroxide. Here we report that cytochrome c is similarly able to catalyze the formation of N-arachidonoyl serine, N-arachidonoyl alanine, and N-arachidonoyl gamma aminobutyric acid from arachidonoyl CoA and the respective amino acids. The identities of the arachidonoyl amino acid products were verified by mass spectral fragmentation pattern analysis. The synthetic reactions exhibited Michaelis-Menten kinetics and continued favorably at physiologic temperature and pH. Spectral data indicate that both cytochrome c protein structure and a +3 heme iron oxidation state are required for the reaction mechanism to proceed optimally. Reactions designed to catalyze the formation of N-arachidonoyl dopamine were not efficient due to the rapid oxidation of dopamine substrate by hydrogen peroxide, consuming both reactants. Finally, under standard assay conditions, arachidonoyl CoA and ethanolamine were found to react spontaneously to form anandamide, independent of cytochrome c and hydrogen peroxide. Accordingly, it was not possible to demonstrate a potential role for cytochrome c in the biosynthetic mechanism for either arachidonoyl dopamine or anandamide. However, the ability of cytochrome c to effectively catalyze the formation of N-arachidonoyl serine, N-arachidonoyl alanine, and N-arachidonoyl gamma aminobutyric acid in vitro highlights its potential role for the generation of these lipid messengers in vivo.

Cytochrome c catalyzes the in vitro synthesis of arachidonoyl glycine.

Long chain fatty acyl glycines are an emerging class of biologically active molecules that occur naturally and produce a wide array of physiological effects. Their biosynthetic pathway, however, remains unknown. Here we report that cytochrome c catalyzes the synthesis of N-arachidonoyl glycine (NAGly) from arachidonoyl coenzyme A and glycine in the presence of hydrogen peroxide. The identity of the NAGly product was verified by isotope labeling and mass analysis. Other heme-containing proteins, hemoglobin and myoglobin, were considerably less effective in generating arachidonoyl glycine as compared to cytochrome c. The reaction catalyzed by cytochrome c in vitro points to its potential role in the formation of NAGly and other long chain fatty acyl glycines in vivo.