Acyl-coenzyme A Oxidase 1 Research Articles

Peroxisomal Acyl-Coenzyme A Oxidase Is a Rate-Limiting Enzyme in a Very Long-Chain Fatty Acid β-Oxidation System

The contents of peroxisomal fatty acid β-oxidation enzymes in three rat hepatoma cell lines, i.e., H4IIEC3 (H4), N1S1, and McA-RH7777 (H7), were measured by immunoblot analysis, and a significant difference in acyl-coenzyme A oxidase (AOX) content became evident. These cell lines were respectively infected with a recombinant virus to express significant amounts of AOX protein. The expressed AOX mainly localized in organelle, supposing peroxisomes, and was catalytically active. The cDNA-expression in H4, N1S1, and H7 cells enhanced 2.6-, 2.2-, and 1.0-fold β-oxidation activity of lignoceric acid, respectively. The enhancement in H4 and NISI cells suggests that AOX is a rate-limiting enzyme in the very-long-chain fatty acid β-oxidation system, in these cell lines.

Molecular Cloning and Functional Expression of a Human Peroxisomal Acyl-Coenzyme A Oxidase

cDNA encoding the human peroxisomal acyl-coenzyme A oxidase (AOX) was cloned and sequenced, The longest cDNA insert isolated has 3083 bases and encodes the entire protein of 661-amino acids, including the carboxyl-terminal sequence (Ser-Lys-Leu) known as a minimal peroxisome-targeting signal. At the amino acid level, the significantly high homology (89%) to rat AOX was found. In the cDNA-expression experiment, significant amount of AOX was accumulated in human skin fibroblast and the expressed AOX was catalytically active, while only a limited amount was found in Zellweger syndrome patient′s fibroblast not having normal peroxisomes.

Induction of peroxisomes by treatment with perfluorooctanoate does not increase rates of H 2O 2 production in intact liver

Increases in acyl coenzyme A (CoA) oxidase activity due to peroxisome proliferation are postulated to cause oxidative stress via elevated production of H 2O 2, leading to DNA damage. These changes are suspected to be responsible for tumor formation caused by non-genotoxic carcinogens which do not bind to DNA but cause proliferation of peroxisomes. However, the activity of the peroxisomal enzyme acyl CoA oxidase assayed in vitro in the presence of excess fatty acyl CoA substrate may not reflect rates of H 2O 2 generation in intact liver where fatty acid supply is carefully controlled in part by delivery of substrate. The purpose of this work was to determine if rates of hepatic H 2O 2 generation were altered in perfused liver and in vivo following induction of H 2O 2-generating acyl CoA oxidase activity. Injection of the potent peroxisome proliferating agent perfluorooctanoate into rats 5 days prior to sacrifice caused an expected 4-fold increase of H 2O 2-generating acyl CoA oxidase activity measured in hepatic homogenates. In contrast, rates of H 2O 2 generation in perfused liver measured spectrophotometrically (660−640 nm) through a lobe of the liver were not altered by perfluorooctanoate treatment (7.3 ± 1.5 vs. 7.8 ± 0.5 μmol gh in livers from untreated control rats). Similar treatment with perfluorooctanoate also increased in vitro acyl CoA oxidase activity 9-fold in livers from deermice; however, rates of elimination of methanoi, a selective substrate for catalase in rodents whose oxidation is limited by the supply of H 2O 2, were not altered significantly in vivo (control, 110 ± 11 μmol gh vs. perfluorooctanoate, 112 ± 32 μmol gh ). Taken together, these data demonstrate that elevation of H 2O 2 formation by acyl CoA oxidase activity measured in vitro is not necessarily associated with increases in rates of H 2O 2 generation in intact perfused liver or in vivo, most likely due to rate-limitation in intact cells by fatty acid supply. These data do not support the hypothesis that the induction of peroxisomes leads to excessive H 2O 2 production and oxidative stress. It follows that alternative hypotheses to explain carcinogenesis caused by peroxisome-proliferating agents need to be considered.

Peroxisome Targeting Signal of Rat Liver Acyl-Coenzyme A Oxidase Resides at the Carboxy Terminus

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus.

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

Sensor for free fatty acids based on acyl coenzyme-a synthetase and acyl coenzyme-a oxidase

The measurement is based on monitoring dissolved oxgyen consumed by the two sequential reactions catalyzed by the enzymes immobilized in photo-cross-linkable poly (vinyl alcohol) resin (PVA-SbQ). A linear correlation was observed between current decrease and 0.3–2.6 mM oleic or palmitic acid. The sensor was stable in use for three days and could be used to monitor the freshness of oily food.