Abstract
Cell-free extracts prepared from rat kidney or liver catalyzed the oxidative deamination of S-adenosyl- L -homocysteine to S-adenosyl-γ-thio-α-ketobutyrate. This reaction was found to be catalyzed by L -amino acid oxidase ( L -amino acid:oxygen oxidoreductase (deaminating), EC 1.4.3.2). In the presence of catalase, 0.48 µmole of oxygen was consumed for each micromole of substrate oxidized, and 1 µmole each of S-adenosyl-γ-thio-α-ketobutyrate and ammonia were formed. In the absence of catalase oxygen consumption was doubled. The pH optimum for S-adenosyl-γ-thio-α-ketobutyrate formation ranged from 8.8 to 9.2 and the Km value for S-adenosyl- L -homocysteine was 2.48 × 10−2 M . At substrate concentrations of 10 m M and at pH 9.0, the ratio of specific activities with S-adenosyl- L -homocysteine to that with L -leucine or with L -methionine was 0.31 and 0.41, respectively. Partially purified L -amino acid oxidase was also found to be active with L -homocysteine and S-adenosyl- L -methionine.
Highlights
In the presence of catalase, 0.48 pmole of oxygen was consumed for each micromole of substrate oxidized, and 1 pmole each of S-adenosyl-y-thio-a-ketobutyrate and ammonia were formed
Tissue Survey-To investigate the formation of S-adenosyl-ythio-a-ketobutyrate from S-adenosyl-L-homocysteine, dialyzed extracts prepared from rat kidney, liver, spleen, heart, small intestine, muscle, brain, and testes were incubated with S
Chromatographic analysis of the various assay mixtures resulted in the isolation of measurable arnounts of S-adenosyl,y-thio-o-ketobutyrate only from those mixtures in which kidney and liver extracts were used
Summary
L-Homocysteine thiolactone-IICl, L-methionine, r,-leucine (methioninc-free), pyridosal phosphate, NA%l), and SXDP rvcre purchased from Calbiochem. Cx-ketobutyrate, cY-ketof;lutarate, and oc-ketoisocaproic acid were obtained from. The cr.keto acids Ivhich n-we not availublc commercially n-erc lxeparcd by the complete oxidation of known amounts of the corresponding amino acids with crystalline n-amino acid osidase from Cro:alus terr~$cus lerrificus venom in the presence of excess bovine liver catalase. Hot the L-amino acid osidase and the cat&se wcrc purchased from l%ochringcr, Mannheim. S-XdenosSl-L-homoc~steilze and S-:Ldel?osyl~3H-L-horno~~steine (adenosine-labclctl) were prepared as outlined previously [4, 5]. 0.75 a Cnits per mg of protein, measured b S-Adenosyl-L-homocysteine.
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