Abstract
Sarcosine oxidation in mammals occurs via a mitochondrial dehydrogenase closely linked to the electron transport chain. An additional H2O2-producing sarcosine oxidase has now been purified from rabbit kidney. A corresponding cDNA was cloned from rabbit liver and the gene designated sox. This rabbit sox gene encodes a protein of 390 amino acids and a molecular mass of 44 kDa identical to the molecular mass estimated for the purified enzyme. Sequence analysis revealed an N-terminal ADP-betaalphabeta-binding fold, a motif highly conserved in tightly bound flavoproteins, and a C-terminal peroxisomal targeting signal 1. Sarcosine oxidase from rabbit liver exhibits high sequence homology (25-28% identity) to monomeric bacterial sarcosine oxidases. Both purified sarcosine oxidase and a recombinant fusion protein synthesized in Escherichia coli contain a covalently bound flavin, metabolize sarcosine, L-pipecolic acid, and L-proline, and cross-react with antibodies raised against L-pipecolic acid oxidase from monkey liver. Subcellular fractionation demonstrated that sarcosine oxidase is a peroxisomal enzyme in rabbit kidney. Transfection of human fibroblast cell lines and CV-1 cells (monkey kidney epithelial cells) with the sox cDNA resulted in a peroxisomal localization of sarcosine oxidase and revealed that the import into the peroxisomes is mediated by the peroxisomal targeting signal 1 pathway.
Highlights
Sarcosine oxidation in mammals occurs via a mitochondrial dehydrogenase closely linked to the electron transport chain
Purification of Sarcosine Oxidase—Rabbit kidney sarcosine oxidase catalyzes the demethylation of sarcosine to glycine, formaldehyde, and H2O2
When the gene for this protein was cloned, we found that its amino acid sequence showed the most homology to the monomeric sarcosine oxidases
Summary
Materials—Sarcosine, L-pipecolic acid, 4-hydroxyphenylacetic acid, protease inhibitors, protein molecular weight standards, and protein standards for isoelectric focusing were from Sigma (Deisenhofen, Germany). The resulting pellet was resuspended in 3.5 volumes (v/w) homogenization buffer and again centrifuged at 600 ϫ g for 10 min Both supernatants were combined and further centrifuged at 5,100 ϫ g for 15 min to pellet the M fraction which contained most of the mitochondria and peroxisomes. This pellet was gently rehomogenized in 6 volumes of homogenization buffer. Butyl-Sepharose CL 4B (Pharmacia, Freiburg, Germany), equilibrated with 1 M potassium phosphate buffer, 0.1 mM EGTA, pH 7.8 (column 1 ϫ 2 cm, 1.6-ml bed volume) was loaded with the enzyme solution (0.47 ml/min) and washed with 15-bed volumes of equilibration buffer containing 1 M pepstatin and 1 M leupeptin. Spectophotometric Characterization of the Flavoprotein—Sarcosine oxidase dissolved in 10 mM potassium phosphate buffer, 0.1 mM EGTA, pH 7.8, was initially scanned in a Shimadzu double beam dual wave-
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