The Electron-transferring Flavoprotein of Primate Liver Mitochondria PURIFICATION OF THE: ESZYME AND RECONSTITUTION OF THE SARCOSINE OXIDASE SYSTEM’ DALE

The oxidation of sarcosine (N-methylglycine) by purified rat liver sarcosine dehydrogenase is known to require the participation of a flavin adenine dinucleotide-dependent protein, the electron-transferring flavoprotein, for efficient transfer of electrons between reduced dehydrogenase and 2,6-dichlorophenolindophenol (l), or cytochrome c (a), or cytochrome b (2). On the basis of spectral evidence, the primary dehydrogenase appears to be a flavoprotein (3). Rat liver is the only known tissue in which such a coupled flavoprotein system involving amino acid oxidases has been described. In a recent communication, however, we reported the solubilization, purification, and characterization of sarcosine dehydrogenase from liver mitochondria of the Rhesus monkey (,2~acaca mulatta) and demonstrated that the activity of the purified enzyme is dependent on a protein separable from the dehydrogenase during an early stage of purification (4). Preliminary evidence indicated that, the dehydrogenase is flavin-dependent. Characterization of the electronmediating protein was rendered difficult by its instability and by the fact that appreciable quantities of the dehydrogenase are required to monitor attempts at further purification. No requirement for flavin was shown for crude preparations of the mediating protein aft,er acid-ammonium sulfat,e precipitation under a variety of conditions (4). The observation that purified rat liver sarcosine dehydrogenase is capable of replacing the purified primate enzyme in the basic assay system has been the basis for tjhc purification and characterization of the electron-mediating protein from extracts of monkey liver mitochondria reported in this paper. Rat liver is the richest known source of the dehydrogenase. ‘This latter enzyme is easily purified, and is stable for many months when kept in the frozen state. It is t,hc purpose of t,his paper to report t.hc purification of the elertron-mediat’ing protein from extracts of monkey liver mitochondria, to present, evidence that the prosthetic group of this protein is flavin adenine dinucleotide, and to show the reconstit,ution of the sarcosine oxidase system with the use of purified monkey liver sarcosine dchydrogenase, the electron-transferring flavoprotein, and a particulate electron transport system essentially devoid of the latter two enzymes. Data relating to the inhibition of reronstitution by electron transport’ chain inhibitors are also reported.