I04 THOMAS P. SINGER 



As to diaphorase, Massey's work [5, 6] has clearly demonstrated that it is 

 part of the a-ketoglutarate oxidase complex and, as such, it is concerned 

 with the reduction of DPN, not the oxidation of DPNH. More recently, 

 Ziegler aiid colleagues [3] have reported the isolation of a lipid-bound 

 DPNH dehydrogenase of very high molecular weight and lipid content 

 and with very interesting properties, and King and Howard reported the 

 extraction of DPNH dehydrogenase from heart muscle mince by treatment 

 with phospholipase [7]. Regarding the former preparation, it remains to 

 be seen whether its high lipid content represents a fimctional component 

 or an impurity. In either case, we felt that it was desirable to isolate the 

 dehydrogenase without recourse to organic solvents, bile salts, or other 

 harsh treatments and in a lipid-free form, so that, as in the case of succinic 

 dehydrogenase, the properties of the protein could be adequately charac- 

 terized. The possible relation of King and Howard's soluble preparation 

 to the enzyme I shall describe will be discussed later on in this paper. 



Our initial work on this problem w^as concerned wuth the linkage of 

 DPNH dehydrogenase to the respiratory chain. It has been known for over 

 five years that, although succinic and DPNH dehydrogenases operate via 

 a common respiratory chain and are interlinked at the oxidation level of 

 cytochrome h, methods which solubilize succinic dehydrogenase from 

 respiratory chain preparations [8] fail to extract DPNH dehydrogenase. 

 Thus, superficially, the bonds holding these two closely related enzymes 

 to the electron transport system appear to be quite different. During the 

 isolation of a-glycerophosphate dehydrogenase from brain mitochondria 

 a few years ago [9], we found that the incubation of brain mitochondria 

 with phospholipase A resulted not only in the extraction of a-glycero- 

 phosphate dehydrogenase in soluble form but also of considerable DPNH 

 dehvdrogenase activity. Following this initial lead, with Drs. Minakami 

 and Ringler, w-e decided to undertake its isolation and characterization [10]. 



Two problems faced us at the outset : the choice of starting material 

 and the assav. In our work on other mitochondrial dehydrogenases it has 

 been shown that these are the paramount factors deciding the success or 

 failure of the isolation. As to starting material, we did not wish to use 

 heart muscle mince or even mitochondria, since, besides the enzyme we 

 were after, they were bound to contain other DPNH dehydrogenases, 

 such as the Straub diaphorase, possibly Mahler's reductase, and any 

 DPNH dehydrogenases arising from microsomal contamination. We 

 decided, therefore, to use the particulate DPNH oxidase (ETP) prepara- 

 tion of Crane, Glenn, and Green [11], since this has been reported to be a 

 purified form of the DPNH dehydrogenase linked to the cytochrome chain, 

 free from numerous contaminating enzymes, particularly diaphorase, in 

 which the oxidation of DPNH is completely antimycin and amytal- 

 sensitive [11]. Thus any soluble preparation isolated from it would be 



