SOLUBILIZATION AND PROPERTIES OF THE DPNH DEHYDROGENASE lOQ 



of ETP or DPNH oxidase preparations to this inhibitor in the flavoprotein- 

 ferricyanide interaction (cf. discussion of Fig. i) suggest that amytal 

 interrupts electron transport between flavoprotein and the respiratory 

 chain, as is also the case in the choline oxidase system of liver [17], and not 

 between DPNH and flavoprotein. Contrary conclusions in the earlier 

 literature were based on the "cross-over technique" which relies on the 

 measurement of the oxidation state of the flavoprotein in the 450-465 mfj. 

 region. As will be documented later in this paper, the application of this 

 technique to DPNH dehydrogenase has some major weaknesses: the 

 diff'erence spectrum is atypical of simple flavoproteins; the extinction 

 coefficient of simple flavoproteins at 465 m^u, is not applicable to this 

 enzyme; and it is not even certain that the flavin is fully reduced in 

 normal catalysis. 



40 



NAJA-NAJA 1:25 



(mm ) 

 Fig. 6. Progress of solubilization of DPNH dehydrogenase by phospholipase 

 A. Aliquots of a DPXH oxidase preparation [11] were incubated at 30^, pH 7-4, 

 with Naja naja venom as a source of phospholipase. The ratios indicated define 

 the mg. weight of venom employed per mg. protein in the particulate preparation 

 (determined by biuret reaction, using a coefficient of 0-095 for i nng. protein per 

 3 ml., I cm. light path, 540 m/w). At various times aliquots were rapidly cooled to 

 o , centrifuged at 105 000 x g for 30 min., and the supernatant solution was assayed. 

 Activities are given in arbitrarv units on the ordinate. 



Turning now to the isolation and characteristics of the dehydrogenase, 

 Fig. 6 shows the progress of solubilization of the enzyme, starting with 

 ETP, at two levels of cobra venom. Compared with brain a-glvcerophos- 

 phate dehydrogenase [9] and choline dehydrogenase from liver [18], the 

 level of phospholipase A (cobra venom) required for extensive solubiliza- 

 tion of DPXH dehydrogenase is quite high and the progress of the reaction 

 under the same conditions is rather slow. These observations are in accord 

 with King and Howard's findings on the conditions necessary for the 

 extraction of the various DPXH oxidizing activities from heart muscle 

 mince [7]. Table I shows the balance of solubilization. It may be noted 

 that the assay is reliable for both particles and the soluble enzyme, since 

 the recovery is satisfactory. By repeating the incubation with a second 



