Isolation of reconstitutively active succinate dehydrogenase in highly purified state.

Existing procedures for the isolation of mammalian succinate dehydrogenase yield preparations of high purity or retain reconstitution activity, but not both. A new procedure is described for the isolation in good yield of virtually homogeneous preparations with full reconstitution activity, and retaining iron-sulfur center 3 and the "low Km" reaction site of ferricyanide. On reincorporation of the soluble enzyme into alkali-treated membranes, the same high turnover number (approximately 21,000/min at 38 degrees) is obtained in catalytic assays as with intact inner membrane preparations.

When succinate dehydrogenase is removed from its natural environment of the inner mitochondrial membrane, it becomes extremely labile, modifications to its structure being mainfested as changes in various activities of the enzyme. In order to investigate more closely what structural modifications may occur and to relate them to changes in catalytic activity, it is highly desirable to have available a soluble form of the enzyme which is essentially pure and as unmodified as possible. Preparations hitherto described do not meet all these criteria.
The first highly purified preparation reported (1) appeared homogeneous by physical criteria, but a major contaminant was later detected on SDS-acrylamide gels. Moreover, it does not restore succinoxidase activity to alkali-treated membranes. King's (2) modification of the l-butanol extraction procedure (3) yields preparations in which nearly all the catalytically active molecules are also reconstitutively active, but the purity is low (-30%) and further purification inevitably results in the loss of reconstitution activity, since the latter survives only for a short period in the soluble state, even in anaerobiosis. Further, when Keilin-Hartree particles are used as the starting material, as recommended (2), the turnover number in the succinate/PMS/DCIP assay is low (-10,000 to ll,OOO), apparently because the starting material contains some inactivated enzyme, which is extracted along with the active enzyme and contributes to the histidyl flavin content on which the turnover number is based (4). In order to circumvent this last problem, ETP, which does not contain inactivated succinate dehydrogenase, has been used as the starting material (4). The l-butanol/succinate-extracted enzyme from this source has a high turnover number (-15,000 at 38"); 85 to 90% of the enzyme molecules are reconstitutively active; nearly all have low K,,, ferricyanide reductase activity; and the enzyme retains the EPR signal attributed to the Fe& nonheme iron cluster (HiPIP or center 3). This signal is not elicited if the enzyme is reconstitutively inactive. The purity of this type of preparation, however, is still low (-30%).
Another useful procedure is that of Davis and Hatefi (51,in which the enzyme is extracted from Complex II by strong perchlorate solutions in the presence of succinate and a thiol. The preparation is essentially pure, but suffers from the fact that its reconstitution activity is low, i.e. only about 15% of the molecules are reported to be capable of recombining with alkali-treated membrane preparations (ETP) and reconstituting succinoxidase activity (6). In confirmation of this, we have found that only about 15% of the enzyme population has low K,,, ferricyanide reductase activity (7), another test for the intactness of the enzyme (8,9).
This communication describes a new method for the rapid isolation of the enzyme which combines the advantages of the King (2) and  procedures and yields an enzyme of high purity with essentially full reconstitution activity.

Complex
II was isolated with minor modifications (11) ofthe methods of Baginsky and Hatefi (12). The Davis-Hatefi preparation was isolated from Complex II by the procedure of these authors (5)

AND DISCUSSION
The dehydrogenase was isolated from Complex II by a procedure based on the steps and apparatus described by King (2) for the purification of the enzyme from Keilin-Hartree particles.

Reconstitutively
Active Succinate Dehydrogenase   activity. This is based on the type of plot shown in Fig. 1 low K,rs ferricyanide reductase activity. which compares the enzyme made by the new method with The advantage of using Complex II as starting material for the perchlorate-extracted enzyme isolated with meticulous preparation of the soluble enzyme is that significant purificacare for maintaining anaerobiosis.
As noted here, the l-bu-tion has been achieved prior to rendering the enzyme soluble, tanol-extracted enzyme has much greater reconstitution activ-whereupon it becomes extremely labile. The anaerobic lity. On comparison with preparations isolated exactly as butanol/succinate extraction procedure applied here serves to described by Davis and Hate6 (5), an even greater (about 7-preserve the extremely labile activity for the few steps then fold) difference is found (6). If the same data are plotted with needed to achieve essentially full purification. The data sugthe abscissa denoting only the moles of enzyme having low gest that the lower reconstitution activity of the Davis-Hatefi K,,, ferricyanide activity, instead of total enzyme, added to preparation may arise during extraction of the enzyme with the membrane, then the two types of preparations give nearly perchlorate.
We feel the most deleterious feature of this coincidental reconstitution activity. This shows once again method may be the introduction of air in the homogenizations that the low K,,, ferricyanide reductase and reconstitution used in the perchlorate extraction. activities are interchangeable measures of the intactness of the dehydrogenase (7-9). It is well known that the turnover number of the enzyme is much higher in mitochondria and in the membrane than in the extracted form, but that reincorporation into the membrane restores the original high turnover number (4). We regard this as an expression of the modulation of the activity by the membrane environment.
In untreated membrane samples, the turnover number at 38" is 21,000 to 23,000 in the PMS/DCIP assay (4). Alkali treatment abolishes this activity. On mixing anaerobically, under conditions previously described (41, an alkali-treated ETP containing -12 nmol of succinate dehydrogenase in the original active state with a substoichiometric amount (4.9 nmol; 62 units of activity) of the highly purified enzyme just described, almost all of the enzyme recombines with the membrane and there is an absolute increase in the total dehydrogenase activity (activity units now equal 86.5 units). The reconstituted membrane, collected by sedimentation, has, within experimental error, the same high turnover number (TN = 21,600) as untreated ETP, while the trace of uncombined enzyme in the supernatant (maintained anaerobic during centrifugation) has a low turnover number in this assay (6,200) and lacks completely 2.