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From a study of the magnetic field dependence of the linear electric field effect (LEFE) in EPR spectroscopy, we demonstrate that iron-sulfur cluster 3 in air-oxidized, beef heart succinate-ubiquinone oxidoreductase (Complex II) is a 3-iron cluster. This suggests that cluster 3 may arise by oxidative degradation from a 4-iron cluster originally present in the enzyme and may be reconverted back into a 4-iron cluster under reducing conditions. Linear electric field effect studies of succinate-reduced Complex II are in accord with the view that cluster 1 is a 2-iron cluster.

The turnover number of succinate dehydrogenase from mammalian heart determined by the spectrophotometric phenazine methosulfate assay, after complete activation, is approximately 21,000 mol of succinate oxidized/min/mol of histidyl flavin at 38 degrees in relatively intact inner membrane preparations and mitochondria. Reconstitutively active soluble preparations, extracted anaerobically in the presence of succinate from inner membrane preparations show turnover numbers of 11,500 to 14,500 and a significantly lower apparent Km for phenazine methosulfate than the parent particles. The decline of both the turnover number and of the Km occurs during the brief period when the enzyme is detached from the membrane. The observed values represent the activities in the soluble extract of both the reconstitutively active and reconstitutively inactive enzyme. The latter may be from 10 to 40% even in the most carefully prepared enzyme; it has a lower turnover number in the phenazine methosulfate assay than the average for the solution and is devoid of catalytic activity in the "low Km" ferricyanide assay (Vinogradov, A. D., Ackrell, B.A.C., and Singer, T.P. (1975) Biochem. Biophys. Res. Commun. 67, 803-809). The reconstitutively active form of the soluble enzyme has a turnover number of at least 15,000 and an equal activity in the low Km ferricyamide assay. When recombined with the membrane the total activity of the enzyme is increased by over 60% and it regains the original turnover number, Km for phenazine methosulfate, and sensitivity of the phenazine methosulfate reductase activity to thenoyltrifluoroacetone, carboxamides, and cyanide. It appears, therefore, that the membrane environment or some component of it exerts a positive modulating influence on the enzyme even in the fully activated state. In certain particulate sources (Keilin-Hartree preparations, Complex II) the enzyme shows lower turnover numbers (11,000 to 12,500) than in more intact inner membranes. This seems to be due to inactivation in the course of preparation and, in the case of Complex II, in part also to loss of the normal membrane environment or of a membrane component, possibly Q-10, during isolation.

The fumarate reductase complex and soluble enzyme from Escherichia coli have been investigated by low temperature magnetic circular dichroism and electron paramagnetic resonance spectroscopies. The results confirm the presence of one [2Fe-2S] cluster and show that the high potential iron-sulfur center is a 3Fe cluster of the type found in bacterial ferredoxins. Since the 3Fe cluster is present in catalytically competent enzyme and does not appear to be involved in any type of cluster conversion under reducing conditions, we conclude that it is an intrinsic component of the functional enzyme. The significance of the results is discussed in relation to the published amino acid sequence and the iron-sulfur cluster composition of bacterial fumarate reductases.

The reoxidation phase of the catalytic cycle of succinate dehydrogenase was studied in Complex II preparations' by the rapid freeze-electron paramagnetic resonance (epr) technique. With the synthetic water-soluble Q1 analog, 2,3-dimethoxy-5-methyl-6-pentyl-1, 4-benzoquinone (DPB), as the oxidant, the observed reoxidation of the epr-detectable components, previously reduced with dithionite or succinate, came to completion within a few milliseconds, well within the turnover time of the enzyme. Only ~80% of Fe-S center 1 and the HiPIP (the high-potential cluster) Fe-S center reacted rapidly with DPB, however; similarly incomplete reactions were observed previously in our studies of the reduction of the enzyme by succinate. The subsequent addition of ferricyanide, which appears to act as a chemical oxidant in these experiments, caused immediate reoxidation of the Fe-S centers and of the free radical. Ferricyanide and phenazine methosulfate (PMS) reoxidized all epr-detectable components in Complex II as well as in reconstitutively active, soluble preparations in'

Riboflavin-requiring mutants of Saccharomyces cerevisiae are able to transport 14C-labeled riboflavin into the cell, although no significant transport is seen in commercial yeast or in the parent strain from which the mutants were derived. Transport activity is greatest in the early to mid-log phase of anaerobic growth and declines sharply in the late log phase. In aerobically grown cells activity is substantially lower at all stages of growth. In the assay devised for its measurement, transport activity shows a sharp pH optimum at pH 7.5, a strong temperature dependence (EA = 23,100 cal/mol), and saturation kinetics with respect to riboflavin (Km = 15 muM), characteristics consistent with a carrier-mediated mechanism. Monovalent inorganic cations, particularly K+ and Rb+, stimulate riboflavin uptake, while certain organic cations are inhibitory. Besides riboflavin only 7-methylriboflavin, 8-methylriboflavin, and 5-deazaflavin have been found to serve as substrates, while lumiflavin, tetraacetylriboflavin, and N10-[4'-carboxybutyl]-7,8-dimethylisoalloxazine do not, although a number of flavin analogs in which the ribityl side chain is modified are good competitive inhibitors of riboflavin uptake. Compounds resembling the ribityl side chain, such as sugars and sugar alcohols, do not inhibit. An apparent inhibition of uptake by D-glucose, D-mannose, and D-fructose, which develops in the course of assay, proved to result from stimulation of an opposing process, the release of riboflavin from the cells.

Succinate dehydrogenase in the deactivated form becomes activated on incubation at mildly acid pH to an extent dependent on the pH, type of anion present, and the concentration of the anion. The equilibrium level established under one set of conditions can be perturbed by changing either the pH or the anion concentration and the resulting level of activation reflects the new set of conditions. The rate of activation is influenced by the temperature, the anion concentration, and the pH when simple anions (e.g. Br-) are used, but the energy of activation is not affected by either pH or anion concentration. The value found for the activation energy (Ea) is 25 Cal per mole. In contrast, when activation of the enzyme is mediated by succinate, malonate, or reduced coenzyme Q10, the Ea value is 31 to 33 Cal per mole. Although this difference in energies of activation suggests that activation by mild acid and anions involves a different mechanism, no difference in the catalytic activity of preparations activated by these different methods is detected.

The effects of iron deficiency on the NADH- and succinate-oxidizing complexes of rat skeletal muscle mitochondria have been investigated. Both systems were similarly affected: activities were about 30% of normal in dehydrogenase, ubiquinone reductase, and oxidase assays, and similar reductions in the concentration of their respective flavin prosthetic groups were also evident in the iron-deficient membranes. Thus, the turnover numbers of the two enzymes were unchanged in iron deficiency. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed similarly reduced levels of those peptide components of Complexes I and II that could be unequivocally distinguished. Soluble beef heart succinate dehydrogenase added to alkaline-treated rat skeletal muscle mitochondrial membranes attached to binding sites exposed by the treatment, forming a hybrid complex indistinguishable from the original skeletal muscle complex, with restoration of succinoxidase and succinate-ubiquinone reductase activities to the levels observed in the original rat membranes. Iron-deficient particles behaved like the normal in these tests. No unfilled binding sites for the enzyme could be detected prior to alkaline treatment. The data are interpreted as indicating that the lower activities of these two respiratory complexes in iron deficiency are due to lower content of the enzymes rather than to the presence of impaired enzymes in the membrane, that only fully competent complexes are present in these membranes, and that iron-deficient complexes are either not assembled or are lost after assembly.

Electron paramagnetic resonance studies of Complex II from the mitochondrial respiratory chain and soluble preparations of succinate dehydrogenase have, for the first time, identified a signal arising from a [4Fe-4S]1+ cluster, S2, in dithionite-reduced samples. Redox titrations, monitored by electron paramagnetic resonance spectroscopy demonstrate that this signal appears at the same midpoint potential as the enhancement of the spin relaxation properties of the [2Fe-2S]1+ center, S1, in both Complex II and reconstitutively active soluble enzyme. The results complement recent magnetic circular dichroism studies of succinate dehydrogenase (Johnson, M. K., Morningstar, J. E., Bennett, D. E., Ackrell, B. A. C., and Kearney, E. B. (1985) J. Biol. Chem. 260, 7368-7378) which assigned cluster S2 as a [4Fe-4S]2+,1+ center and provide evidence for spin interaction between the paramagnetic reduced forms of centers S1 and S2.

When succinate dehydrogenase contains oxalacetate in firmly bound form, activity cannof the enzyme results in dissociation of oxalacetate and activation of the enzyme. The course of reductive titrations appears the same whether or not the enzyme contains oxalacetate, and complete reduction as monitored by bleaching of chromophoric groups requires the incorporation of 6 to 7 reducing equivalents in either case. The stoichiometry is that expected from the non-heme iron and flavin content of the enzyme. Activation of the enzyme during reductive titrations occurs predominantly with the incorporation of the second pair of electrons, while determination of activation levels at various poised potentials shows that the group involved is reduced with the uptake of 2 H+ and 2 e-. These characteristics are consistent with titration of the flavin moiety rather than non-heme iron groups. Thus it appears that activation is concurrent with the reduction of flavin to the hydroquinone form. From the measured half-reduction potential for activation, that of the flavin in an oxalacetate-free enzyme has been estimated at -90 to -60 mv at pH 7.

Reconstitutively active, soluble preparations of succinate dehydrogenase have been examined for the stoichiometric relationship of the covalently bound flavin to the electron paramagnetic resonance (epr) signals of the iron-sulfur centers known to be present in membrane-bound forms of the enzyme. The Fe-S center ( g = 2.01), which is paramagnetic in the oxidized state (the high-potential Fe-S cluster of the enzyme, HiPIP) and which was identified in earlier studies as a component of succinate-ubiquinone reductase (Beinert et al. , 1975, Eur. J. Biochem. 54 , 185), can be demonstrated in reconstitutively active, soluble preparations (0.20 to 0.35 equiv/mol of enzyme) at a significantly higher level than in enzyme preparations extracted without succinate. Studies of the kinetics of the reduction of the epr-detectable Fe-S centers revealed no difference between reconstitutively active and inactive preparations in the behavior of centers 1 and 2 and, of the oxidized Fe-S center detected in reconstitutively active preparations, only a part or none at all was reduced by succinate at catalytically significant rates. The failure to demonstrate the HiPIP center in stoichiometric amounts even when the enzyme is extracted and purified under anaerobic conditions in the presence of succinate may be due to the experimental conditions needed to convert this center to the paramagnetic state for observation, i.e., the obligatory use of artificial electron acceptors, such as ferricyanide. A procedure devised to circumvent this problem by incorporating the soluble enzyme into alkali-treated membranes resulted in a reconstituted preparation which was more stable to oxidizing agents. After incorporation into the membrane, between 50 and 100% of the HiPIP center was then detectable, suggesting a relationship between this Fe-S component and reconstitution activity. It has also been noted that, under aerobic conditions, the rates of decay of this epr signal, the reconstitution activity, and the catalytic activity of soluble preparations with low concentrations of ferricyanide as electron acceptor go parallel and may, indeed, be functions of the integrity of the same property of the protein. During this time, about half of the phenazine methosulfate reductase activity also declines, and the process is accompanied by a major increase in K m for phenazine methosulfate. The content of the two ferredoxin-type Fe-S cehters, one of which is reduced by the substrate (center 1) and the other by dithionite but not by succinate (center 2), in reconstitutively active preparations did not differ significantly from that of reconstitutively inactive, soluble preparations. On the basis of these data, the notion that the ferredoxin-type Fe-S centers or signals of reconstitutively active enzyme have properties very different from those of other preparations is considered lacking in experimental support.

In most procedures for the isolation of membrane-bound or soluble succinate dehydrogenase, the enzyme is obtained largely in the deactivated form. As judged by the quantity of oxalacetate enzymatically determined in perchlorate extracts, the deactivated form contains tightly bound oxalacetate in 1:1 proportion to the enzyme (equated with histidyl flavin content) in particles. A somewhat lower ratio has been obtained for soluble preparations of the enzyme. The oxalacetate is bound very tightly: it is not displaced by succinate or malonate in the cold, nor released by precipitation or gel exclusion of the enzyme, and it does not react with malate dehydrogenase. It is released, however, on denaturation of the enzyme with perchloric acid or under conditions which favor conversion of the enzyme to the activated form. If the enzyme is freed from oxalacetate by activation with bromide and gel exclusion, treatment of the oxalacetate-free fully active enzyme with very low concentrations of l- or d-malate or oxalacetate leads to deactivation and concomitant formation of tightly bound oxalacetate in the same molar ratio found in the enzyme as isolated from heart mitochondria. Dissociation of the complex formed in this manner requires the same experimental conditions as those needed for dissociation of the complex as found in the isolated enzyme. The activation energies for activation and for release of oxalacetate are the same but vary with the type of activator used. The first order rate constant for activation by a variety of agents is about twice that measured for the dissociation of tightly bound oxalacetate. Moreover, with some activators oxalacetate release shows a considerable lag behind recovery of catalytic activity. It is suggested that the presence of oxalacetate in the deactivated enzyme is not the cause of its lack of catalytic activity but a consequence of the deactivated conformation. Further observations are described which suggest that a deactivation-activation cycle may also occur in the absence of oxalacetate.

Reconstitutively active and inactive succinate dehydrogenase have been investigated by low temperature magnetic circular dichroism (MCD) and EPR spectroscopy and room temperature CD and absorption spectroscopy. Reconstitutively active succinate dehydrogenase is found to contain three spectroscopically distinct Fe-S clusters: S1, S2, and S3. In agreement with previous studies, MCD and CD spectroscopy confirm that center S1 is a succinate-reducible [2Fe-2S]2+,1+ center. The MCD characteristics of center S2 identify it as a dithionite-reducible [4Fe-4S]2+,1+ similar to those in bacterial ferredoxins. EPR power saturation studies and the weakness of the EPR signal from reduced S2 indicate that there is a weak magnetic interaction between centers S1 and S2 in their paramagnetic, S = 1/2, reduced states. Center S3 is identified both by the form of the MCD spectrum and the characteristic magnetization behavior as a reduced [3Fe-xS] center in both succinate- and dithionite-reduced reconstitutively active succinate dehydrogenase. Arguments are presented in favor of centers S2 and S3 being separate centers rather than interconversion products of the same cluster. Reconstitutively inactive succinate dehydrogenase is found to be deficient in center S3. These results resolve many of the controversies concerning the Fe-S cluster content of succinate dehydrogenase and reconcile published EPR data with analytical and core extrusion studies. Moreover, they indicate that center S3 is a necessary requirement for reconstitutive activity and suggest that it is able to sustain ubiquinone reductase activity as a [3Fe-xS] center.

Summary Reductive titrations and rapid kinetic studies are reported on extensively or completely activated, particulate and solubilized succinate dehydrogenase (SD) preparations. There is one iron-sulfur (Fe−S) center of the ferredoxin type present per flavin which is reduced by succinate, but even in activated preparations at most 60% of these centers were reduced within the turnover time of the enzymes. Flavin semiquinone formation does not precede or significantly lag behind the reduction of the Fe−S centers. On reduction of the soluble enzymes an accumulation of semiquinone is observed. No qualitative difference between the behavior of preparations containing 4 Fe or 8 Fe per flavin was found. Succinate-ubiquinone reductase (Complex II) contains an Fe−S component with properties of high-potential Fe−S proteins (See Ruzicka and Beinert, Biochem. Biophys. Res. Communs. this issue). It occurs at a concentration close to that of the bound flavin and has been observed to be reduced by succinate at approximately the same rate as the ferredoxin type (g=1.94) component. With dithionite, reduction of additional Fe−S groups (0.2 to 0.5 per flavin) is observed but the significance of this is uncertain.

The reduced nicotinamide dinucleotide dehydrogenase of the respiratory chain has been further purified by gradient centrifugations at pH 10. The best preparations obtained are approximately 70% pure by sedimentation analysis and show a single flavoprotein component. The molecular weight is estimated to be of the order of 550,000, and the turnover number 800,000 per minute at 30 degrees. Provisional data are given for the NADH dehydrogenase content of heart mitochondria, Keilin-Hartree preparations, and electron transport particles. It is shown that NADH dehydrogenase contributes to the acid-extractable flavin of these preparations only to a very minor extent. (Author)

1. 1. The spontaneous, reversible inactivation of the l -amino acid oxidase of snake venoms has been compared with reversible processes which regulate the activity of other enzymes. 2. 2. Since no change in the size, shape, electrophoretic mobility or solubility of the oxidase has been detected upon extensive inactivation of the enzyme, the process has been interpreted as involving only a limited alteration of the configuration of the native enzyme rather than an extensive unfolding. 3. 3. The mechanism of this reversible process has been interpreted as follows. The active enzyme is in reversible equilibrium with an inactive form, which differs from the active form by the presence of a new acidic group not present in the native enzyme, or an acidic group whose dissociation constant changes by about two pH units as a result of structural alterations in the protein during the inactivation step. The probable nature of this ionizing group has been discussed and its possible identity with an imidazole nitrogen, located at the active center of the enzyme, has been pointed out. 4. 4. On the basis of the above hypothesis, a kinetic development has been presented to account for the fact that the rate of reactivation of the enzyme is a function of pH while the rate of inactivation is not. 5. 5. It has been suggested that the inactivation process may involve the reversible breakage of one or two strong covalent linkages or of several weaker hydrogen bonds and that at least one of these may be joined to an imidazole ring at the active center of the enzyme. 6. 6. The possible nature of the ionizing groups at the active center of the enzyme is discussed from considerations of the influence of pH on the activity and stability of the enzyme.

Summary Succinate dehydrogenase (SD) in intact, respiring mitochondria undergoes activation and deactivation in response to the metabolic state of the mitochondria. Highest SD activity is observed in state 4 and lowest in state 2 or in the presence of uncouplers, while in state 3 the level of activation is intermediate and varies with the nature of the substrate. Transition from the active to inactive (unactivated) state of SD occurs in mitochondria with a lower energy of activation (10 Kcal/mole) than in soluble or membrane preparations (33 to 36 Kcal/mole). In addition to activation by succinate and CoQ 10 H 2 , the enzyme in mitochondria is uniquely activated by ATP (or a compound in equilibrium with it), a process which has not been previously observed in submitochondrial particles and is oligomycin-insensitive. In tightly coupled mitochondria reversible activation by all these agents may occur concurrently, but experimental conditions are described to study the action of each type of activator independently.

The respiratory chain-linked reduced nicotinamide adenine dinucleotide dehydrogenase of S. cerevisiae and C. utilis in mitochondrial and in membrane preparations has been investigated. Among methods tested, the highest activities for the dehydrogenase are obtained in the NADH-coenzyme Q1 reaction in S. cerevisiae, while the NADH-ferricyanide reaction provides the best activity determination for the enzyme from C. utilis. In several respects, including specificity for electron acceptor and substrate and inhibition by amytal, rotenone, and piericidin A, the dehydrogenase from C. utilis resembles its counterpart in mammalian mitochondria much more closely than does the enzyme from S. cerevisiae. The latter enzyme appears to be extensively solubilized on digestion of membrane preparations with N. naja venom phospholipase A, as is the case with the mammalian enzyme, whereas the dehydrogenase from C. utilis is not extracted by this treatment. The failure of phospholipase A to extract the C. utilis enzyme is not due to unusual phosphatide composition as shown by phosphatide analyses and by the fact that lecithin isolated from C. utilis mitochondria is readily hydrolyzed by phospholipase A, but may be due to the inaccessibility of the phospholipase to mitochondrial lipids in situ. NADH dehydrogenase from both types of yeasts is extensively inactivated by the heat-acid-ethanol method previously used by other workers for the isolation of “NADH dehydrogenase” from S. cerevisiae. During the O2-induced mitochondrial biogenesis in S. cerevisiae the respiratory chain-linked enzyme is formed considerably more rapidly than the overall NADH oxidase system; thus, the assembly of the components may be the rate-limiting step. On exposure of aerobic cells of S. cerevisiae to high glucose concentration, NADH dehydrogenase appears to undergo active destruction.

1. 1. The reversible inactivation of l-amino acid oxidase, previously described for the enzyme of moccasin venom, occurs in all species of Crotalidae venoms and in many Viperidae venoms, while Elapidae do not at all undergo this type of inactivation. This indicates a basic difference in the protein component of the enzyme from Crotalidae and from Elapidae. 2. 2. The l-amino acid oxidase activity of 23 species of venoms has been determined under conditions where the enzymes are stable. 3. 3. The following univalent anions protect the enzyme from inactivation to about the same extent: Cl−, Br−, F−, NO3+, CNS−, N3+, and ClO4+; I−, which is somewhat inhibitory to the enzyme, protects less effectively. The singly charged phosphate, metaphosphate, and arsenate anions do not prevent the inactivation. None of the doubly or triply charged anions or undissociated acids tested were protective. The cationic constituents of buffers are without effect on the enzyme at the concentrations tested. 4. 4. In dilute solutions of phosphate and related anions the inactivation and its reversal proceed in the same manner as in distilled water. Phosphate enhances the extent of inactivation only at high concentrations. 5. 5. The effect of the protective univalent anions is on the stability of the enzyme; they are not required for activity. The differences in the behavior of various anions are ascribed to selective binding of certain anions by the protein at those sites where bonds are ruptured during the reversible inactivation process.

The amino acid sequence of the coenzyme binding site of beef heart succinate dehydrogenase is serine-histidine-threonine-valine-alanine. Flavin adenine dinucleotide is covalently bound to a ring nitrogen of histidine through the 8α position of the isoalloxazine ring system.

1. 1. L-Cysteinesulfinic dehydrogenase, the enzyme which catalyzes the dehydrogenation of L-cysteinesulfinic acid, requires a coenzyme for activity. The coenzyme requirement is not fulfilled by known vitamins, coenzymes, or related substances, but boiled yeast extract is a good source of the cofactor. 2. 2. The new coenzyme, provisionally named coenzyme III, has been extensively purified from baker's yeast. When the dehydrogenase oxidizes cysteinesulfinic acid in the presence of the purified coenzyme, the latter is reduced to a substance with an absorption spectrum characteristic of dihydropyridine nucleotides. Upon addition of L-cysteic acid, the reduced spectrum disappears, indicating reversal of the dehydrogenation. 3. 3. The purified coenzyme is rapidly destroyed by 0.1 N acids at 100° and by nucleotide pyrophosphatase but is not acted upon by 5-nucleotidase or phosphodiesterase. The heavy metal salts of coenzyme III are water soluble. In charcoal chromatography it behaves very much like nicotinamide mononucleotide. 4. 4. On the basis of its behaviour to chemical reagents and to hydrolytic enzymes, the structure of coenzyme III is tentatively suggested to be nicotinamide-ribose-(5)-pyrophosphate. 5. 5. Some of the salient properties of L-cysteinesulfinic dehydrogenase have been investigated. 6. 6. On the basis of a detailed study of the electron transport from coenzyme III to O2, it is concluded that a diaphorase (or cytochrome reductase), concerned with the reoxidation of coenzyme III, is present in the extracts in the apoenzyme form. This protein requires a second new coenzyme, which is not identical with FMN or FAD, but is likely to be a flavin. The latter cofactor is present in yeast extract and is readily separated from coenzyme III. 7. 7. It is pointed out that coenzyme III also functions as the prosthetic group of dehydrogenases of animal origin and that it appears to be of wide distribution.

The fact that the d -α-hydroxy acid dehydrogenase is formed best under anaerobic conditions of growth and disappears on environmental adaptation of the cells to O2, while l (+) lactic dehydrogenase is absent in anaerobic cells and is synthesized during O2 adaptation, has repeatedly led to suggestions in the literature that d -α-hydroxy acid dehydrogenase is a precursor of l (+) lactic dehydrogenase. In the present study it is shown that the disappearance of d -α-hydroxy acid dehydrogenase during O2 adaptation is the consequence of the fact that the enzyme is rapidly destroyed in yeast cells once active growth ceases and that the destruction is as rapid on anaerobic incubation of the cells as aerobically. Thus the destruction of d -α-hydroxy acid dehydrogenase can proceed without any synthesis of l (+) lactic dehydrogenase. Further, under suitable conditions, extensive synthesis of l (+) lactic dehydrogenase occurs without the disappearance of d -α-hydroxy acid dehydrogenase . These facts and the extensive differences in constitution and catalytic properties between d -α-hydroxy acid dehydrogenase and l (+) lactic dehydrogenase suggest that a precursor-product relationship between the two enzymes is highly unlikely.

The disappearance of succinate dehydrogenase activity and of protein-bound histidyl flavin were studied in aerobic yeast cells incubated with high glucose concentrations. The decay of succinate dehydrogenase activity, covalently bound flavin, and of respiration is prevented by cycloheximide but not by chloramphenicol. During this decay there is a large increase in mitochondrial phospholipase D activity; the appearance of this enzyme is also prevented by cycloheximide. It seems possible, therefore, that the formation of phospholipase D may be important in triggering the disappearance of covalently bound flavin, succinate dehydrogenase, and of other mitochondrial enzymes during glucose repression of aerobic yeast cells.

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.