Reduced Phenazine Methosulfate Research Articles

Isolation and Characterization of Hydrophobic Proteins (H Proteins) in the Membrane Fraction of Bacillus subtilis

Cytoplasmic membranes of Bacillus subtilis, grown in complex medium containing glucose, were fractionated into three membrane subfractions [light band (1.155 - 1.158 g/cm3); medium band (1.181 - 1.183 g/cm3); heavy band (1.21 - 1.25 g/cm3)] by sucrose density gradient centrifugation. Among these subfractions, the light and medium bands consisted mainly of membranes but the heavy band consisted of an irregular arrangement or aggregate of small globular protein components of 5 - 8 nm in diameter. We named this H-protein. H-protein formed trilamellar unit membrane structure when combined with lipid. In pulse-labeling and pulse-chase experiments with radioactive leucine, it was found that H-protein consisted of the newest membrane protein synthesized in the cells and the label incorporated into H-protein was shifted into light and medium band of the membranes during the chase. Cytochromes were not found in H-protein. However, when H-protein was incubated with haem alpha and protohaem, these compounds were incorporated into the apoproteins of the cytochromes present in H-protein and form cytochromes a and b. Cytochromes were also formed in H-protein which were isolated from the cells grown in the presence of haemin (haemin-grown H protein). Succinate dehydrogenase activity was increased about 4-fold by combining H-protein or haemin-grown H protein with lipid. H-protein had no cytochrome oxidase activity; however, haemin-grown H protein was found to have some of the activity and this was increased about 4-fold by combining the protein with lipid. Haemin-grown H protein was also found to form succinate: cytochrome c oxidoreductase when combined with lipid and vitamin K2. On the other hand, succinate oxidase was required for the addition of lipid, vitamin K2 and cytochrome c. NADH oxidase was also found in haemin-grown H protein and was activated about 9-fold in constituted reaction systems. Vesicles formed by haemin-grown H protein and lipid, could accumulate alanine and proline by addition of NADH or reduced phenazine methosulfate. Alanine and proline was also accumulated into the vesicles when transport energy was supplied as a membrane potential introduced by K+-diffusion via valinomycin. These results would indicate that H-protein contains the apoprotein of cytochromes, and a carrier involved in the active transport of alanine and proline.

Energy-dependent binding of dansylgalactoside to the lac carrier protein: direct binding measurements.

High specific activity 6'-N-[3H]dansyl)aminohexyl 1-thio-beta-D-galactopyranoside (Dns6-Gal) has been synthesized, and its binding to Escherichia coli membrane vesicles measured directly by flow dialysis. With ML 308-225 vesicles containing the lac carrier protein, specific binding is not detected in the absence of D-lactate or reduced phenazine methosulfate. In the presence of these electron donors, binding is observed, and the binding constant and number of binding sites are approximately 4 muM and 1.5 nmol/mg of membrane protein, respectively. These values are in excellent agreement with those obtained by fluorescence titration. p-Chloromercuribenzenesulfonate, which directly inactivates the lac carrier protein, and carbonylcyanide m-chlorophenylhydrazone, which collapses the membrane potential, cause release of bound Dns6-Gal. Moreover, significant binding is not observed with membrane vesicles that are devoid of the lac carrier protein. The results provide qualitative and quantitative confirmation of previous studies which indicate that changes in dansylgalactoside fluorescence observed on "energization" of membrane vesicles reflect binding of the probe to the lac carrier protein.

Localization of D-lactate dehydrogenase in native and reconstituted Escherichia coli membrane vesicles

In the preceding paper the preparation and characterization of antiserum to purified D-lactate are described. In this paper the effects of the antibody on D-lactate dehydrogenase activity and D-lactate-dependent active transport in native Escherichia coli ML 308-225 membrane vesicles and ML 308-225dld-3 vesicles reconstituted with D-lactate dehydrogenase are described. The results demonstrate that D-lactate dehydrogenase is inaccessible to antibody in native ML 308-225 vesicles, but readily accessible to antibody in reconstituted dld-3 vesicles. The findings indicate that D-lactate dehydrogenase is located on the inner surface of native ML 308-225 vesicles and on the outer surface of reconstituted dld-3 vesicles. The results with the native vesicle preparations also provide further evidence that virtually none of the vesicles is inverted or sufficiently damaged to allow access of antibody to D-lactate dehydrogenase. In addition, experiments are presented which demonstrate that an impermeable electron carrier, reduced 5-N-methylphenazonium-3-sulfonate, drives active transport in native ML 308-225 vesicles as well as its permeable analogue reduced phenazine methosulfate. Thus, reduction of the respiratory chain from either side of the vesicle membrane is able to drive active transport. Ca2+, Mg2+-stimulated ATPase is also inaccessible to antibody in ML 308-225 vesicles unless the preparation is subjected to ultrasonic sound, incubated in Tris buffer at pH 9.0, or homogenized vigorously. Moreover, as opposed to D-lactate dehydrogenase and cytochrome b1, ATPase is readily lost from the membrane during the preparation of vesicles.

Active transport of calcium in inverted membrane vesicles of Escherichia coli.

Accumulation of (45)Ca(++) was found to occur in membrane vesicles of E. coli prepared by lysis with a French pressure cell. The uptake occurs by active transport, requiring an energy source. Substrates of the electron transport chain, including D-lactate, reduced phenazine methosulfate, and NADH, stimulated accumulation, but this effect was blocked by the addition of cyanide. ATP could also stimulate accumulation, and this effect was blocked by dicyclohexylcarbodiimide. Uncouplers of oxidative phosphorylation inhibited the accumulation driven by either type of energy source. Accumulation of calcium is rapid, reaching the steady-state plateau within 1 min. Addition of phosphate to the assay buffer results in a prolongation of the reaction, allowing for the time-dependent accumulation of calcium for as long as 30 min. Vesicles prepared by lysis with a French pressure cell exhibit almost no ability to accumulate proline, while vesicles prepared by the method of Kaback transport proline but exhibit little energy-dependent transport of calcium. It is suggested that the accumulation of calcium in these vesicles, which are believed to be inverted, reflects a system that in vivo is responsible for the active extrusion of calcium from the cells.

Mechanism of aromatic hydroxylation: Properties of a model for pyridine nucleotide-dependent flavoprotein hydroxylases

A model (NADH-phenazine methosulfate-O 2) formally similar to pyridine nucleotide-dependent flavoprotein hydroxylases catalyzed the hydroxylation of several aromatic compounds. The hydroxylation was maximal at acid pH and was inhibited by ovine Superoxide dismutase, suggesting that perhydroxyl radicals might be intermediates in this process. The stoichiometry of the reaction indicated that a univalent reduction of oxygen was occurring. The correlation between the concentration of semiquinone and hydroxylation, and the inhibition of hydroxylation by ethanol which inhibited semiquinone oxidation, suggested the involvement of phenazine methosulfate-semiquinone. Activation of hydroxylation by Fe 3+ and Cu 2+ supported the contention that univalently reduced species of oxygen was involved in hydroxylation. Catalase was without effect on the hydroxylation by the model, ruling out H 2O 2 as an intermediate. A reaction sequence, involving a two-electron reduction of phenazine methosulfate to reduced phenazine methosulfate followed by disproportionation with phenazine methosulfate to generate the semiquinone, was proposed. The semiquinone could donate an electron to O 2 to generate O 2 which could be subsequently protonated to form the perhydroxyl radical.

A Transport System for C4-Dicarboxylic Acids in Isolated Membrane Preparations fromEscherichia coli

To investigate the mechanism of succinate transport system in Escherichia coli, the isolated membranes were prepared from E. coli W2252 and T5, a mutant defective in succinate uptake derived from W2252. Uptakes of 14C-substrates by W2252 and T5 membranes and the dilution of accumulated radioactivity by unlabeled C4-dicarboxylic acids, indicated that C4-dicarboxylic acids in the tricarboxylic acid cycle are transported by the same system in E. coli which requires a suitable energy source such as NADH, D-lactate or reduced phenazine methosulfate. The uptakes of succinate by W2252 membranes were inhibited by an anaerobic incubation or some of the inhibitors of electron transport chain. Difference spectra of reduced versus oxidized membranes from W2252 and T5 indicated the reduction of flavoproteins and cytochromes by dithionite, NADH or D-lactate. From these results it was concluded that the uptake of the C4-dicarboxylic acids in isolated membranes is coupled to an electron transport chain involving a specific dehydrogenase system.

Deoxycytidine uptake by isolated membrain vesicles from Escherichia coli K 12

1. 1. Two different deoxycytidine-uptake systems were found in isolated membrane vesicles of Escherichia coli K 12 cells: one was not significantly inhibited by guanine nucleosides, the other was markedly inhibited by these nucleosides. In isolated membrane vesicles of a showdomycin-resistant mutant (Shm r-001), the former system was no longer detectable but the latter was found to remain active. 2. 2. The deoxycytidine uptake by these systems was stimulated by reduced phenazine methosulfate, d(-)-lactate, succinate, and NADH, but not by ATP, phosphoenolpyruvate, or phosphoribosyl pyrophosphate. The reduced phenazine methosulfate-stimulated uptake of deoxycytidine by these systems was markedly inhibited by sodium azide. 3. 3. Among sulfhydryl reagents tested, N-ethylmaleimide did not significantly inhibit the uptake of deoxycytidine by these systems, even at very high concentrations; p-chloromercuribenzoic acid did inhibit uptake. 4. 4. The antibiotic showdomycin strongly inhibited the uptake of deoxycytidine by membrane vesicles of Escherichia coli K 12 but only slightly inhibited that by membrane vesicles of the mutant (Shm r-001). Further, showdomycin produced a rapid efflux of deoxycytidine from previously loaded membrane vesicles of Escherichia coli K 12. These results suggest that showdomycin is transported by the deoxycytidine transport system which is not inhibited by guanine nucleosides in isolated membrane vesicles of Escherichia coli K 12.

Transport of lactate and succinate by membrane vesicles of Escherichia coli, Bacillus subtilis and a pseudomonas species.

Transport of d‐lactate, l‐lactate and succinate in isolated membrane vesicles of Escherichia coli, Bacillus subtilis and a fresh‐water Pseudomonas species occurs actively by specific transport systems which are linked to the electron transport chain. These substrates are not transported by the membrane vesicles unless an electron donor (reduced phenazine methosulfate, NADH, d‐lactate, l‐lactate, or succinate) is present in the reaction mixture; in the presence of an electron donor, the substrates or their oxidized forms are concentrated several‐fold inside the membrane vesicles; and inhibitors of the electron transport chain markedly inhibit this electron‐donor mediated transport. In addition, transport of pyruvate in membrane vesicles of E. coli, but not in those of B. subtilis and the Pseudomonas sp. is stimulated by reduced phenazine methosulfate. Activity of the membrane‐bound lactate and succinate dehydrogenases is not essential for the transport of lactate and succinate, since inhibitors of these activities have little effect on reduced phenazine methosulfate‐ or NADH‐energized transport of these substrates; and the Km values for oxidation of these substrates by the membrane vesicles are 30‐ to 80‐fold higher than the Km values for their transport. d‐Lactate and l‐lactate cause strong inhibition of the transport of each other in the three organisms, suggesting that the two substrates share a common transport system. In general, transport of these substrates is not inhibited by dicarboxylic acids. In contrast, transport of succinate is strongly inhibited by other dicarboxylic acids in the three organisms, suggesting that these substrates may share a common transport system. The transport systems for lactate and succinate are repressed by glucose.

A Transport System for C4-Dicarboxylic Acids in Isolated Membrane Preparations from Escherichia coli

To investigate the mechanism of succinate transport system in Escherichia coli, the isolated membranes were prepared from E. coli W2252 and T5, a mutant defective in succinate uptake derived from W2252. Uptakes of 14C-substrates by W2252 and T5 membranes and the dilution of accumulated radioactivity by unlabeled C4-dicarboxylic acids, indicated that C4-dicarboxylic acids in the tricarboxylic acid cycle are transported by the same system in E. coli which requires a suitable energy source such as NADH, D-lactate or reduced phenazine methosulfate. The uptakes of succinate by W2252 membranes were inhibited by an anaerobic incubation or some of the inhibitors of electron transport chain. Difference spectra of reduced versus oxidized membranes from W2252 and T5 indicated the reduction of flavoproteins and cytochromes by dithionite, NADH or D-lactate. From these results it was concluded that the uptake of the C4-dicarboxylic acids in isolated membranes is coupled to an electron transport chain involving a specif...

Transport of Succinate in Escherichia coli: II. CHARACTERISTICS OF UPTAKE AND ENERGY COUPLING WITH TRANSPORT IN MEMBRANE PREPARATIONS

Abstract Membrane vesicles prepared from a strain of Escherichia coli which lacks membrane-bound succinate dehydrogenase and fumarate reductase activity are capable of accumulating succinate in the presence of d(-)lactate or reduced phenazine methosulfate as electron donors. The steady state, intramembranal concentration of succinate reaches 30- to 50-fold of the external concentration. Both the initial and the steady state rates are higher with ascorbate-phenazine methosulfate than with d(-)lactate. ATP, ADP, and various other metabolic intermediates do not stimulate succinate transport. Magnesium ions are required for maximum uptake. Monovalent cations, such as Na+, K+, or Li+ also stimulate transport to varying degrees. With ascorbate-phenazine methosulfate as electron donor the Michaelis constant for succinate is about 10 µm, and the inhibition constants for fumarate and l-malate are 12 µm and 22 µm, respectively. Both the ascorbate-phenazine methosulfate-dependent and d(-)lactate-dependent uptake of succinate are inhibited by energy poisons, uncouplers, and various other respiratory chain inhibitors. Compounds known to interfere with phosphorolytic and transphosphorylation reactions are without significant effect on transport. Amongst the thiol reagents, N-ethylmaleimide and showdomycin do not inhibit succinate uptake even at very high concentrations, but p-chloromercuribenzoate, Hg++ and Ag++ are very potent inhibitors. Both N-ethylmaleimide and p-chloromercuribenzoate, however, strongly inhibit d-lactate oxidation. From these observations, and from oxamate inhibition data, it is concluded that electrons are coupled to succinate transport directly at the initial step of lactate dehydrogenation. It is proposed that succinate uptake is mediated by alternate reduction and oxidation of a succinate carrier in membranes. Membranes prepared from strains carrying genes dct A or dct B are defective in the uptake of succinate. It thus appears that at least two genetic loci are involved in the formation of the succinate transport system in the membranes.

Amino Acid Transport in Membrane Vesicles of Bacillus subtilis

Abstract Membrane vesicles of Bacillus subtilis actively transport amino acids in the presence of electron donors. The effectiveness of each physiological electron donor depends on the presence of the necessary membrane proteins that allow substrate oxidation and couple it to amino acid transport. Some of these proteins are present in all membranes while others (e.g. oxidizing glycerol phosphate) appear only in certain growth media (containing glycerol). Physiological electron donors are NADH, NADPH, l-α-glycerol phosphate, l-lactate, and succinate. NADH stimulates amino acid uptake at the highest initial rate whereas glycerol phosphate is the most effective electron donor since it energizes the transport of the largest number of amino acid molecules per nmole of substrate oxidized per min. Reduced phenazine methosulfate (PMS) is a very efficient nonphysiological electron donor which energizes amino acid transport at a high initial rate. At the membrane concentrations usually employed (1 to 5 mg of membrane protein per ml), NADH, NADPH, and reduced PMS consume oxygen so rapidly that special oxygenation is required to maintain the uptake of amino acids at a high rate. This uptake stops when the energy source has been oxidized; the accumulated amino acid then leaks out at a rate of 8% per min. With reduced (by ascorbate) PMS as electron donor, the Km and Vmax values of the uptake of 18 amino acids were determined. The Km values range from 10-5 to 10-6 m whereas the Vmax values vary widely for the different amino acids. Competition experiments have shown the presence of at least nine different amino acid transport carriers in the vesicles. The energy for active transport appears to be produced in the cytochrome-linked electron transport chain: all electron donors effective in amino acid transport cause the reduction of the membrane-bound cytochromes. The electron transport inhibitors antimycin, 2-heptyl-4-hydroxyquinoline-N-oxide, cyanide, and azide also inhibit amino acid transport, for all energy sources. Rotenone and oligomycin inhibit only the lactate-energized transport. Under conditions under which amino acids are transported no net ATP synthesis has been observed.

The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen

The reduction of nitro blue tetrazolium (NitroBT) with NADH mediated by phenazine methosulfate (PMS) under aerobic conditions was inhibited upon addition of superoxide dismutase. This observation indicated the involvement of superoxide aninon radical (O2−) in the reduction of NitroBT, the radical being generated in the reoxidation of reduced PMS. Similarly, the reduction of NitroBT coupled to D-amino acid oxidase-PMS system under aerobic conditions was also inhibited by superoxide dismutase. A simple method for detecting superoxide dismutase is described.

L-serine transport in membrane vesicles of Bacillus subtilis energized by NADH or reduced phenazine methosulfate

L-serine transport in membrane vesicles of Bacillus subtilis energized by NADH or reduced phenazine methosulfate

Light-induced electron transfer reactions and adenosine triphosphate formation by Rhodospirillum rubrum chromatophores

The photooxidation of reduced phenazine methosulfate (PMS) by chromatophores from Rhodospirillum rubrum in a terminal reaction with added ubiquinone as an electron acceptor is primarily a nonphosphorylating reaction. In the presence of reduced PMS (ubiquinone absent), chromatophores can also cycle electrons through a nonphosphorylating as well as a phosphorylating system. The photooxidation of reduced PMS is a monophasic, first-order reaction while the subsequent dark reduction of oxidized PMS by endogenous components of the chromatophores is biphasic and has kinetics consistent with two first-order reactions of different rate constants. Comparison of the rates of PMS turnover and ATP formation show a decreasing efficiency for PMS-stimulated photophosphorylation with increasing light intensity, indicating that more electrons cycle via the nonphosphorylating pathway at higher intensities. Changing the percentage of reduced PMS present in reaction mixtures from less than 10 to about 80 had little effect on the rate of PMS-stimulated cyclic photophosphorylation. The photooxidation of horse heart ferrocytochrome c by chromatophores has been coupled to the reduction of various quinones, including ubiquinones, plastoquinone, α-tocopheryl quinone, vitamine K 3, and trimethyl- p-benzoquinone. Under anaerobic conditions these reactions are completely reversible in the dark. Oxidized ubiquinone-2, but not ubiquinone-6, was found to be a potent inhibitor of photophosphorylation. In the reduced form both ubiquinones behaved similarly, and show some inhibition with fresh preparations and stimulation with aged chromatophore fragments. An active reduced ubiquinone-cytochrome c reductase activity was demonstrated. This reaction was sensitive to antimycin A, but no associated ATP forming capacity was observed. ATP formation was not associated with the rapid photooxication of ferrocytochrome c with either oxygen or ubiquinone acting as the electron acceptor. Previously observed increases in ATP formation under these conditions can be explained by the role of reduced cytochrome c in stimulating cyclic photophosphorylation.

Purification and Mechanism of Action of a Steroid Δ-5β-Dehydrogenase

Abstract A soluble Δ4-5β-dehydrogenase has been isolated from extracts of Pseudomonas testosteroni grown on a steroid-containing medium. This enzyme introduces the Δ4-double bond into C19 and C21 steroids in which the A:B ring fusion has the cis configuration. The enzyme has been purified about 100-fold from acetone powders of the microorganism by a procedure involving precipitation of nucleic acids with protamine; fractionation with ammonium sulfate; chromatography on diethylaminoethyl-cellulose; and calcium phosphate gel adsorption. The purified enzyme oxidized about 6.5 µmoles of 5β-androstane-3,17-dione per min per mg of protein. Phenazine methosulfate is an efficient artificial acceptor in the Δ4-5β-dehydrogenase reaction. Various quinones, notably 1,2- and 1,4-naphthoquinone, 1,4-benzoquinone, menadione, and coenzyme Q10, were able to replace phenazine methosulfate in the reaction, but other quinones and acceptors of other chemical types were inactive. A simple aerobic assay for steroid Δ-dehydrogenase activity is described. It depends upon measurement of the nonenzymatic reduction of cytochrome c (at 550 mµ) by the reduced phenazine methosulfate formed in the enzymatic oxidation. An interesting blank reaction occurs between phenazine methosulfate and cytochrome c in the absence of enzyme or steroid. The rate of this reaction is markedly accelerated by light and at high pH. The reaction rate is proportional to the concentration of phenazine methosulfate, but independent of that of cytochrome c. These findings suggest that a product of the light-catalyzed dismutative decomposition of the dye is the reductant of cytochrome c. Treatment with acid ammonium sulfate inactivated the enzyme almost completely, and a substantial portion of the original activity could be restored by addition of a low concentration (Km = 0.33 µm) of flavin mononucleotide but not by flavin adenine dinucleotide.

The role of a reaction center in photochemical activities of bacterial chromatophores.

Abstract Strain PM-8 of Rhodopseudomonas spheroides is unable to grow photosynthetically even though it generates a normal complement of bacteriochlorophyll and carotenoids when grown aerobically in the dark. This mutant strain fails to show any of the light reactions, observable in the photosynthetically competent parent strain, that signal the operation of photochemical reaction centers. These light-induced effects include absorbancy changes, fluorescence transients, and an electron-spin-resonance signal. The pigment P870 is missing in strain PM-8. Bacterial chromatophores have been observed to catalyze a variety of photochemical electron-transfer reactions involving externally added substances such as cytochrome and quinones. Chromatophores from strain PM-8 prove to be inert or extremely inefficient in catalyzing the following reactions: (I) oxidation of reduced tetramethylphenylenediamine or of mammalian cytochrome c coupled with reduction of an endogenous substance; (2) oxidationi of reduced phenazine methosulfate or cytochrome c coupled with reduction of the ubiquinones UQ2 and UQ6. The same reactions are catalyzed efficiently (with quantum requirements of the order of 1–3 quanta per electron transfer) by chromatophores from photosynthetically competent strains of R. spheroides, even when the major bacteriochlorophyll component of the chromatographores has been converted to bacteriopheophytin.

Absence of light-induced absorbancy changes in a mutant of Rhodopseudomonas spheroides unable to grow photosynthetically

Strain PM-8 of Rhodopseudomonas spheroides is unable to grow photosynthetically even though it generates a normal complement of bacteriochlorophyll and carotenoids when grown aerobically in the dark. This mutant strain fails to show any of the light reactions, observable in the photosynthetically competent parent strain, that signal the operation of photochemical reaction centers. These light-induced effects include absorbancy changes, fluorescence transients, and an electron-spin-resonance signal. The pigment P870 is missing in strain PM-8.Bacterial chromatophores have been observed to catalyze a variety of photochemical electron-transfer reactions involving externally added substances such as cytochrome and quinones. Chromatophores from strain PM-8 prove to be inert or extremely inefficient in catalyzing the following reactions: (I) oxidation of reduced tetramethylphenylenediamine or of mammalian cytochrome c coupled with reduction of an endogenous substance; (2) oxidationi of reduced phenazine methosulfate or cytochrome c coupled with reduction of the ubiquinones UQ2 and UQ6. The same reactions are catalyzed efficiently (with quantum requirements of the order of 1–3 quanta per electron transfer) by chromatophores from photosynthetically competent strains of R. spheroides, even when the major bacteriochlorophyll component of the chromatographores has been converted to bacteriopheophytin.

Enzymes catalyzing sequential reactions in mouse brain and liver supernatant fractions: II. The use of phenazine methosulfate.

1 . Spectrophotometric and manometric studies of pertinent non-enzymic solutions showed that reduced diphosphopyridine nucleotide is rapidly oxidized by phenazine methosulfate. The reduced phenazine methosulfate (PMS) is gradually oxidized by molecular oxygen. H2O2 is formed. A portion of this H2O2 is spontaneously split into H2O and O2 2. In mouse brain and liver supernatants, with PMS as dye and reduced diphosphopyridine nucleotide (DPNH) as substrate, the same reactions occur. In addition, the formed H2O2 is split by catalase demonstrated to be present in the supernatants used. Th oxygen taken up was a direct measure of the DPNH oxidized. 3. Pyruvate was the main competing electron acceptor studied. When it was added to, or formed by the supernatants, the oxygen uptake during DPNH-oxidation through PMS was reduced, depending on the relative concentration of the electron transferring systems present. In that case the amount of oxygen consumed, when added to the amount of lactate accumulated, was a direct measure of the DPNH oxidized. 4. When oxidized diphosphopyridine nucleotide (DPN) and an appropriate substrate were added to supernatants, the reoxidation by PMS of the DPNH formed by the DPN-requiring dehydrogenase components of a given enzyme group was measured. The oxygen uptake was a measure of the amount of DPNH formed, and thus a measure of the enzymic breakdown of the substrate by the enzyme system under study. 5. When the metabolism of a substrate by the enzyme system under study included the formation of pyruvate, the PMS-dependent oxygen uptake, added to the lactate accumulated, was a measure of the activity of the enzyme system. 6. Homogenized brain tissue yielded a supernatant with fragmented enzyme groups lacking functional flavoprotein. By contrast, as shown previously, this tissue yielded a mitochondrial fraction which retained all of the enzynes necessary for an integrated glucose-metabolizing enzyme system. 7. Homogenized liver tissue yielded a supernatant with flavoprotein linked enzyme systems less fragmented than those found in brain supernatants. At the same time it yields a mitochondrial fraction with an incomplete glycolytic enzyme system. 8. The data support the thesis proposed previously, that the enzymes found in the supernatant fraction of brain and liver cells represent mainly enzymes washed from the mitochondria, and therefore complementary to those retained by these organelles.