Submitochondrial particles, preparation

The mitochondrial complex that carries out ATP synthesis is called ATP synthase or sometimes FjFo-ATPase (for the reverse reaction it catalyzes). ATP synthase was observed in early electron micrographs of submitochondrial particles (prepared by sonication of inner membrane preparations) as round, 8.5-nm-diameter projections or particles on the inner membrane (Figure 21.23). In micrographs of native mitochondria, the projections appear on the matrixfacing surface of the inner membrane. Mild agitation removes the particles from isolated membrane preparations, and the isolated spherical particles catalyze ATP hydrolysis, the reverse reaction of the ATP synthase. Stripped of these particles, the membranes can still carry out electron transfer but cannot synthesize ATP. In one of the first reconstitution experiments with membrane proteins, Efraim Racker showed that adding the particles back to stripped membranes restored electron transfer-dependent ATP synthesis. 

The high affinity of Con A for cell surface oligosaccharides has also facilitated the immobilization of various cells including those of yeast [131], red blood cells [126, 128] and Trichosporon cutaneum [132]. An early study has also described the co-immobilization of enzymes and living cells using Con A [133]. More recently Habibi-Rezaei and Nemat-Gorgani [134] immobilized submitochondrial particles prepared from beef liver mitochondria on Con A support for continuous catalytic transformations involving succinate-cytochrome c reductase. 

Many methods have been employed to break mitochondrial membranes into submitochondrial particles that retain an ability to catalyze some of the reactions of the chain.61 For example, the Keilin-Hartree preparation of heart muscle is obtained by homogenizing mitochondria and precipitation at low pH.62 Tire resulting particles have a low cytochrome c content and do not carry out oxidative phosphorylation. 

One of the most instructive fractionation procedures is the preparation of submitochondrial particles (SMPs). The particles are produced by soni-cation (see Experiment 4) and centrifugation. The pellet, which sediments between 12,000 and 100,000 X g after sonication, defines the submitochondrial particle fraction. Submitochondrial particles are actually chunks of inner membrane that have undergone circularization and inversion. In other words, the membrane has been turned inside-out. Essentially all of the components for electron transport are still present however, matrix enzymes are largely removed. 

Students will isolate intact mitochondria from beef heart and fractionate them to prepare submitochondrial particles. Each fraction will be characterized by protein estimation by the biuret method and measurement of malate dehydrogenase and monoamine oxidase activity. 

Complex II. Preparations of complex II catalyze the oxidation of succinate by either PMS (at 7,nax) or ubiquinone at a rate of about 45-55 /imoles/min/mg protein at 38°. Thus the turnover number in either reaction is about 10,000. It has been shown by Ziegler and Doeg (153) that the PMS reductase activity of complex II is the same in the presence or absence of added ubiquinone-2 (complex II preparations are essentially devoid of bound ubiquinone). This is not in agreement with the conclusions of others on ubiquinone effect in submitochondrial particles (see, however. Section III,A,4). 

Baginsky and Hatefi (155, 156) showed that loss of reconstitution activity appears to be related to a damage in the iron-sulfur system of the enzyme which is not detectable by assay for iron and labile sulfide content. They obtained a preparation of succinate dehydrogenase from complex II which exhibited no reconstitution activity but had an iron labile sulfide flavin ratio close to 8 8 1. They were then able to reactivate this enzyme for reconstitution by treating it with NajS, ferrous ions, and mercaptoethanol, essentially in the same manner as apoferredoxin had been previously converted to ferredoxin (181, 18Z). The reactivated preparation was able to reconstitute with alkali-treated submitochondrial particles or complex II. Analyses showed that the preparation had acquired additional iron and labile sulfide, but control experiments indicated that reconstitution activity was not a spurious effect. The reactiva-... 

Fra. 33. Activation of succinate dehydrogenase by NADH. A preparation of phos-phorylating submitochondrial particles (ETPh) (succinoxidase activity = 1.18 /unoles succinate per min per mg at 30°) was washed by centrifugation in a sucrose-Tris-Mg buffer (pH 7.4) and resuspended in the same buffer at 1 mg of protein/ml. Antimycin A (1 nmole/mg protein) was added to slow the rate of aerobic oxidation of NADH, followed by 0.25 mAf NADH. Oxidation of the latter at 23° was monitored spectro-photometrically at 340 nm (dashed line). Samples were removed periodically and assayed immediately for succinate dehydrogenase activity in the presence of 033 mg of PMS/ml (solid line). At 16 min a second aliquot of 035 mM NADH was added. From Gutman et al. (197). 

Studies with beef-heart submitochondrial particles initiated in Green s laboratory in the mid-1950s resulted in the demonstration of ubiquinone and of non-heme iron proteins as components of the electron-transport system, and the separation, characterisation and reconstitution of the four oxidoreductase complexes of the respiratory chain. In 1960 Racker and his associates succeeded in isolating an ATPase from submitochondrial particles and demonstrated that this ATPase, called F, could serve as a coupling factor capable of restoring oxidative phosphorylation to F,-depleted particles. These preparations subsequently played an important role in elucidating the role of the membrane in energy transduction between electron transport and ATP synthesis. 

Submitochondrial particles (membranes from washed sonicated mitochondria) prepared from juice vesicles of Hamlin oranges harvested in September contained KCN-insensitive respiratory activity (46% of total) using a substrate mixture containing 0.05 M raalate, 0.05 M succinate, 0.01 M glutamate and 0.01 M TPP (21). 

This activity contrasts to the small (3%) KCN-insensitive respiratory activity observed when NADH was used as substrate with the mitochondria (Table I). To calculate the contribution of each oxidase to the total oxygen uptake, the titration method of Bahr and Bonner (25) was used, ( -uptake of submitochondrial particles (membranes) prepared from juice vesicles of Hamlin oranges harvested in September and January was measured in the presence and absence of ImM KCN titrated with a series of SHAM concentrations using the malate, succinate, glutamate substrate mixture (Table II). 

Fig. 21. Characteristics of NADH oxidation by submitochondrial particles from C. utilis during transformation from exponential to stationary phase. Candida utilis was grown in 1.5% (v/v) ethanol in a fermentor at 30°. Cells were harvested at the times shown for isolation of mitochondria and preparation of submitochondrial particles. NADH oxidase activity is expres.sed as microatoms of oxygen per min per mg protein at 30°. NADH dehydrogenase activity is expressed as micromoles of NADH oxidized per min per mg particle protein at 25° at with respect to Fe(CN). " sensitivity to piericidin A (0.5 nmole/mg protein) is expressed as percent inhibition of NADH oxidase and turbidity is given as absorbance at 650 nm in 1 cm light path. The pH was maintained during growth by automatic addition of 6 N KOH (pH-stat) at 5.0 until 25 hr, after which no further acid development occurred but the pH rose to between 5.0 and 62. From Grossman et al. (ISS).

Since bacterial membranes, like submitochondrial particles, oxidize NADH in a ratio NADH/O equal to 1, it follows that / in this particular case will be 0.00389/0.00251 = 1.55. Table III shows the dependence of the correction factor on pH and temperature for respiratory enzymes of rate heart submitochondrial particles and membrane preparations from Citrobacterfreundi, when Hb02 or HbCPA were used as oxygen donors. It... 

The treatment of submitochondrial particles, which are prepared from the inner mitochondrial membrane by sonication and which have the orientation of their membrane reversed (See Figure 18.26, which shows a structure analogous to a submitochondrial particle), with urea removes subunits. When these treated particles are incubated in air with an oxidizable substrate and calcium ion, the concentration of calcium inside the particles increases. 

Obviously, both views are incompatible. One possibility is that the diffusion gradient of matrix aspartate, observed at 4°C, does not exist at 37°C. Also, the assumption that the for matrix aspartate can be measured in submitochondrial particles may be incorrect because of alterations in properties of the carrier during particle preparation. In fact, the of aspartate transport in these particles was... 

Purified preparations of cytochrome oxidase are unstable and researchers have to deal, as a rule, with submitochondrial particles including, together with cytochrome oxidase, a part of the lipid membrane. The enzyme contains heme and copper in equimolar quantities as prosthetic groups. It apparently reacts with cytochrome c due to electrostatic interaction interaction with oxygen is limited by the latter s rate of diffusion. 

When mitochondria are disrupted into small vesicles or fragments with detergents, or by sonic oscillation, some enzymes and enzyme systems remain associated with the particles, while some others are recovered in the soluble phase. The cytochromes and the flavoproteins of the respiratory chain are exclusively recovered in the membrane fractions and seem to be firmly bound to the membrane. The ability to couple oxidation to phosphorylation is usually lost upon fragmentatioiu however, if the submitochondrial particles are prepared very carefully, they can... 

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