Submitochondrial particle

The spheres removed from SMPs do not support ATP synthesis but do hydrolyze ATP to ADP and phosphate. Thus, ATP synthesis is carried out by Fq/Fi—ATPase (ATP synthase). The subscript o in Fq indicates that it contains the site at which a potent antibiotic inhibitor, oligomycin, binds and inhibits oxidative phosphorylation. Oligomycin does not bind Fi-ATPase and does not inhibit ATP hydrolysis to ADP and phosphate. 

The fully active SMPs can be reconstituted by adding Fi-ATPase to depleted vesicles under appropriate conditions. 

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FIGURE 21.9 Typical visible absorption spectra of cytochromes, (a) Cytochrome c, reduced spectrum (b) cytochrome c, oxidized spectrum (c) the difference spectrum (a) minus (b) (d) beef heart mitochondrial particles room temperature difference (reduced minus oxidized) spectrum (e) beef heart submitochondrial particles same as (d) but at 77 K. a- and /3- bauds are labeled, and in (d) and (e) the bauds for cytochromes a, h and c are indicated. 

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. 

Controversial results of oxygen radical detection in mitochondria have been described in the literature. Owing to experimental difficulties many authors were obliged to work with sub-mitochondrial particles instead of the whole mitochondria. However, it is quite possible that oxygen radical production by submitochondrial particles may be artificially enhanced due to exposure to oxygen. On the other hand, some analytical methods of superoxide detection such as cytochrome c reduction cannot be used due to the direct reduction of cytochrome by mitochondrial components. 

Usually, mitochondrial superoxide production is registered only after the incubation of submitochondrial particles with respiratory inhibitors, first of all, rotenone and antimycin. Under such conditions, superoxide production may achieve about 1 nmol 1 1 min 1 per mg of... 

As a rule, oxygen radical overproduction in mitochondria is accompanied by peroxidation of mitochondrial lipids, glutathione depletion, and an increase in other parameters of oxidative stress. Thus, the enhancement of superoxide production in bovine heart submitochondrial particles by antimycin resulted in a decrease in the activity of cytochrome c oxidase through the peroxidation of cardiolipin [45]. Iron overload also induced lipid peroxidation and a decrease in mitochondrial membrane potential in rat liver mitochondria [46]. Sensi et al. [47] demonstrated that zinc influx induced mitochondrial superoxide production in postsynaptic neurons. 

As described earlier, superoxide is a well-proven participant in apoptosis, and its role is tightly connected with the release of cytochrome c. It has been proposed that a switch from the normal four-electron reduction of dioxygen through mitochondrial respiratory chain to the one-electron reduction of dioxygen to superoxide can be an initial event in apoptosis development. This proposal was supported by experimental data. Thus, Petrosillo et al. [104] have shown that mitochondrial-produced oxygen radicals induced the dissociation of cytochrome c from bovine heart submitochondrial particles supposedly via cardiolipin peroxidation. Similarly, it has been found [105] that superoxide elicited rapid cytochrome c release in permeabilized HepG2 cells. In contrast, it was also suggested [106] that it is the release of cytochrome c that inhibits mitochondrial respiration and stimulates superoxide production. 

LOX catalyzed the oxidation of arachidonoylphosphatidylcholine at both carbon-12 and carbon-15. Later on, it has been found [21] that reticulocyte lipoxygenase oxidized rat liver mitochondrial membranes, beef heart submitochondrial particles, rat liver endoplasmic membranes, and erythrocyte plasma membranes without preliminary release of unsaturated acids by phospholipases. 

Schnurr et al. [22] showed that rabbit 15-LOX oxidized beef heart submitochondrial particles to form phospholipid-bound hydroperoxy- and keto-polyenoic fatty acids and induced the oxidative modification of membrane proteins. It was also found that the total oxygen uptake significantly exceeded the formation of oxygenated polyenoic acids supposedly due to the formation of hydroxyl radicals by the reaction of ubiquinone with lipid 15-LOX-derived hydroperoxides. However, it is impossible to agree with this proposal because it is known for a long time [23] that quinones cannot catalyze the formation of hydroxyl radicals by the Fenton reaction. Oxidation of intracellular unsaturated acids (for example, linoleic and arachidonic acids) by lipoxygenases can be suppressed by fatty acid binding proteins [24]. 

Calculated from data presented in Ref. [53] (see text). bSMP are submitochondrial particles. 

Argese, E., C. Bettiol, A.V. Ghirardini, M. Fasolo, G. Giurin, and P.F. Ghetti. 1998. Comparison of in vitro submitochondrial particle and Microtox assays for determining the toxicity of organotin compounds. Environ. Toxicol. Chem. 17 1005-1012. 

The mitochondrial respiratory parameters have also been employed to determine the toxicity of surfactants, including anionic (LAS), nonionic (NPEO) and their metabolites, sulfophenyl carboxylates (SPCs), NP and nonylphenoxy carboxylate (NPECi) [37]. The system employed was the in vitro response of submitochondrial particles from beef heart. The EC50 toxicity calculated as the reduction rate of NAD+ ranged from 0.61 mg L-1 for a commercial LAS mixture to 18 000 mg L-1 for SPCs, and 1.3 mg L-1, 8.2 and 1.8mgL 1 for NPEOio, NPECi and NP, respectively. These results indicate that from the toxicity perspective, LAS is the compound demanding increased attention, while for NPEO, the parental compound and the metabolites must be quantified. 

To explain how H+ transfer occurred across the membrane Mitchell suggested the protons were translocated by redox loops with different reducing equivalents in their two arms. The first loop would be associated with flavoprotein/non-heme iron interaction and the second, more controversially, with CoQ. Redox loops required an ordered arrangement of the components of the electron transport system across the inner mitochondrial membrane, which was substantiated from immunochemical studies with submitochondrial particles. Cytochrome c, for example, was located at the intermembranal face of the inner membrane and cytochrome oxidase was transmembranal. The alternative to redox loops, proton pumping, is now known to be a property of cytochrome oxidase. 

Gustavson, K.E., Svenson, A., and Harkin, J.M. Comparison of toxicities and mechanism of action of / -alkanols in the submitochondrial particle and the Vibrio fischeri bioluminescence (Microtox ) bioassay. Environ. Toxicol. Chem., 17(10) 1917-1921, 1998. 

The thiocyanate ion-selective electrode [21,247] This has a heterogeneous membrane containing AgCNS. It is used for the determination of the activity of SCN in suspensions of submitochondrial particles and chromatophores from Rhodospirillum rubrum in order to measure the membrane potential of these particles [200], as well as for other applications. 

Moreno-Sanchez, R. et al. (1999) Inhibition and uncoupling of oxidative phosphorylation by nonsteroidal antiinflammatory drugs study in mitochondria, submitochondrial particles, cells, and whole heart. Biochemical Pharmacology, 57 (7), 743-752. 

Submitochondrial particles usually have the matrix side of the inner membrane on the outside... 

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. 

Chance and associates employed spectrophotometry on intact mitochondria or submitochondrial particles to investigate both the sequence of carriers and the sites of phosphorylation. Using the dual wavelength spectrophotometer, the light absorption at the absorption maximum (Aniax) of a particular component was followed relative to the absorption at some other reference wavelength (Aref). The principal wavelengths used are given in Table 18-6. From these measurements the state of oxidation or reduction of each one of the carriers could be observed in the various states and in the presence of inhibitors. The... 

Many of the biochemical processes that generate chemical energy for the cell take place in the mitochondria. These organelles contain the biochemical equipment necessary for fatty acid oxidation, di- and tricarboxylic acid oxidation, amino acid oxidation, electron transport, and ATP generation. In this experiment, a mitochondrial fraction will be isolated from beef heart muscle. The mitochondria will be analyzed for protein content and fractionated into submitochondrial particles. Each fraction will be analyzed for malate dehydrogenase and monoamine oxidase activities. 

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. 

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