Mitochondria preparation

The major pathway probably proceeds via D-galacturonic acid and L-galactono-1,4-lactone, as these are converted to L-ascorbic acid by mitochondria prepared from peas and mung beans, but neither L-gulono-1,4-... 

McEnery, M. W., Buhle, E. L.,Jr, Aebi, U., andPedersen, P. L. (1984). Proton ATPase of rat liver mitochondria Preparation and visualization of a functional complex using the novel zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate./. Biol. Chem. 259, 4642-4651. 

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). 

Each of these assays has drawbacks associated with them. The major obstacle for developing rapid screens for inhibitors of cytochrome c release from mitochondria is that enriched mitochondria have a finite time in which they can be used. We are in the process of testing mitochondria preparations to determine the stability of the mitochondria with respect to use in the cytochrome c release assay. Mitochondria stored on ice for 4 h (the longest time we have tested at the time of this writing) can be used in the cytochrome c release assay. Therefore, it is possible to run multiple assay cycles with one preparation of mitochondria. The short format cytochrome c release assay is not affected by cell permeability, makes no assumptions about the mechanism of action, does not use cultured cells, is targeted at the process to be inhibited (i.e., cytochrome c release from mitochondria), and is a colormetric assay easily monitored by spectrophotometric plate readers. This assay would, therefore, increase the chances of detecting lead compounds to be subsequently modified to increase potency, cell permeability, and pharmacological efficacy. 

Suzuki, K., Mizuno, Y., Yamauchi, Y., Nagatsu, T. and Yoshida, M. (1992) Selective inhibition of complex I by N-methylisoquinolinium ion and Af-methyl-l,2,3,4-tetrahydro-isoquinoline in isolated mitochondria prepared from mouse brain, y. Neurol. Sci. 109 219—223. 

It is hoped that further work on the plant mitochondria preparations studied by Young and Conn (25) may help to clarify the possible role of GSH in plant respiration. Preliminary experiments have shoAvn that the mitochondria from avocados catalyze oxidation of ascorbic acid by O2, and that GSH is also oxidized by O2 when a trace of ascorbic acid is added to the particles. Some evidence has also been obtained that the particles catalyze a reduction of dehydroascorbic acid by citrate in the presence of TPN and GSH. Further work on these systems is in progress. Experiments are also in progress to determine whether oxidative phosphorylations occur during reactions of the type described. 

Juglone also affects uptake of molecular oxygen by excised roots of maize and inhibits state m oxidation rate in mitochondrial respiration [97]. The inhibition of state III uptake (ADP-dependent respiration) by purified mitochondria preparations is commensurate with the inhibition observed in vivo. Exposing soybean seedlings to juglone also causes changes in the activities of root peroxidases and the overall lignification of the roots [98]. 

We have therefore determined lipoamide dehydrogenase activity in mitochondria prepared from the livers and brains of some experimented animals. ... 

Wood C, Jalil MNR, McLaren I, et al. Carnitine long-chain acyltransferase and oxidation of palmitate, palmitoyl coenzyme A and palmitoylcamitine by pea mitochondria preparations. Planta 1984 161 255-260... 

Fig. 2. Identification of fMet on subunit 9 of oligomycin-sensitive ATPase. Sac-charomyces cerevisiae, strain 4D, was grown on SSA 3% lactate, labeled with PH]formate for 6 h, and mitochondria prepared as previously described. Preparation of chloroform-methanol (CM) fractions was done as described by Sierra and Tzagoloff. Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis was done by the method of Weber and Osborn, with 8% acrylamide for 11 h at 5 mA per gel. The 12-cm gels were cut into 1-mm slices and counted. Different patterns were normalized thru mobility of protein standards and tracking dye. (A) Final purified CM fraction after several ether...

The kinetic and equilibrium parameters of L-malate, succinate, citrate, and a-oxoglutarate uptake have been determined in mitochondria isolated from respiratory-competent cells grown under conditions of aerobic derepression, aerobic and anaerobic catabolite repression, and inhibition of mitochondrial protein synthesis, and also in mitochondria prepared from a respiratory-deficient cytoplasmic petite strain. The activity and kinetic characteristics of the systems were similar in all cases. It may be concluded that the protein components of these transport systems are coded entirely by nuclear DNA and are synthesized on the cytoplasmic ribosomes. 

We have recently reported that adenine nucleotide uptake of mitochondria prepared from a cytoplasmic petite strain and from cells grown in the presence of erythromycin was not sensitive to atractylate inhibition. It was inferred that a product of mitochondrial protein synthesis was required for atractylate sensitivity. Kolarov and Klingenberg have... 

Figure 12A shows typical polysome structures released by detergent extraction of purified mitochondria prepared without the use of EDTA and with a buffer containing Mg +. These structures are to be compared with free cytoplasmic polysomes prepared from the postmitochondrial supernatant fraction without the use of detergent (Fig. 12C). That some membrane material is still associated with Triton-X-100-extract ed mitochondria-bound polysomes is suggested by the fact that rapidly sedimenting material comprising predominantly larger polysome structures appears in the gradient following treatment of the Triton-extracted preparation with deoxycholate (Fig. 12B). No similar effect is observed on the optical density profile of free cytoplasmic polysomes prepared without the use of detergent (Fig. 12C).

The results presented in Fig. 17 show that mitochondria prepared in the presence of Mg from a log-phase culture of cells had a buoyant density in sucrose of 1.249 g/ml (Fig. 17A). However, if these mitochondria were washed with buffer containing 2 mM EDTA, a condition which will remove ribosomes from membranes, the mitochondrial density decreased markedly to 1.208 g/ml (Fig. 17C). When a log-phase culture of cells was starved for 1 h at 30°C, the mitochondria isolated in the presence of Mg were less dense (Fig. 17B) than mitochondria isolated from log-phase cells (Fig. 17A). In addition, mitochondria prepared from stationary-phase cells had a buoyant density of about 1.21 g/ml, considerably less than that of mitochondria prepared from log-phase cells. 

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