Mitochondrial preparation

If the mechanism of superoxide production in microsomes by NADPH-cytochrome P-450 reductase, NADH-cytochrome b5 reductase, and cytochrome P-450 is well documented, it cannot be said about microsomal hydroxyl radical production. There are numerous studies, which suggest the formation of hydroxyl radicals in various mitochondrial preparations and by isolated microsomal enzymes. It has been shown that the addition of iron complexes to microsomes stimulated the formation of hydroxyl radicals supposedly via the Fenton... 

Between 1955 and 1960 various sub-mitochondrial preparations were developed to give vesicles comprising only sealed inner mitochondrial membranes. Cooper and Lehninger used digitonin extraction Lardy and Kielley Bronk prepared sub-mitochondrial particles by sonication. At this time, too, Racker and his colleagues isolated Fq/F1 particles from mitochondria and showed that a separated FI particle behaved as an ATPase. The F0 portion had no enzymic properties but conferred oligomycin sensitivity on the FI ATPase. The orientation of these sub-mitochondrial vesicles (inside-out or vice-versa) was shown by the position in electron micrographs of the dense (FI) particles which in normal intact mitochondria project into the matrix and so define the surface of the inner mitochondrial membrane. 

Mustafa et o/. were unable to detect lung lipid peroxides after shortterm or subacute ozone exposures (0.8-2.0 ppm), but noted their presence after in vitro exposures of mitochondrial preparations. Dowell et a/. saw no evidence of lipid peroxidation in an alveolar macrophage preparation obtained from rabbits acutely exposed to ozone at up to 10 ppm. 

TABLE III. Inhibition by Selected Allelochemlcals of Malate OxJ atlon by Intact Mung Bean Mitochondria and of Mg -ATPase Activity of Freeze-thawed Mung Bean Mitochondrial Preparations ... 

Oxidation of Initiated Habilitate Pakrritate uniformly labeled with tritium (3H) to a specific activity of 2.48 X 10 counts per minute (cpm) per micromole of palmitate is added to a mitochondrial preparation that oxidizes it to acetyl-CoA. The acetyl-CoA is isolated and hydrolyzed to acetate. The specific activity of the isolated acetate is 1.00 X 107 cpm//xmol. Is this result consistent with the /1-oxidation pathway Explain. What is the final fate of the removed tritium ... 

B 9. A student group completed the biuret assay on their mitochondrial preparation. Assume that the following absorbance values were obtained as described in Table E10.2. 

It has been reported that vitamin Kj and several of the vitamin K2 homologues are capable of restoring electron transport in solvent-extracted or irradiated bacterial and mitochondrial preparations. Other reports suggest that vitamin K is concerned with the phosphorylation reactions accompanying oxidative phosphorylation The capacity of these compounds to exist m several forms, e.g., quinone, quinol. chromanol, etc., appears to strengthen the proposal that links them to oxidative phosphorylation. Information has suggested that vitamin K acts to induce prothrombin synthesis. Since prothrombin has been shown to be synthesized only by liver parenchymal cells m the dog, it would appear that the proposed role for vitamin K is not specific for only prothrombin synthesis, but applicable to other proteins. 

In 1960, Rafter (32) first described an activity in mouse liver mitochondrial preparations which was capable of catalyzing the hydrolysis of PPi and the transfer of a phosphoryl group from PPi to glucose to produce glucose-6-P [reaction (3a)]. This same activity was also observed... 

Small amounts of activity which often have been reported for mitochondrial preparations appear to result from contamination of such preparations with small amounts of microsomes, as indicated above (37, 39). 

A number of important biochemical reactions occur intramitochondrially and some of these are described below. One major drawback to the study of energy metabolism in cestodes is the failure, due to technical difficulties, to produce isolated mitochondrial preparations of the purity which can be obtained from mammalian tissues. Generally, mitochondrial fractions of cestode origin are contaminated with other cellular components such as glycogen, endoplasmic reticulum and various membranes, and some of the data obtained with such mitochondria should, therefore, be treated with caution. 

An acyl-CoA transferase has been detected in sonicated mitochondrial preparations from Spirometra mansonoides (643). 

There are a number of complications in the interpretation of such data. The mitochondria must be permeable to cations other than H+. If this were not the case, then H+ extrusion would lead to an increase in Ai/f, which would diminish further H+ extrusion. Incorporation of K+ and addition of the ionophore valinomycin to the mitochondrial preparation prevents increases in Ai/>. 

Addition of DCCD (dicyclohexylcarbodiimide) to mitochondrial preparations decreases the rates of both ATP synthesis and electron transport. Only the latter process can be restored to normal levels upon addition of 2,4-dinitrophenol. How can these observations be explained ... 

The pattern of inhibition of 17a-hydroxylase exhibited by metyrapone lends further support to the hypothesis that cytochrome P450 is involved in the enzyme system319 whilst the effect of administration of spironolactone on the cytochrome P450 content and 17a-hydroxylase activity in adrenal tissue has shown that decreases in both of these factors occur only in animals that produce predominantly cortisol rather than corticosterone.320 18-Hydroxylation of deoxycorticosterone has been demonstrated in rat and bovine mitochondrial preparations and in reconstituted systems obtained from these fractions.321 In all cases, 18-hydroxylation was accompanied by 11/8-hydroxylation, and the study indicated that very similar types of cytochrome P450 were involved in both hydroxylation systems. 

Sprinson and coworkers [30] conducted the methylmalonyl-CoA mutase reaction in deuterium oxide using a crude mitochondrial preparation. The presence of methylmalonyl-CoA epimerase insured that (1) all substrate molecules incorporated one atom of deuterium into position 2, and (2) in the course of the reaction the (2R)-epimer of methylmalonyl-CoA was continuously supplied by epimerization of the (25)-epimer, which was in turn generated by the enzymic carboxylation of propionyl-CoA. Alkaline hydrolysis of the product and subsequent purification furnished succinic acid which was mainly monodeuterated (70% 2H,-, 15% 2H2-labelled and 13% unlabelled species). A positive ORD curve revealed its (5) configuration indicating stereochemical retention for the AdoCbl-dependent rearrangement (Fig. 22). No plausible explanation could be offered for the formation of doubly deuterated and unlabelled species. Essentially the same results were later obtained with a highly purified mutase preparation from Propionibacterium sher-manii (J. Retey, unpublished). 

These results were similar to results of work done in plants, which showed that propionate is metabolized to acetate (20,21) by mitochondrial preparations. 

Yuan, C., Acosta, D., Jr. (2000). Effect of cocaine on mitochondrial electron transport chain evaluated in primary cultures of neonatal rat myocardial cells and in isolated mitochondrial preparations. Drug Chem. Toxicol. 23 339-48. 

Add 20 4mM dinitrophenol to the sample chamber using a Hamilton syringe. At this point the quality of the mitochondrial preparation can be ascertained. Mitochondria in good condition give a substantially higher rate of oxygen consumption when they are uncoupled with dinitrophenol. Explain this observation. 

The traditional way to determine OXPHOS enzyme activities is by spectrophotometry. Several assays have been described for all 5 OXPHOS complexes. The assays are performed in homogenates of tissue samples or cultured cells, in crude mitochondria-enriched 600 g supernatants of tissue/cell homogenates, or in mitochondrial preparations from 14000 g pellets derived from 600 g supernatants. Obviously, the higher the... 

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