Milligram Of Mitochondrial Protein Research Articles

Stimulation of Mitochondrial Calcium Uptake by Light during Growth of Corn Shoots

Ca(2+) uptake in mitochondrial fractions, isolated on Percoll discontinuous density gradients, from light- and dark-grown corn (Zea mays L. var W64A x W182E) shoots was characterized by dual wavelength spectroscopy and the Ca(2+)-sensitive dye murexide. In light-grown seedlings, the rate of mitochondrial Ca(2+) uptake was about 40 nanomoles per minute per milligram of mitochondrial protein. A portion of the Ca(2+) uptake required an exogenous supply of ATP (65%) while the remaining 35% was the respiratory substrate-dependent reaction. Ruthenium red (2 micromolar) completely inhibited both ATP- and substrate-dependent reactions. There was no detectable Ca(2+) efflux from the mitochondria with the inhibitor. When the mitochondrial fraction was prepared from the dark-grown shoots, the rate of uptake, in particular the ATP-dependent reaction, was greatly reduced. The dark treatment caused a reduction in mitochondrial Ca content which is largely due to the reduction of Ca associated with the mitochondrial membrane rather than to a reduction of Ca in the soluble matrix.

Binding of Butyl Gallate to Isolated Mung Bean Mitochondria

The binding of radioactively labeled butyl gallate to sucrose gradient-purified mung bean (Vigna radiata L.) mitochondria was studied. Titrations showed the binding of [(14)C]butyl gallate to the mitochondria consisted of both reversible and irreversible components. The reversible component bound with a dissociation constant of approximately 1 micromolar which was comparable to the observed inhibition constant for the inhibition of the alternative pathway by butyl gallate. The reversible binding of labeled butyl gallate was also prevented by addition of excess, unlabeled salicylhydroxamic acid. The concentration of binding sites associated with reversible butyl gallate binding was around 0.5 nanomole per milligram of mitochondrial protein. These results were consistent with the reversible binding site being associated with the butyl gallate site of inhibition of the cyanide-resistant, alternative electron transfer pathway in mung bean mitochondria. In addition to the reversible butyl gallate binding site, a nonspecific, irreversible association of butyl gallate with the mitochondrial membrane was observed. The latter binding did not readily saturate at high butyl gallate concentrations and was not correlated with butyl gallate inhibition of the alternative pathway.

Reye's syndrome: Plasma-induced alterations in mitochondrial structure and function

Preincubation of rat liver mitochondria with plasma from Reye's syndrome (RS) patients induces a transient stimulation of the State 4 respiratory rate of the oxidation of NAD-linked substrates which is followed by inhibition. A loss of nearly 90% of the intramitochondrial NAD + and NADP + is also seen. The respiratory rate cannot be stimulated upon subsequent addition of ADP, but can be fully restored upon the addition of either NAD + or succinate (plus rotenone). The degree of effectiveness depends on the incubation time and the ratio of RS-plasma/mitochondrial protein. The RS-plasma effects can be eliminated by an inhibitor of mitochondrial Ca 2+ transport (ruthenium red) or by a Ca 2+ chelator (ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid). Control plasma at a concentration of 2 mg dry wt per milligram of mitochondrial protein, or 30 μ m Ca 2+ gives no effect, but can reproduce the RS-plasma effects completely when a minute amount of allantoin (10 −11 mol/mg mitochondrial protein) is also present. We conclude that allantoin and Ca 2+ can increase the permeability of mitochondrial membrane, and may be the key components responsible for the mitochondrial injuries produced by RS-plasma.

Changes in mitochondrial DNA in cardiac hypertrophy in the rat.

We studied DNA (mtDNA) replication in adult female rat hearts undergoing hypertrophy secondary to constriction of the ascending aorta. MtDNA was measured in isolated mitochondria by a fluorometric method adapted for that purpose. The conditions for removal of contaminating nuclear DNA were developed, and the purity of the mtDNA was assessed from its molecular conformation (open and closed circles) and by renaturation-kinetic analysis. The mtDNA concentration in mitochondria, expressed as micrograms of DNA per milligram of mitochondrial protein, increased 2, 4, and 7 days postoperatively by 21, 73, and 98%, respectively. Similar results were obtained when mtDNA was expressed per nonomole of cytochrome a. The population of replicative intermediates of mtDNA was analyzed by electron microscopy. In normal hearts, we observed molecular forms characteristic of animal mtDNA, such as circular monomers and dimers, catenated molecules, D-loops, expanded D-loops, and gapped molecules. D-loop frequency, which was near 50% in the mtDNA of control hearts, was markedly reduced to 5-7% in hypertrophying hearts. This result indicates that the increase in replicative flux of mtDNA is associated with the removal of a block in the conversion of D-loops to other intermediates.

Effect of palmitoyl-CoA and beta-oxidation of fatty acids on the kinetics of mitochondrial citrate transporter.

The kinetics of the hepatic mitochondrial citrate transporter were studied using 1,2,3-benzene tricarboxylate and the inhibitor-stop technique at 8 degrees C. The apparent Km for this transporter was 250 muM and the maximum velocity was 2 nmol of citrate transported per minute per milligram of mitochondrial protein. This apparent Km was increased when hepatic mitochondria were preincubated with both L-palmitoylcarnitine and CoA-SH but not with either alone. This rise in apparent Km was accompanied by a rise in the acid insoluble CoA-SH content. Removal of mitochondrial acid insoluble CoA by "defatted albumin" resulted in a parallel decrease in the apparent Km. The apparent Km for the citrate transporter was increased after coupled beta-oxidation of L-palmitoylcarnitine or octanoate without a detectable increase in acid insoluble CoA. Inhibition of beta-oxidation of L-palmitoylcarnitine by the D-derivative prevented the rise in the apparent Km. Preincubation with ATP resulted in an increase in this apparent Km. When L-palmitoylcarnitine oxidation occurred without ATP accumulation (hexokinase, glucose, ADP, and inorganic phosphate) the apparent Km for the citrate transporter increased two- to threefold. Therefore, the apparent Km for the citrate transporter varied directly with the acid insoluble CoA content. In addition, this Km was increased as a result of beta-oxidation of fatty acids but the mechanism was not solely attributable to a rise in acid insoluble CoA or ATP. The physiological implications of these findings are discussed.

Direct counting and sizing of mitochondria in solution.

Resistive particle counting has been developed for the accurate sizing and counting of mitochondria in solution. The normal detection limit with a 30 micro aperture is 0.48 micro diameter, or 0.056 micro(3) particle volume The mean volume of rat liver mitochondria was 0.42 micro(3) or 0.93 micro in diameter. The average value for numbers of particles per milligram of mitochondrial protein was 4.3 x 10(3), and per gram of rat liver was about 11 x 10(10). These values compare satisfactorily with those derived by light microscopy and electron microscopy. The mean volume for mitochondria from rat heart was 0 60 micro(3) and from rat kidney cortex, 0.23 micro(3). These values agree within 15% of those determined by electron microscopy of whole tissue. Mitochondrial fragility and contaminating subcellular organelles were shown to have little influence on the experimentally determined size distributions The technique may be applied to rapid swelling studies, as well as to estimations of the number and size of mitochondria from animals under different conditions such as liver regeneration and hormonal, pathological, or drug-induced states Mitochondrial DNA, RNA, cytochrome c-oxidase, cytochrome (a / a(3)), and iron were nearly constant per particle over large differences in particle size. Such data may be particularly valuable for biogenesis studies and support the hypothesis that the net amount per particle of certain mitochondrial constituents remains constant during mitochondrial growth and enlargement

Influence of a Low Casein Diet on Malate Dehydrogenase Activity of Rat Liver Mitochondria and Homogenate Disrupted by Various Treatments

It has been shown previously in this laboratory that the structure of rat liver mitochondria was changed by a low casein diet. In the present experiments, by measuring malate dehydrogenase activity, the degree of disruption by various destructive treatments on liver mitochondria from rats fed a low casein diet was investigated. The low casein diet contained 4% casein, and the control diet, 25%. Mitochondrial structure was disrupted by three methods: dilution with water, sonic disintegration, and addition of Triton X-100. Malate dehydrogenase activity per milligram of mitochondrial protein was higher in rats fed the low casein diet when the mitochondrial structure was disrupted by dilution with water and by addition of Triton X-100. However, when it was disrupted by sonic disintegration, the activity was almost the same in rats in both diet groups.

Respiration of Isolated Liver Mitochondria from Chickens Fed “Carbohydrate-free” Diets

One-day-old chickens were fed either a glucose control diet or diets in which all nonprotein calories were provided by either soybean oil or soybean oil fatty acids. Since feeding these “carbohydrate-free” diets has previously been found to reduce the liver mitochondrial NAD/NADH ratio without altering the ATP/ADP ratio in liver extracts, the possibility was considered that either the rate or efficiency of oxidative phosphorylation in liver mitochondria was adversely affected by feeding these diets. The rates of oxygen uptake per milligram of mitochondrial protein (in the presence and absence of added ADP) and the P/O ratios were measured in isolated mitochondria using either succinate, malate, α-ketoglutarate or citrateas electron donors. These parameters did not change as a function of diet. There was, however, an increase in the rate of NADH oxidation in uncoupled mitochondria when the “carbohydrate-free” diets were fed, and an increase in mitochondrial protein in response to feeding the soybean oil fatty acid diet. It is concluded that the change in the NAD/NADH ratio in liver mitochondria in response to feeding the “carbohydrate-free” diets is not directly attributable to a simple mitochondrial lesion.

Effect of exercise on α-glycerophosphate dehydrogenase activity in skeletal muscle

The level of activity of cytochrome oxidase, expressed per gram of fresh muscle, doubled in the gastrocnemius muscles of rats subjected to a strenuous program of treadmill running. The concentration of cytochrome c also increased twofold. In contrast, the levels of activity of mitochondrial and of eytoplasmic α-glycerophosphate dehydrogenase per gram of muscle were unaffected by the exercise. The protein content of the mitochondrial fraction from gastrocnemius muscles was increased approximately 60% in the exercising group. As a result, when cytochrome oxidase activity was expressed per milligram of mitochondrial protein, the difference between the exercising and sedentary groups was no longer significant. On the other hand, when mitochondrial α-glycerophosphate dehydrogenase activity was expressed per milligram of mitochondrial protein the value for the exercising group was significantly lower than that for the sedentary controls. These findings provide evidence that the adaptive response to prolonged exercise involves a change in the composition of the cristae of skeletal muscle mitochondria, rather than a simple uniform increase in size or number.

Oxygen consumption and respiratory pigments of mitochondria of the inner medulla of the dog kidney

Mitochondria of the inner medulla and of the cortex of the dog kidney were isolated and compared. The oxygen consumption of the medullary mitochondria was found to be inhibited to a greater degree than cortical mitochondria when the osmolality of the medium was raised by addition of sodium chloride. The respiratory pigments of medullary mitochondria have the same general pattern of distribution as is found in cortial mitochondria, but the actual amount of pigments per milligram of mitochondrial protein is less. In addition, kidney cortex contains approx. 15 times as much mitochondrial material per unit weight as does inner medualla. These findings provide further, though indirect, support for the view that the renal medulla relies largely upon anaerobic rather than aerobic pathways for its energy needs.