Well-coupled Mitochondria Research Articles

Procedures of Mitochondria Purification and Gene Expression to Study Alternative Respiratory and Uncoupling Pathways in Fruits.

We describe detailed procedures to get intact and well-coupled mitochondria from a variety of fruit species such as papaya (Carica papaya), guava (Psidium guajava), tomato (Solanum lycopersicum), and strawberry (Fragaria x ananassa) as well as the protocols to assess the capacities of AOX and UCP pathways in intact fruit mitochondria. The procedures presented here were tested for the species mentioned above; their use with other types of fruits must be tested for yield of intact and active mitochondria. This is possible from individual adjustments. Strict care during extraction, including the use of osmotic protectants (i.e., mannitol/sucrose) and antioxidants (i.e., cysteine, ascorbate) at defined concentrations, are critical factors to ensure mitochondrial integrity and to obtain higher yields. The mitochondria purified using the discontinuous Percoll gradients described here can be used for the analysis of the capacity of alternative respiration and uncoupling pathways in fruits. In addition, protocols for quantitative and semiquantitative PCR applicable for the analysis of AOX and UCP gene expression in fruits are shown. Microarray and RNA-seq data from public databases are also valuable for the analysis of AOX and UCP genes. In both cases having the sequences of genes or cDNAs to be used in primer design or probe identification is necessary.

Preparation of purified perikaryal and synaptosomal mitochondrial fractions from relatively small hypothalamic brain samples

In order to measure the activity of neuronal mitochondria, a representative proof of neuronal processes, physiologically relevant mitochondrial samples need to be gained as simply as possible. Existing methods are, however, either for tissue samples of large size and/or homogenous microstructures only, or are not tested for mitochondrial function measurements. In the present article we describe a gradient fractionation method to isolate viable and well-coupled mitochondria from relatively heterogeneous histological microstructures such as the hypothalamus. With this new method, we are able to isolate a sufficient amount of functional mitochondria for determination of respiratory activity, in a short period of time, using affordable equipment.•Verified by electron microscopy, our method separates highly enriched and well-preserved perikaryal and synaptosomal mitochondria. Both fractions contain minimal cell debris and no myelin. Respiratory measurements (carried out by Clark-type electrode) confirmed undisturbed mitochondrial function providing well-evaluable records. The demonstrated protocol yields highly viable mitochondrial subfractions within 3h from small brain areas for high-precision examinations. Using this procedure, brain regions with relatively heterogeneous histological microstructure (hypothalamus) can also be efficiently sampled.•Up to our present knowledge, our method is the shortest available procedure with the lowest sample size to gain debris-free, fully-viable mitochondria.

Determination of the H+-ATP Synthase and Hydrolytic Activities

The H+-ATP synthase of the inner mitochondrial membrane utilizes the proton gradient generated by the respiratory chain to synthesize ATP. Under depolarizing conditions, it can function in reverse by hydrolyzing ATP to generate a proton gradient. The protocols presented here allow the facile determination of both the synthetic and hydrolytic activities of the H+-ATP synthase in isolated mitochondria and in permeabilized mammalian cells. Since the protocol requires the isolation of polarized and well-coupled mitochondria, first we describe the protocol for mitochondrial isolation from mouse tissues. Second, we describe the protocol for measuring the ATP synthetic activity as end-point and kinetic modes in isolated mitochondria and in permeabilized cells. Finally, we describe the protocol for the determination of the ATP hydrolytic activity of the enzyme in isolated mitochondria.

Lazy or old: mitochondria decline in vivo in human muscle similarly with inactivity and age

Here we tested the role of lifestyle vs. age using innovative noninvasive tools to measure key mitochondrial functional properties in vivo. Mitochondrial coupling (ATP/O2 or P/O), and phosphorylation capacity (ATPmax) were assessed in human vastus lateralis muscle (VL). Oxidative capacity was biochemically assessed in vitro from biopsies taken from the same site as the in vivo measures. The chronic activity level was measured using a physical activity questionnaire, VO2max test (active >40 ml O2 kg-1 min-1) and verified using a triaxial accelerometer. Subjects were screened to be healthy and disease free and were on average 30 yrs old for the active (n=9) and sedentary (n=18) subjects and 68 yrs old for the elderly (n=30). Mitochondrial coupling in active controls (2.3+0.20, mean+SE) agreed with well-coupled mitochondria (2.3–2.5) but both sedentary (P/O=1.41+0.09) and elderly groups (P/O=1.69+0.11) were substantially uncoupled. Similarly, active subjects had a higher ATPmax (1.11+0.08 mM ATP/sec) than sedentary (0.67+0.03) or aged groups (0.61+0.04). Oxidative capacity was reduced in both groups by 25% relative to active adults. These results demonstrate that mitochondrial properties in vivo drop substantially in sedentary adults and the old. Thus inactivity may be as important as age in the mitochondrial decline found in the elderly. Supported by NIH AG 030226, AR 41928, RC2AG036606, 1P30 DK072476.

Preserved Coupling of Oxidative Phosphorylation But Decreased Mitochondrial Respiratory Capacity in IL-1β-Treated Human Peritoneal Mesothelial Cells

The peritoneal mesothelium acts as a regulator of serosal responses to injury, infection, and neoplastic diseases. After inflammation of the serosal surfaces, proinflammatory cytokines induce an "activated" mesothelial cell phenotype, the mitochondrial aspect of which has not previously been studied. After incubation of cultured human peritoneal mesothelial cells with interleukin (IL)-1beta for 48 h, respiratory activity of suspended cells was analyzed by high-resolution respirometry. Citrate synthase (CS) and lactate dehydrogenase (LDH) activities were determined by spectrophotometry. Treatment with IL-1beta resulted in a significant decline of respiratory capacity (p < 0.05). Respiratory control ratios (i.e., uncoupled respiration at optimum carbonyl cyanide p-trifluoromethoxyphenylhydrazone concentration divided by oligomycin inhibited respiration measured in unpermeabilized cells) remained as high as 11, indicating well-coupled mitochondria and functional integrity of the inner mitochondrial membrane. Whereas respiratory capacities of the cells declined in proportion with decreased CS activity (p < 0.05), LDH activity increased (p < 0.05). Taken together, these results indicate that IL-1beta exposure of peritoneal mesothelial cells does not lead to irreversible defects or inhibition of specific components of the respiratory chain, but is associated with a decrease of mitochondrial content of the cells that is correlated with an increase in LDH (and thus glycolytic) capacity.

Isolation and some biochemical properties of porcine pancreas mitochondria.

Morphologically and functionally intact mitochondria were isolated from the porcine pancreas using a conventional, differential centrifugation method. The homogenate of the porcine pancreas made in a medium containing 0.25 M sucrose, 10 mM Tris-Cl, pH 7.4, 0.5% bovine serum albumin and 1 mM EDTA, in the presence or in the absence of dibucaine and trypsin inhibitor, was centrifuged for 10 min at 700xg. The supernatant was centrifuged for 10 min at 7000xg, and the pellet was washed three times. Trypsin activity of mitochondria isolated in the absence of dibucaine and trypsin inhibitor was as low as that of mitochondria isolated in the presence of dibucaine and trypsin inhibitor, and a major part of the activity remained inactivated. Phospholipase A2 activity of the former mitochondria was as low as that of the latter, and remained unchanged up to 8-10 hr at 4 degrees C. The presence of bovine serum albumin and EDTA in the respiration medium was absolutely required to obtain good respiratory controls of those mitochondria. These data suggest that well-coupled mitochondria can be obtained from the pancreas by a conventional isolation procedure without activating the major part of trypsin.

Pathway of glutamate oxidation and its regulation in the HuH13 line of human hepatoma cells

Well-coupled mitochondria were isolated from a HuH13 line of human hepatoma cells and human liver tissue. The liver mitochondria showed a feeble glutamine oxidation activity in contrast to the hepatoma mitochondria, whereas they utilized glutamate well for the oxidative phosphorylation. In the liver mitochondria, glutamate was oxidized via the routes of transamination and deamination. On the other hand, glutamate oxidation was initiated preferentially via transamination pathway in the tumor mitochondria. In the liver mitochondria, bicarbonate nearly at a physiological concentration inhibited oxygen uptake with glutamate as substrate. The interaction of bicarbonate with the pathway of glutamate oxidation occurred primarily at the level of succinate dehydrogenase, due to competitive inhibition of the enzyme by the compound. In contrast to the liver mitochondria, glutamate oxidation was not affected by bicarbonate in the tumor mitochondria. These results indicate that the aberrations in the glutamate metabolism and its regulation observed in the hepatoma mitochondria may be favorable to the respiration utilizing glutamine and/or glutamate as an energy source.

A simple method for preparing physiologically active mitochondria from plant leaves rich in oils and phenolics

Amberlite XAD-7, a nonionic polyacrylate adsorbent, was found to be a very effective protectant for isolating mitochondria from tissues rich in oils and phenolics. Physiologically active, well-coupled mitochondria were successfully prepared from young green leaf tissues of citrus, apple, pear and tobacco.

Characteristics of potassium ion transport in pea cotyledon mitochondria isolated by a zonal technique.

Methodology was developed for measurement of K+ transport in pea cotyledon mitochondria isolated by zonal centrifugation. The valinomycin-mediated transport process required respiratory energy from Krebs cycle intermediates and was completely reversible in the absence of oxygen. The rate and extent of uptake were dependent on the presence of proton-conducting anions (with acetate plus phosphate being most effective), well-coupled mitochondria, and on the K+ concentration.

Preparation and properties of mitochondria from lactating rat mammary gland in particular relation to lipogenesis

1. 1. Well-coupled mitochondria have been prepared from lactating rat mammary gland. 2. 2. The mitochondria can accumulate pyruvate via a specific carrier system to the same extent as can liver mitochondria. Transport of pyruvate across the mitochondrial membrane is facilitated by the passage of citrate or malate in the opposite direction.

Isolation of bovine adrenal cortex mitochondria from the zona glomerulosa

Isolation procedures for the bovine adrenal cortex mitochondria from the zona glomerulosa are described. Special care was taken to avoid contamination of the mitochondria from the zona fasciculata. Bovine adrenal cortex mitochondria in the zona glomerulosa in situ had a tubular or tubulo-vesicular form of cristae when they were examined in the electron microscope, whereas they showed a condensed form when isolated in sucrose-Tris or sucrose-Tris-EDTA medium. By contrast, mitochondria in the zona fasciculata and zona reticularis, either in situ or isolated in a sucrose-Tris medium showed a vesicular form of cristae, whereas they showed a condensed form when isolated in a sucrose-Tris-EDTA medium. Well-coupled mitochondria were obtained from the zona glomerulosa when they were isolated in a sucrose-Tris-EDTA medium.