Mitochondrial Enrichment Research Articles

Critical Role of Mitochondrial Glutathione in the Survival of Hepatocytes during Hypoxia

Hypoxia is known to stimulate reactive oxygen species (ROS) generation. Because reduced glutathione (GSH) is compartmentalized in cytosol and mitochondria, we examined the specific role of mitochondrial GSH (mGSH) in the survival of hepatocytes during hypoxia (5% O2). 5% O2 stimulated ROS in HepG2 cells and cultured rat hepatocytes. Mitochondrial complex I and II inhibitors prevented this effect, whereas inhibition of nitric oxide synthesis with Nomega-nitro-L-arginine methyl ester hydrochloride or the peroxynitrite scavenger uric acid did not. Depletion of GSH stores in both cytosol and mitochondria enhanced the susceptibility of HepG2 cells or primary rat hepatocytes to 5% O2 exposure. However, this sensitization was abrogated by preventing mitochondrial ROS generation by complex I and II inhibition. Moreover, selective mGSH depletion by (R,S)-3-hydroxy-4-pentenoate that spared cytosol GSH levels sensitized rat hepatocytes to hypoxia because of enhanced ROS generation. GSH restoration by GSH ethyl ester or by blocking mitochondrial electron flow at complex I and II rescued (R,S)-3-hydroxy-4-pentenoate-treated hepatocytes to hypoxia-induced cell death. Thus, mGSH controls the survival of hepatocytes during hypoxia through the regulation of mitochondrial generation of oxidative stress.

Seminal quality correlates with mitochondrial functionality

Oligozoospermia is an important manifestation of male subfertility and very little attention has been paid to study a possible relationship between the total number of ejaculated spermatozoa and mitochondrial functionality. In this work we report a direct correlation between spectrophotometrically measured mitochondrial enzyme activities (citrate synthase and respiratory complex I, II, I+III, II+III and IV) and seminogram parameters (sperm motility, vitality and cell concentration). In addition, total ejaculated spermatozoa correlate much better with the nuclear-encoded citrate synthase and complex II than with the mitochondrial-encoded complex I, III and IV activities. Furthermore, total number of spermatozoa has a significant but negative correlation with the ratios of complex I, complex III and complex IV to complex II (and citrate synthase). These ratios are significantly higher in aged subjects emphasizing the physiological relevance of this observation. These results suggest that the simultaneous increase of the number of ejaculated spermatozoa and the mitochondrial enrichment of citrate synthase and complex II are both parallel responses to the same regulatory events.

Fission yeast Msp1 is a mitochondrial dynamin-related protein.

We recently identified Msp1p, a fission yeast Schizosaccharomyces pombe dynamin-related protein, which is essential for the maintenance of mitochondrial DNA. The Msp1p sequence displays typical features of a mitochondrial protein. Here we report in vitro and in vivo data that validate that prediction. We demonstrate that the targeting sequence of Msp1p is processed by recombinant mitochondrial processing peptidase and that Msp1p is imported into S. pombe mitochondria in vitro in the presence of cellular extracts. We show that the first 109 residues of Msp1p encompass a functional peptide signal that is sufficient to direct chimera to mitochondria. Immunofluorescence studies indicate that Msp1p staining colocalises with a mitochondrial marker and electron microscopy shows that the protein is located inside the mitochondria. Mitochondrial enrichment and fractionation further confirm that localisation and show that Msp1p is anchored to the matrix side of the mitochondrial inner membrane. Finally, we report that overexpression of the Msp1 protein results in gross alteration of the mitochondrial structure and function. All together our results suggest that Msp1p is an essential component for mitochondrial maintenance.

Mitochondrial DNA enrichment for species identification and evolutionary analysis

Nucleic acid-based species identification often targets the mitochondrial encoded cytb gene. However, polymerase chain reaction (PCR)- restriction fragment length polymorphism (RFLP) analysis using universal primers sometimes leads to ambiguous results, which are due to the presence of nuclear encoded pseudo-cytb genes. Such ambiguities were succesfully avoided using a newly developed method for the enrichment of mitochondrial DNA. In addition, a mitochondrial cytb-specific PCR system was designed allowing the unambiguous identification of game meat.

Hepatic stellate cell activation in genetic hemochromatosis: Lobular distribution, effect of increasing hepatic iron and response to phlebotomy: [formula omitted] Liver Unit, Queensland Institute of Medical Research and 1Department of Pathology, Royal Brisbane Hospital, Brisbane, QLD., AUSTRALIA, 4029

Iron overload is a key contributor to the pathogenesis of multiple disorders including the sideroblastic anemias. The specific iron compounds present in tissues or cells that are the target of iron deposition remain poorly understood, but there is evidence that some forms are magnetically active. We have developed a simple and specific method to purify iron-overloaded red blood cells using magnetic affinity columns. Here we describe this method and characterize purified Sod2-deficient siderocytes.RBC derived from mice transplanted with Sod2-deficient hematopoietic stem cells served as a source of iron-laden cells. Purification was based upon the observation that iron deposits in Sod2-deficient cells are “magnetically susceptible” and allow for retention of iron-laden cells in a strong magnetic field. Peripheral blood from Sod2-deficient chimeric mice was passed through magnetic separation columns; iron-overloaded cells were eluted and characterized by flow cytometry, Western blot, and microscopy.We were able to purify 2.8% of the total red cells as iron-laden siderocytes. The magnetically purified Sod2-deficient cells were predominantly identified as reticulocytes. They had numerous siderotic granules, produced enhanced levels of reactive oxygen species, and showed increased protein oxidative damage, mitochondrial enrichment, and mitochondrial hyperpolarization.Our method can be used to purify iron-laden cells as well as iron-associated subcellular fractions prepared from iron-loaded tissues, allowing elucidation of the structure, location, and protein composition of such iron deposits. This data will help develop our understanding of the pathogenesis of SA and other disorders characterized by iron overload.

Characterization of a mitochondrial NADP-dependent isocitrate dehydrogenase in axes of germinating sunflower seeds

An NADP-dependent isocitrate dehydrogenase (NADP-IDH) activity was measured in a Percoll purified mitochondrial fraction from axes of germinating sunflower ( Helianthus annuus, L.) seeds. Fraction enrichment in mitochondria was revealed by marker enzyme activities and by electron microscopy which showed no contamination by other organelles and indicated that no fragmentation of mitochondria had occurred during their preparation. More than 95% of the NADP-IDH activity was latent; it was found to be different from the mitochondrial NAD-IDH activity by competition assay. Mitochondrial NADP-IDH was electrophoretically and immunologically different from the NAD-specific and the cytosolic NADP-specific isoforms. The enzyme activity from mitochondrial matrix extract was eluted from Superose 12 with a molecular weight of 70 kDa. A possible physiological role of the mitochondrial NADP-IDH is discussed.

Age-dependent impairment of mitochondrial function in primate brain.

It has been hypothesized that some of the functional impairments associated with aging are the result of increasing oxidative damage to mitochondrial DNA that produces defects in oxidative phosphorylation. To test this hypothesis, we examined the enzymes that catalyze oxidative phosphorylation in crude mitochondrial preparations from frontoparietal cortex of 20 rhesus monkeys (5-34 years old). Samples were assayed for complex I, complex II-III, complex IV, complex V, and citrate synthase activities. When enzyme activities were corrected for citrate synthase activities (to account for variable degrees of mitochondrial enrichment), linear regression analysis demonstrated a significant negative correlation of the activities of complex I (p < 0.002) and complex IV (p < 0.03) with age but no significant change in complex II-III or complex V activities. Relative to animals 6.9 +/- 0.9 years old (n = 7), the citrate synthase-corrected activity of complex I was reduced by 17% in animals 22.5 +/- 0.9 years old (n = 6) (p < 0.05) and by 22% in animals 30.7 +/- 0.9 years old (n = 7) (p < 0.01). Similar age-related reductions in the activities of complexes I and IV were obtained when enzyme activities were corrected for complex II-III activity. These findings show an age-associated progressive impairment of mitochondrial complex I and complex IV activities in cerebral cortices of primates.

Calcium pools in sea urchin eggs: Roles of endoplasmic reticulum and mitochondria in relation to fertilization

A preparation of sea urchin eggs permeabilized with digitonin (40 μM for 2.5 min) was used to study the kinetic characteristics of the two cellular compartments suspected to play a key role in cellular calcium transfer during fertilization: an ATP-dependent Ca 2+ pool (Km = 0.47 μM; Vm = 0.48 nmol/min.mg protein) probably located in the endoplasmic reticulum and a mitochondrial Ca 2+ pool (Km = 1.50 μM; Vm = 0.12 nmol/min.mg protein). Fertilization triggered a decrease in the rate of ATP dependent uptake by the non-mitochondrial pool (Km = 0.59 μM; Vm = 0.15 nmol/min.mg protein) while it transiently increased the Ca 2+ uptake into mitochondria (2 min post-fertilization: Km = 2.20 μM; Vm = 0.40 nmol/min.mg protein). Microanalysis studies performed on quickly frozen, freeze substituted and embedded eggs showed a transient Ca 2+ enrichment of mitochondria soon after fertilization thus suggesting that mitochondria behave as a Ca 2+ sink at fertilization. Results are discussed in relation to the role of endoplasmic reticulum and mitochondria in handling free calcium during the early period following sea urchin egg fertilization.

Subcellular distribution and activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase in the southern armyworm, Spodoptera eridania

AbstractIn mid‐fifth‐instar larvae of the southern armyworm, Spodoptera eridania, the subcellular distribution of four antioxidant enzymes—superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX), and glutathione reductase (GR)—were examined. Two‐thirds (4.26 units ·mg protein−1) of the SOD activity was found in the cytosol, and one‐thirds (2.13 units ·mg protein−1) in the mitochondria. CAT activity was unusually high and not restricted to the microsomal fraction where peroxisomes are usually isolated. The activity was distributed as follows: cytosol (163 units) mitochondria (125 units) and microsomes (119 units). Similar to CAT, the subcellular compartmentalization of both GPOX and GR was unusual. No activity was detected in the cytosol, but in mitochondria and microsomes, GR levels were 5.49 and 3.09 units. Although GPOX activity exhibited 14–16‐fold enrichment in mitochondria and microsomes, respectively, over the 850g crude homogenate, the level was negligible (mitochondria = 1.4 × 10−3 units; microsomes = 1.6 × 10−3 units), indicating that this enzyme is absent. The unusual distribution of CAT has apparently evolved as an evolutionary answer to the absence of GR from the cytosol, and the lack of GPOX activity.

Method to Obtain a Chlorophyll-free Preparation of Intact Mitochondria from Spinach Leaves

Mitochondria from green leaves of spinach have been prepared using a three-step procedure involving differential centrifugation, partition in an aqueous dextran polyethylene glycol two-phase system and Percoll gradient centrifugation. The mitochondrial fractions after the different steps of purification were compared. The final mitochondrial preparation was totally free from chloroplast material measured as chlorophyll content. The enrichment of mitochondria in relation to peroxisomes and microsomes was approximately 12 and 33 times, respectively, based on NAD:isocitrate dehydrogenase activity, glycolate oxidase activity, and NADPH:cytochrome c oxidoreductase activity. The apparent intactness of the inner and the outer mitochondrial membranes was higher than 90% as measured by latency of enzyme activities. The mitochondria showed high respiratory rates with respiratory control and the ADP/O ratios approached the theoretical limits.

Preparatiön of mitochondria from gree leaves of spinach by differential centrifugation and phase partition

Mitochondria from green leaves of spinach ( Spinacia oleracea L) have been prepared by a preparation procedure using differential centrifugation and partition in an aqueous dextran-poly(ethylene glycol) two-phase system. The enrichment of mitochondria in relation to chloroplast material was 200–400 times based on NAD: isocitrate dehydrogenase (IDH) activity and chlorophyll content. The protein/chlorophyll ration in the preparation was approximately 100. The apparent intactness of both the inner and the outer membrane was about 90%, as measured by latency of enzyme activities. The mitochondria oxidized NADH (respiratory control 2.1), succinate (1.6), glycine (3.8) and malate (3.0). The mitochondrial preparation was morphologically characterized by electron microscopy.

Profound muscle weakness associated with axis cylinder ballooning. A case report.

Multifocal central white matter axonal swelling characterized electron microscopically by focal axonal mitochondrial and membraneous body enrichment occurred in a 52 year old man who presented with the chief complaint of severe progressive muscular weakness accompanied by disorientation. The clinical and pathological findings in this patient appear unique and are presented in order to broaden our concepts of axis cylinder pathology.

Formation of yeast mitochondria. 3. Biochemical properties of mitochondria isolated from a cytoplasmic petite mutant.

A number of biochemical properties of mitochondria from a cytoplasmic petite mutant ofSaccharomyces cerevisiae with an extremely high adenine plus thymine content have been studied. When such particles are isolated by means of standard procedures developed for use with wild-type yeasts they are grossly contaminated by non-mitochondrial membrane fragments. Further enrichment of mitochondria is achieved by non-equilibrium centrifugation in sucrose gradients. Throughout this purification procedure the particles can be shown to retain an outer limiting, as well as a non-cristate inner membrane. In many of their morphological and physical features (size, shape, buoyant density) they resemble mitochondria isolated from the wild type. Although enzymes of the respiratory chain are absent from the mutant particles, their content ofl-malate dehydrogenase, NADP-dependent isocitrate dehydrogenase, and ATPase is comparable to that found in the wild type. The mitochondrial ATPase in this mutant strain is cold stable, oligomycin insensitive, Dio-9 sensitive, and susceptible to inhibition by the F1 inhibitor of beef heart. The enzyme can be rendered cold labile by its detachment from the membrane, followed by fractionation with protamine sulfate and ammonium sulfate. The existence of mutant particles that are incapable of function in oxidation and phosphorylation but resemble their functional homologues in many other ways raises the possibility that mitochondria are required in the cellular economy for purposes not directly linked to oxidative phosphorylation and electron transport. This hypothesis has led us to suggest that, contrary to models currently under discussion, mitochondria did not evolve as a consequence of endosymbiosis. We propose as an alternative that the mitochondrial organelle evolved as a means of improvement of existing subcellular structures in the primordial (perhaps eukaryotic) cell. Partial autonomy may thus constitute a relatively recent modification; the present-day mitochondrial genome had its origin in nuclear DNA and may have been amplified in a manner not unlike the amplification of ribosomal RNA cistrons in developing oocytes ofXenopus.