Mitochondrial structure and function are disrupted by standard isolation methods

Mitochondria regulate critical components of cellular function via ATP production, reactive oxygen species production, Ca(2+) handling and apoptotic signaling. Two classical methods exist to study mitochondrial function of skeletal muscles: isolated mitochondria and permeabilized myofibers. Whereas mitochondrial isolation removes a portion of the mitochondria from their cellular environment, myofiber permeabilization preserves mitochondrial morphology and functional interactions with other intracellular components. Despite this, isolated mitochondria remain the most commonly used method to infer in vivo mitochondrial function. In this study, we directly compared measures of several key aspects of mitochondrial function in both isolated mitochondria and permeabilized myofibers of rat gastrocnemius muscle. Here we show that mitochondrial isolation i) induced fragmented organelle morphology; ii) dramatically sensitized the permeability transition pore sensitivity to a Ca(2+) challenge; iii) differentially altered mitochondrial respiration depending upon the respiratory conditions; and iv) dramatically increased H(2)O(2) production. These alterations are qualitatively similar to the changes in mitochondrial structure and function observed in vivo after cellular stress-induced mitochondrial fragmentation, but are generally of much greater magnitude. Furthermore, mitochondrial isolation markedly altered electron transport chain protein stoichiometry. Collectively, our results demonstrate that isolated mitochondria possess functional characteristics that differ fundamentally from those of intact mitochondria in permeabilized myofibers. Our work and that of others underscores the importance of studying mitochondrial function in tissue preparations where mitochondrial structure is preserved and all mitochondria are represented.     

Separation of cell organelles in density gradients based on their permeability characteristics

The buoyant density of intracellular organelles is dependent in part on the nature of the buffer composition of the density gradient and the permeability characteristics of the organelle membrane to the constituents of this buffer. Therefore, knowledge of the transport properties of different organelles allows the design of density gradients useful for their purification. We have used this approach to significantly decrease mitochondrial contamination of pancreatic zymogen granules in a one-step purification procedure on a 40% Percoll density gradient. These gradients, prepared with isoosmotic sucrose, yield a narrow band of zymogen granules and mitochondria. However, by substitution of sucrose with salts to which mitochondria but not zymogen granules are permeable, the densities of mitochondria are altered to give a significant separation. For example, the incorporation of 100 mM sodium succinate in the Percoll gradient can produce a 70% reduction in mitochondrial contamination. The increased ionic strength has an additional beneficial effect on zymogen granule yield by 5-10%. The recognition and utilization of transport pathways in organelle membranes is the principal feature of this technique and should prove to be widely applicable to other isolation procedures.     

Lipid Synthesis and Ultrastructure of Isolated Barley Chloroplasts

The cell organelle contents of chloroplast preparations made from barley leaves with salt and sucrose isolation media at pH 6 and 8 were determined and compared with the acetate incorporating activity of these preparations. A chloroplast preparation obtained with 0.5 m sucrose at pH 8 gave the highest number of intact chloroplasts (with envelope and stroma), the lowest number of contaminating mitochondria, and the highest activity in light dependent acetate incorporation into lipids. In the preparations observed, the light induced lipid synthesizing capacity correlates well with the percentage of intact chloroplasts. It is suggested that the intact chloroplasts are responsible for the light induced lipid synthesis of the preparations and that the synthesizing enzymes are localized in the chloroplast stroma. Acetate is mainly incorporated into palmitic and oleic acids. The low yield of intact chloroplasts and of light induced lipid synthesis in preparations isolated at pH 6 seem to result from the action of galactolipid lipase(s).     

Effect of KCl Medium on Malate Oxidation in Mitochondria of Sugar Beet Taproot

Isolated mitochondria were obtained from growing and stored sugar beet (Beta vulgaris L.) taproots. These preparations were used to monitor the mitochondrial matrix volume and malate oxidation after the replacement of sucrose with KCl in the reaction medium. The transfer of mitochondria from sucrose-containing isolation medium to the isoosmotic KCl solution initiated spontaneous or energy-dependent (in the presence of respiratory substrate) swelling whose kinetic parameters (the initial rate and amplitude) were virtually independent of the plant age. At the same time, effects of KCl-induced swelling on oxidative and phosphorylating activities of mitochondria were age-dependent. In mitochondria from growing taproots, K⁺ ions stimulated nonphosphorylating malate oxidation, thereby decreasing the respiratory control ratio and the ADP/O coefficient. The incubation of mitochondria from stored taproots in KCl solution induced a short-term activation and subsequent progressive inhibition of malate oxidation but did not inhibit the oxidation of exogenous NADH. The inhibition of malate oxidation was not released by adding ADP or uncouplers and was enhanced in the presence of valinomycin. The swelling of mitochondria in KCl solutions did not impair the integrity of mitochondrial membranes and did not preclude stimulation of malate oxidation by exogenous NAD. It is supposed that the KCl-induced inhibition of respiration is related to a large increase in the matrix volume and a drastic decrease in the concentration of a coenzyme NAD. Previous studies with isolated mitochondria from stored taproots showed that the mitochondrial NAD level was a rate-limiting factor of malate oxidation assayed in the sucrose-containing media. A possible role of K⁺-transporting mechanisms in regulation of mitochondrial matrix volume and metabolic activity of plant mitochondria is discussed.     

Glucagon stimulation of liver mitochondrial CO2 fixation utilizing pyruvate generated inside the mitochondria.

Glucagon administration to the intact rat has been shown to stimulate pyruvate metabolism in liver mitochondria, presumably by increasing pyruvate transport into the organelle. In this report, we used alanine in place of pyruvate to examine the possibility that glucagon might stimulate pyruvate carboxylation per se independent of its postulated action on pyruvate transport. In agreement with previous reports, injection of a low dose of glucagon (50 micrograms/kg of rat) increased respiration, ATP synthesis, pyruvate decarboxylation, and CO2 fixation in liver mitochondria subsequently isolated. When alanine was used as a substrate, CO2 fixation, but not decarboxylation, was increased in liver mitochondria isolated from glucagon-treated rats. Pyruvate accumulation under these conditions was significantly lower in the glucagon-treated rat preparation. When mitochondria were incubated in a HCO3- -deficient buffer, pyruvate accumulation was identical in both preparations. The addition of a pyruvate transport inhibitor, alpha-cyanohydroxycinnamate (0.5 mM), inhibited CO2 fixation with pyruvate by 70%, but had no effect when alanine was used. Our data therefore suggest that glucagon stimluates mitochondrial pyruvate carboxylation independent of its possible action on pyruvate transport.     

Testing for diffusion limitations in salt-activated enzyme catalysts operating in organic solvents

The dramatic activation of serine proteases in nonaqueous media resulting from lyophilization in the presence of KCl is shown to be unrelated to relaxation of potential substrate diffusional limitations. Specifically, lyophilizing subtilisin Carlsberg in the presence of KCl and phosphate buffer in different proportions, ranging from 99% (w/w) enzyme to 1% (w/w) enzyme in the final lyophilized solids, resulted in biocatalyst preparations that were not influenced by substrate diffusion. This result was made evident through use of a classical analysis whereby initial catalytic rates, normalized per weight of total enzyme in the catalyst material, were measured as a function of active enzyme for biocatalyst preparations containing different ratios of active to inactive enzyme. The active enzyme content of a given biocatalyst preparation was controlled by mixing native subtilisin with subtilisin preinactivated with PMSF, a serine protease inhibitor, and lyophilizing the enzyme mixture in the presence of different fractions of KCl and phosphate buffer. Plots of initial reaction rates as a function of percent active subtilisin in the biocatalyst were linear for all biocatalyst preparations. Thus, enzyme activation (reported elsewhere to be as high as 3750-fold in hexane for the transesterification of N-Ac-L-Phe-OEt with n-PrOH) is a manifestation of intrinsic enzyme activation and not relaxation of diffusional limitations resulting from diluted enzyme preparations. Similar activation is reported herein for thermolysin, a nonserine protease, thereby demonstrating that enzyme activation due to lyophilization in the presence of KCl may be a general phenomenon for proteolytic enzymes.     

Dissociation of cytochrome c from the inner mitochondrial membrane during cardiac ischemia

Mitochondria isolated from ischemic cardiac tissue exhibit diminished rates of respiration and ATP synthesis. The present study was undertaken to determine whether cytochrome c release was responsible for ischemia-induced loss in mitochondrial function. Rat hearts were perfused in Langendorff fashion for 60 min (control) or for 30 min followed by 30 min of no flow ischemia. Mitochondria isolated from ischemic hearts in a buffer containing KCl exhibited depressed rates of maximum respiration and a lower cytochrome c content relative to control mitochondria. The addition of cytochrome c restored maximum rates of respiration, indicating that the release of cytochrome c is responsible for observed declines in function. However, mitochondria isolated in a mannitol/sucrose buffer exhibited no ischemia-induced loss in cytochrome c content, indicating that ischemia does not on its own cause the release of cytochrome c. Nevertheless, state 3 respiratory rates remained depressed, and cytochrome c release was enhanced when mitochondria from ischemic relative to perfused tissue were subsequently placed in a high ionic strength buffer, hypotonic solution, or detergent. Thus, events that occur during ischemia favor detachment of cytochrome c from the inner membrane increasing the pool of cytochrome c available for release. These results provide insight into the sequence of events that leads to release of cytochrome c and loss of mitochondrial respiratory activity during cardiac ischemia/reperfusion.     

Expression of Classical Mitochondrial Respiratory Responses in Homogenates of Rat Forebrain

Respiratory studies of brain mitochondria have, in general, been limited to purified preparations. Conventional procedures for mitochondrial isolation yield relatively small and potentially selected subfractions of mitochondria. Examination of respiratory responses of homogenates of rat forebrain indicated that key respiratory properties of mitochondria are fully expressed in these preparations. In a high K+ buffer, comparable to those commonly used for purified mitochondria, forebrain homogenates exhibited many of the characteristics of oxygen uptake by "free" mitochondria: requirement for both pyruvate and malate for maximal respiration, stimulation (over threefold) by ADP, stimulation by uncoupling agent [carbonyl cyanide m-chlorophenylhydrazone (CCCP)], but little effect of digitonin. In a modified Krebs-Ringer phosphate buffer (a physiological buffer), respiratory responses were primarily due to mitochondria enclosed in synaptosomes: respiration with glucose was markedly stimulated by CCCP, further stimulated by pyruvate, and extensively inhibited by digitonin (which disrupts the cholesterol-rich synaptosomal membranes). Studies with purified mitochondria and synaptosomes supported the specificity of these responses. These data indicate that classical mitochondrial responses are expressed in whole brain homogenates and, under appropriate conditions, provide functional measures of the total pools of free and synaptosomal mitochondria.     

Capillary electrophoresis monitors changes in the electrophoretic behavior of mitochondrial preparations

The presence of electrical charges on the surface of an organelle is the source of the organelle's electrophoretic mobility. Recently, we reported that capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) can be used to determine the electrophoretic mobility of individual mitochondria. Here, we describe the use of CE-LIF to monitor changes in the electrophoretic mobility distributions of: (i). mitochondria isolated from cultured NS-1 mouse hybridoma cells disrupted by nitrogen cavitation or mechanical homogenization; (ii). mitochondria isolated from rat liver and purified by gradient centrifugation before and after being frozen in liquid nitrogen; and (iii). mitochondria chemically transformed into mitoplasts. These results indicate that the organelle electrophoretic mobility observed by researchers is affected by preparation procedures and that CE-LIF is a complementary technique for monitoring the quality of mitochondrial preparations.     

Morphology and ATP-ase of isolated mitochondria

Changes in the morphology of rat liver mitochondria brought about by different methods of isolation and the concomitant changes in ATP-ase activity were studied. The morphology was investigated with the electron microscope. It was found that the ATP-ase activity of the isolated mitochondria cannot be readily correlated with the morphology of the mitochondria. The ATP-ase found in these preparations was latent, resembling the enzyme described in mitochondria prepared in 0.25 M sucrose. In confirmation of earlier results the use of 0.88 M sucrose yielded preparations with a higher initial ATP-ase than did other methods. Preparation in 0.25 M sucrose resulted in round, swollen mitochondria of which 30 to 40 per cent appeared to have lost a substantial part of the mitochondrial matrix. Preparations in 0.44 to 0.88 M sucrose contained mainly rod-shaped mitochondria plus a small amount of another type of swollen mitochondria. The matrix of mitochondria isolated in 0.88 M sucrose was highly condensed. By the use of 0.44 M sucrose adjusted to pH 6.2 with citric acid, it was possible to isolate, for the first time, mitochondria closely resembling those in situ and containing latent ATP-ase.     

Rapid step-gradient purification of mitochondrial DNA

A convenient modification of the step gradient (CsCl/ethidium bomide) procedure is described. This rapid method allows isolation of covalently closed circular DNA separated from contaminating proteins, RNA and chromosomal DNA in ca. 5 h. Large scale preparations can be performed for circular DNA from eukaryotic organelles (mitochondria). The protocol uses organelle pelleting/NaCl-sarcosyl incubation steps for mitochondria followed by a CsCl step gradient and exhibits yields equal to the conventional procedures. It results in DNA sufficiently pure to be used for restriction endonuclease analysis, subcloning, 5-end labeling, gel retention assays, and various types of hybridization.     

Steroidogenesis in the zona glomerulosa of the adrenal cortex I. Isolation and some properties of mitochondria from the zona glomerulosa of the bovine adrenal cortex

Isolation procedures for mitochondria from the zona glomerulosa of the bovine adrenal cortex are described and the properties of the mitochondria thus prepared are compared with those isolated from the zona fasciculoreticularis. The cristal membranes of mitochondria in the zona glomerulosa in situ are tubular or tubulovesicular, whereas those of mitochondria in the zona fasciculoreticularis in situ are vesicular. When mitochondria are isolated from the former zone, they invariably showed the condensed configuration regardless of isolation media, whereas those isolated from the latter zone in an ST medium showed the orthodox configuration. When Ca²⁺ was added to mitochondria isolated either from the zona glomerulosa or the zona fasciculoreticularis in an STE medium in the condensed configuration, a transition from the condensed to the orthodox configuration took place; the cristal membranes of mitochondria from the zona glomerulosa became tubular or tubulovesicular and those of mitochondria from the zona fasciculoreticularis became vesicular. Contaminations of mitrochondria of the zona glomerulosa with other cellular organelles were examined using various marker enzymes. There was no difference in cytochrome content between mitochondria of the two zones specified above. The coupling efficiency of mitochondria of the zona glomerulosa was found to be remarkably effected by temperature during the isolation procedures. Effects of various substrates, isolation media, and bovine serum albumin on the coupling efficiency of mitochondria of the glomerulosa are also described.     

Effects of Postdecapitative Ischemia on Mitochondrial Respiration in Brain Tissue Homogenates

Mitochondria isolated from ischemic brain characteristically show changes in respiratory function. As conventional procedures for mitochondrial isolation yield a subpopulation of the total population and require extensive manipulation, it is unclear to what extent these changes are representative of mitochondria in the unfractionated tissue. We previously showed that the oxygen uptake by unfractionated forebrain homogenates, measured under two different sets of incubation conditions, provided information on some aspects of the respiratory activity of both the free and synaptosomal pools of mitochondria. Forebrain homogenates from animals subjected to 30 min of postdecapitative ischemia exhibited large reductions in oxygen uptake rates measured in a high K+ (mitochondrial) buffer in the presence of either ADP (44% of control values) or an uncoupling agent (45% of control values). These reductions in respiratory activity were comparable to alterations observed under the same conditions for mitochondria isolated from the ischemic brains. Similar alterations were seen in homogenates from three subregions: neocortex, hippocampus, and striatum. In a physiological buffer, in which oxygen uptake by homogenates largely resulted from activity of mitochondria within synaptosomes, there was little or no change in basal glucose-supported rates (79-96% of control values) and small reductions in maximal rates (63-81% of control values) measured in the presence of an uncoupling agent. These results suggest that alterations of respiratory function seen in isolated free mitochondria provide appropriate estimates of the dysfunction in the total free mitochondrial pool but that synaptosomal mitochondria may be less affected. Measurements of respiratory function of isolated synaptosomes from ischemic tissue provided further support for the relative preservation of synaptosomal mitochondria during ischemic insult.     

A critical comparison between two classical and a kit‐based method for mitochondria isolation

Numerous protocols for isolation of mitochondria are available. Here, three methods for the isolation of intact mitochondria from mouse liver tissues are compared with regard to yield, purity and activity. Mitochondria were isolated by sucrose density gradient ultracentrifugation, free‐flow electrophoresis or a commercially available kit‐based method. Our analyses show that the sophisticated (and most expensive) free‐flow electrophoresis method enables isolation of intact mitochondria with an enrichment of approximately 70%. Using the classical density centrifugation method is very laborious and time‐consuming, but delivers about 57% intact mitochondria. Using standard laboratory equipment in a quick and simple procedure, the kit provides approximately 50% intact mitochondria, suitable for most standard investigations.     

Fine-Structure Analysis of Intercellular and Intracellular Mitochondrial Diversity in Saccharomyces cerevisiae

Crosses were made between haploid wild-type and suppressive petite strains of bakers' yeast to obtain zygotes for analysis of mitochondrial heterogeneity. Wild-type x petite zygotes contained about 40% noncristate mitochondria when immediate mating mixtures were examined. The frequency of defective mitochondria had decreased to an average of 9.2% in 1-week-old zygote isolate cultures, and to 4.4% in slant cultures 1.5 years after initial zygote isolation. The latter value was not significantly different from values obtained with wild x wild zygotes of either age. The noncristate mitochondria were of two types: one lacking inner membrane invaginations or elaborations and the other containing concentrically arranged loops of inner membrane. The significance of these two types of respiration-deficient mitochondria is unknown. The gradual decrease in frequency of noncristate mitochondria, perhaps due to selection pressures in mixed chondriomes, was discussed as a further indication of the semiautonomous nature of the yeast organelle.     

Yolk platelets in artemia embryos: are they really storage sites of immature mitochondria?

We have used semi-quantitative polymerase chain reaction (PCR) technology to determine the mitochondrial DNA (mtDNA) content of yolk platelets isolated from embryos of the brine shrimp, Artemia franciscana, and ultrastructural analysis of yolk platelet formation to determine whether these organelles contain mitochondria as reported previously. Using six different isolation and purification protocols, we found one yolk platelet preparation to be devoid of mtDNA, while four yolk platelet preparations contained mtDNA ranging from 16.4 to 85 pg/10(6) yolk platelets. One preparation contained 600 pg mtDNA per 10(6) yolk platelets. Based on our PCR analyses, the mtDNA component of Artemia yolk platelets represented 0.16-4.5% of the total DNA isolated from the platelets. We calculated that Artemia yolk platelets contain, on average, approximately 1.78 molecules of mtDNA/platelet. Direct analysis of mtDNA in "free" mitochondria isolated from yolk platelet-free preparations of Artemia embryos and newly hatched larvae yielded 0.76-0.80 ng/animal. Based on these values, the mtDNA content of yolk platelets was approximately 0.2% of total mtDNA in Artemia embryos. Microscopic analysis of yolk platelet formation during oogenesis in Artemia failed to show the inclusion of mitochondria during the assemblage of yolk platelets. The "mitochondria-like" structures that appear in yolk platelets during their utilization lack the well defined inner and outer membranes characteristic of mitochondria making it unlikely that the yolk platelet inclusions are mitochondria. Our results from PCR technology and ultrastructure analysis demonstrate that mtDNA in yolk platelets of Artemia franciscana embryos is a minor component of the total mtDNA in the embryo, and they fail to support the notion that yolk platelets in Artemia are a major source of immature mitochondria for development.     

Cytochrome b559 content in isolated photosystem II reaction center preparations.

The cytochrome b559 content was examined in five types of isolated photosystem II D1-D2-cytochrome b559 reaction center preparations containing either five or six chlorophylls per reaction center. The reaction center complexes were obtained following isolation procedures that differed in chromatographic column material, washing buffer composition and detergent concentration. Two different types of cytochrome b559 assays were performed. The absolute heme content in each preparation was obtained using the oxidized-minus-reduced difference extinction coefficient of cytochrome b559 at 559 nm. The relative amount of D1 and cytochrome b559alpha-subunit polypeptide was also calculated for each preparation from immunoblots obtained using antibodies raised against the two polypeptides. The results indicate that the cytochrome b559 heme content in photosystem II reaction center complexes can vary with the isolation procedure, but the variation of the cytochrome b559alpha-subunit/D1 polypeptide ratio was even greater. This variation was not found in the PSII-enriched membrane fragments used as the RC-isolation starting material, as different batches of membranes obtained from spinach harvested at different seasons of the year or those from sugar beets grown in a chamber under controlled environmental conditions lack variation in their alpha-subunit/D1 polypeptide ratio. A precise determination of the ratio using an RC1-control sample calibration curve gave a ratio of 1.25 cytochrome b559alpha-subunit per 1.0 D1 polypeptide in photosystem II membranes. We conclude that the variations found in the reaction center preparations were due to the different procedures used to isolate and purify the different reaction center complexes.     

An efficient procedure for isolation of nuclei from plant protoplasts

Summary The present communication describes an easy, efficient and rapid method for isolation of nuclei from plant protoplasts. Release of nuclei is accomplished by disruption of protoplasts in an appropriate buffer containing a very low concentration (0.01%) of the detergent Triton X-100. The pH of the nuclei isolation buffer (5.3) played a critical role in the recovery of stable nuclei in large numbers. Supplementation of buffer (10 mM MES) with spermine (0.1 mM), dithiothreitol (2.5 mM), ethylenediaminetetraacetic acid (2.5 mM) and Nad and KCl (10 mM each) improved nuclear yield and quality. With the method developed it is possible to routinely recover 95% nuclei from the protoplasts within 30 minutes. The nuclear preparations are of high purity with little detectable cytoplasmic contamination and no clumping of the nuclei. The structural integrity of the nuclei has been assessed and confirmed by Nomarski differential interference contrast optics and ultrastructural observations.     

Isolation and characterization of peroxisomes from the renal cortex of beef, sheep, and cat

The isolation and characterization of highly purified and structurally well-preserved peroxisomes from the renal cortex of different mammalian species (beef, sheep, and cat) is reported. Renal cortex tissue was homogenized and a peroxisome-enriched light mitochondrial fraction was prepared by differential centrifugation. This was subfractionated by density-dependent banding on a linear gradient of metrizamide (1.12-1.26 g/cm3) using a Beckman VTi 50 vertical rotor. Peroxisomes banded at a mean density of 1.225 cm3. Ultrastructural morphometric examination revealed that peroxisomes made up 97 to 98% of the isolated fractions. By biochemical analysis the contamination with marker enzymes of mitochondria and lysosomes was extremely low. The specific activity of catalase was enriched, depending on the species, between 28- and 38-fold over the homogenate. Peroxisome preparations from all three species exhibited a high but varying level of activity for cyanide-insensitive lipid beta-oxidation. In beef and sheep preparations a small amount of esterase activity cosediments with peroxisomes. These peroxisomes show distinct structural membrane associations with smooth elements of ER. Urate oxidase, a marker enzyme for rat liver peroxisomes, is found only in peroxisomes prepared from beef kidney cortex, with sheep and cat preparations being negative. This correlated with the occurrence of polytubular inclusions in the beef kidney peroxisomes. The large size and the angular shape of isolated peroxisomes as well as the presence of paracrystalline matrical inclusions imply that the majority of peroxisomes are derived from the epithelial cells of the proximal tubule of the kidney cortex. The significant differences found in the characteristics of the renal peroxisomes in three different species investigated, demonstrate the remarkable adaptability and plasticity of this organelle.     

Mitochondria and apicoplast of Plasmodium falciparum: behaviour on subcellular fractionation and the implication

The mitochondrion and the apicoplast of the malaria parasite, Plasmodium spp. is microscopically observed in a close proximity to each other. In this study, we tested the suitability of two different separation techniques--Percoll density gradient centrifugation and fluorescence-activated organelle sorting--for improving the purity of mitochondria isolated from the crude organelle preparation of Plasmodium falciparum. To our surprise, the apicoplast was inseparable from the plasmodial mitochondrion by each method. This implies these two plasmodial organelles are bound each other. This is the first experimental evidence of a physical binding between the two organelles in Plasmodium.