ULTRASTRUCTURAL TRANSFORMATION IN MITOCHONDRIA ISOLATED FROM KIDNEYS OF NORMAL AND LEAD-INTOXICATED RATS

Mitochondria isolated from kidneys of lead-intoxicated rats have been shown to have decreased oxidative and phosphorylative abilities. The purpose of this study was to determine whether these abnormal mitochondria would undergo ultrastructural transformation during controlled respiration in the absence of phosphate acceptor (State IV), as previously demonstrated for normal liver mitochondria. It was first shown that normal rat kidney mitochondria transforms from a condensed ultrastructural conformation to an orthodox conformation after 5 min of State IV respiration with pyruvate-malate substrate. Reversal to a condensed conformation follows stimulation of respiration with adenosine diphosphate (ADP). A large portion of kidney mitochondria from lead-poisoned rats do not change from condensed to orthodox conformation during State IV respiration. Other mitochondria do transform to the orthodox form but they rapidly degenerate. State IV respiration decreases as these few orthodox mitochondria disintegrate. The conclusion is that those mitochondria that do not undergo change in ultrastructure have impairment of electron transport, and that those that do become orthodox have increased membrane lability and undergo degeneration.     

ENERGY-LINKED ULTRASTRUCTURAL TRANSFORMATIONS IN ISOLATED LIVER MITOCHONDRIA AND MITOPLASTS : Preservation of Configurations by Freeze-Cleaving Compared to Chemical Fixation

An investigation was carried out in which microsamples of isolated rat liver mitochondria and freshly prepared mitoplasts in defined energy states were freeze-cleaved. Parallel microsamples were fixed with osmium tetroxide and with glutaraldehyde followed by osmium tetroxide as previously used in this laboratory for the preservation of energy-linked mitochondrial configurations. The details of the orthodox configuration of energized mitochondria and the condensed configuration of de-energized mitochondria, as revealed previously by chemical fixation, are confirmed in this report for nonfixed, freeze-cleaved mitochondria. The precise agreement in preservation of configuration obtained by the physical fixation of rapid freezing and by chemical fixation establishes unequivocally that mitochondria undergo energy-linked ultrastructural transformation between the condensed and the orthodox configurations which are thus natural structural states related to the metabolic activity of the mitochondrion. Configurations observed by freeze-cleaving and by chemical fixation reveal that mitoplasts also undergo a specific and dramatic ultrastructural transformation with the induction of oxidative phosphorylation. The transformation appears to be isovolumetric and therefore is thought to be mediated through energized conformational activity in the surface electron-transport membrane of the mitoplast. Passively swollen, spherical, osmotically active mitoplasts could not be fixed rapidly enough by chemical fixatives as normally used without altering the spherical form. In this special case preservation of configurational form required rapid freezing or chemical fixatives of low osmolar concentration.     

ION-INDUCED ULTRASTRUCTURAL TRANSFORMATIONS IN ISOLATED MITOCHONDRIA : The Energized Uptake of Calcium

The energized uptake of low levels of Ca²⁺ in the presence and absence of phosphate by isolated rat liver mitochondria, and the perturbation effected by this activity on ultrastructural and metabolic parameters of mitochondria have been investigated. In the presence of phosphate, low levels of Ca²⁺ are taken up by mitochondria and result in various degrees of ultrastructural expansion of the inner mitochondrial compartment. This indicates that low levels of Ca²⁺ in the presence of phosphate, are accumulated in an osmotically active form into the water phase of the inner compartment. The first clearly observable quantitative increase in the volume of the inner compartment occurs after the accumulation of 100 nmoles Ca²⁺/mg protein. An accumulation of 150–200 nmoles Ca²⁺/mg protein, which is equivalent to the osmolar concentration of endogenous K⁺, is required to effect a doubling of the volume of the inner compartment. This degree of osmotic perturbation occurs as mitochondria transform from a condensed to an orthodox conformation. The osmotically induced orthodox conformation differs from the mechanochemically induced orthodox conformation previously described, in that its development is concomitant with a marked decrease in acceptor control and oxidative phosphorylation efficiency and it fails to transform to a condensed conformation in response to addition of ADP. In the absence of added phosphate, a maximum of 190 nmoles Ca²⁺/mg protein was found to be taken up by mitochondria (state 6). Ca²⁺ is apparently bound under state 6 conditions since the uptake does not effect an ultrastructural expansion of the inner compartment. Phosphate added after state 6 Ca²⁺ binding, however, results in an immediate ultrastructural expansion of the inner compartment. The addition of phosphate to mitochondria in the absence of exogenous Ca^2- fails to effect an osmotic ultrastructural transformation. Under state 6 conditions, the binding of between 40 and 190 nmoles Ca²⁺/mg protein results in the formation of dense matrix inclusions which appear to be composed of tightly packed, concentrically oriented membranes. Under conditions in which the bound Ca²⁺ is subsequently released, there is a concomitant loss in the density of these matrix inclusions, leaving behind morphologically distinct membrane whorls in the mitochondrial matrix.     

Some effects of Ca2+, Mg2+, and Mn2+ on the ultrastructure,light-scattering properties,and malic enzyme activity of adrenal cortex mitochondria

The ultrastructure and 90 ° light-scattering capacity of adrenal cortex mitochondria have been examined under conditions which lead to an activation of malic enzyme activity in these mitochondria. After isolation, the mitochondria display an aggregate ultrastructure which does not resemble the vesicular (orthodox) form normally seen in vivo. Under conditions of malic enzyme activation (presence of malate, NADP⁺, Mg²⁺ and 1 mm Ca²⁺), the ultrastructure reverts to a vesicular form as seen in vivo. Of these required components, only Ca²⁺ affects the ultrastructure. The ultrastructural transformation from the aggregate to the orthodox form is always accompanied by a decrease in the 90 ° light-scattering capacity. When produced by Ca²⁺, transformation requires energy-dependent Ca²⁺ uptake if an oxidizable substrate is present. In the absence of substrate, the transformation occurs as an apparent energy-independent effect. Mn²⁺ can substitute for Ca²⁺ only in the presence of substrate. In de-energized mitochondria, Mn²⁺ prevents the effects of Ca²⁺. The activation of malic enzyme is always preceded by a decrease in light scattering and transformation to the orthodox ultrastructure; however, the presence of the orthodox form is not a sufficient condition since subsequent chelation of free Ca²⁺ fails to reverse either the decrease in light scattering or ultrastructural transformation but does reverse the enzyme activation. In addition, levels of Mn²⁺ which effectively depress light-scattering capacity and produce the orthodox form, fail to activate malic enzyme significantly. The data are discussed as they relate to Ca²⁺-induced damage in mitochondria.     

A study of rapid mitochondrial structural changes in vitro by spray- freeze-etching

The spray-freeze-etching technique has been used to study energy-linked mitochondrial structural changes in rat liver mitochondria incubated in vitro. The technique involves spraying the suspension of mitochondria into liquid propane at -190 degrees C, and does not require the use of cryoprotectants or chemical fixatives. The results confirmed that freshly isolated mitochondria have a condensed matrix and that this expands at the expense of the outer compartment to give the orthodox configuration when the mitochondria are incubated in a K+ medium in the presence of substrate and phosphate. Addition of adenosine diphosphate (ADP) caused a rapid shrinkage of the matrix compartment, and the time-course and extent of this shrinkage has been measured quantitatively by coupling a rapid sampling device to the spray-freezing apparatus. These data show that for orthodox mitochondria the onset of phosphorylation is accompanied by a reduction of 30% in the matrix volume in 20's, and there is no evidence that the decrease in matrical volume affects the phosphorylation efficiency. These results suggest that natural ionophores in the mitochondrial inner membrane make it permeable enough to permit a rapid readjustment of matrix volume after the addition of ADP, and that the associated ion movement does not cause uncoupling of oxidative phosphorylation.     

ULTRASTRUCTURAL BASES FOR METABOLICALLY LINKED MECHANICAL ACTIVITY IN MITOCHONDRIA : II. Electron Transport-Linked Ultrastructural Transformations in Mitochondria

Isolated mitochondria are capable of undergoing dramatic reversible ultrastructural transformations between a condensed and an orthodox conformation. These two conformations are the extremes in ultrastructural organization between which structually and functionally intact mitochondria transform during reversible respiratory cycles. It has been found that electron transport is required for the condensed-to-orthodox ultrastructural transformation which occurs in mitochondria under State IV conditions, i.e., under conditions in which exogenous substrate is present and ADP is deficient. Inhibition of State IV electron transport at the cyanide-, antimycin A-, or Amytal-sensitive sites in the respiratory chain results in inhibition of this transformation. Resumption of electron transport in initially inhibited mitochondrial systems, initiated by channeling electrons through pathways which bypass the inhibited sites, results in resumption of the ultrastructural transformation. The condensed-to-orthodox transformation is DNP insensitive and, therefore, does not require participation of the coupling enzymes of the energy-transfer pathway. It is concluded that this ultrastructural transformation is manifest by the conversion of the chemical energy of electron transport directly into mechanical work. The reversed ultrastructural transformation, i.e., orthodox-to-condensed, which occurs during ADP-activated State III electron transport, is inhibited by DNP and parallels suppression of acceptor control and oxidative phosphorylation. Mechanochemical ultrastructural transformation as a basis for energy transfer in mitochondria is considered with respect to the results presented.     

Ultrastructural transformations in bean inner mitochondrial membranes

The ultrastructure of inner membrane-matrix mitochondria isolated from bean (Phaseolus vulgaris) shoots was examined in different metabolic states. Gross ultrastructural transformations analogous to the condensed-to-orthodox configurational changes reported in mammalian mitochondria are observed on transistion from nonrespiring to respiring metabolism. With the induction of oxidative phosphorylation, the particles remain in the orthodox configurational state. The reverse orthodox-to-condensed configurational changes observed in mammalian preparations does not occur. Optically monitored absorbancy studies with bean particles show a substrate-supported P_i-induced swelling under the same conditions that induce the condensed-to-orthodox ultrastructural transformation. The swelling is associated with the net uptake of K⁺ and P_i as well as a small P_i-induced respiratory stimulation. When phosphorylation is initiated with these swollen particles, the optically monitored volume remains unchanged. Thus a positive correlation exists between the ultrastructural configuration and the osmotic volume changes, which supports the conclusion that configurational changes reflect internal osmotic adjustments.     

ULTRASTRUCTURAL BASES FOR METABOLICALLY LINKED MECHANICAL ACTIVITY IN MITOCHONDRIA : I. Reversible Ultrastructural Changes with Change in Metabolic Steady State in Isolated Liver Mitochondria

By means of a new "quick-sampling" method, micropellets of mouse liver mitochondria were rapidly prepared for electron microscopy during the recording of steady state metabolism. Reversible ultrastructural changes were found to accompany change in metabolic steady states. The most dramatic reversible ultrastructural change occurs when ADP is added to systems in which only phosphate acceptor is deficient, i.e., during the State IV to State III transition as defined by Chance and Williams. After 15 min in State IV, mitochondria display an "orthodox" ultrastructural appearance as is usually observed after fixation within intact tissue. On transition to State III, a dramatic change in the manner of folding of the inner membrane takes place. In addition, the electron opacity of the matrix increases as the volume of the matrix decreases, but total mitochondrial volume does not appear to change during this transition. This conformation is called "condensed." Isolated mitochondria were found to oscillate between the orthodox and condensed conformations during reversible transitions between State III and State IV. Various significant ultrastructural changes in mitochondria also occur during transitions in other functional states, e.g., when substrate or substrate and acceptor is made limiting. Internal structural flexibility is discussed with respect to structural and functional integrity of isolated mitochondria. Reversible changes in the manner of folding of the inner membrane and in the manner of packing of small granules in the matrix as respiration is activated by ADP represent an ultrastructural basis for metabolically linked mechanical activity in tightly coupled mitochondria.     

Mitochondrial ultrastructural transformations in P. lividus eggs stimulated by ammonia

In mitochondria of P. lividus eggs the transition from the condensed to the orthodox form is accomplished in about 45 min after fertilization. Similar ultrastructural transformations are induced by exposure to ammonia that is known to cause a partial egg activation.     

Mitochondrial regulation in sea urchins. I. Mitochondrial ultrastructure transformations and changes in the ADP:ATP ratio at fertilization.

Mitochondrial ultrastructural transformations have been examined in intact eggs and embryos from three sea urchins, Arbacia punctulata, Strongylocentrotus purpuratus, and Lytechinus pictus. Following fertilization, naturally occurring ultrastructural transformations (designated as condensed to orthodox) were observed to occur in mitochondria of all three families. The ratios of the soluble ADP and ATP pools were examined in eggs of S. purpuratus before and after fertilization in order to test whether the ultrastructural transformations reflect a decrease in relative size of the ADP pool following fertilization. Our data indicate that there is a decrease in the ADP:ATP ratio at fertilization. These findings and their implications are discussed with respect to presently accepted theories on mitochondrial regulation.     

Effect of valinomycin on mitochondrial ultrastructure and function of intact Ehrlich ascites tumor cells

Ultrastructural changes in the mitochondria of intact Ehrlich ascites tumor cells were observed after stimulation by valinomycin of the energy-dependent transport of K⁺ into mitochondria. The mitochondria in cells taken directly from the animal displayed an orthodox configuration. After repeated washings of the cells, the mitochondria were converted to the ‘condensed’ or ‘aggregated’ state. The addition of valinomycin resulted in a transformation of mitochondria from the condensed to orthodox and markedly swollen forms. Alterations in cell size, O₂ uptake, and K⁺ content accompanied the changes in mitochondrial morphology.     

THE MORPHOLOGY OF THE SWELLING PROCESS IN RAT LIVER MITOCHONDRIA

Through the use of combined spectrophotometric and electron microscope techniques, large amplitude swelling of rat liver mitochondria has been described as an ordered sequence of ultrastructural transitions. Prior to the actual swelling, mitochondria undergo two major conformational changes: condensed to twisted form and twisted to orthodox form. This sequence is independent of (a) the nature of swelling agents and (b) the time of onset of swelling. Agents that delay the onset of swelling act to increase the duration of the twisted conformation. Agents that prevent extensive swelling hold mitochondria in intermediate conformations. Gross swelling, immediately preceded by a decrease in electron opacity of the matrix, involves the rupture of the outer membrane and expansion of the inner compartment of the mitochondrion.     

Photosynthetic Activity of Chloroplasts Isolated From Bermudagrass (Cynodon dactylon L.), a Species With a High Photosynthetic Capacity

Chloroplasts have been isolated from bermudagrass (Cynodon dactylon L.) leaves and assayed for photophosphorylation and electron transport activity. These chloroplasts actively synthesize adenosine triphosphate during cyclic electron flow with phenazine methosulfate and noncyclic electron flow concurrent with the reduction of such Hill oxidants as nicotinamide adenosine dinucleotide phosphate, cytochrome c, and ferricyanide. Apparent Km values for the cofactors of photophosphorylation have been determined to be 5 × 10⁻⁵ M for phosphate and 2.5 × 10⁻⁵ M for adenosine diphosphate. The influence of light intensity on photophosphorylation has been studied and the molar ratio of cyclic to noncyclic phosphorylation calculated. It is concluded that the high photosynthetic capacity of bermudagrass leaves probably could be supported by the photophosphorylation capacities indicated in these chloroplast studies and the anomalous lack of data in chlorolast studies on the production of sufficient reductant for CO₂ assimilation at high light intensities has been noted.     

Atractyloside Inhibition of Adenine Nucleotide Translocation in Mitochondria from Hypocotyls of Vigna sinensis cv. Seridó

The translocation of adenine nucleotides into mitochondria isolated from hypocotyls of Vigna sinensis (L.) Savi cv. Serido was examined as a function of oxidative phosphorylation. Mitochondria membrane integrity was assessed by respiratory control and ADP:O ratios. A kinetic analysis of the translocation of adenosine diphosphate into the mitochondria revealed that the mechanism of translocation obeys classical Michaclis-Menten kinetics with a Km of 25 μM for adenosine diphosphate. At moderate ratios of atraetyloside to adenosine diphosphate (lower than 0.03), atractyloside appears to be a competitive inhibitor of the translocation process, with a Ki of 0.4 μM. However, non-linear kinetic parameters are observed with ratios higher than 0.06. A concentration of 2.5 μM atractyloside is sufficient to reduce the translocation of 100 μm ADP by 50%. This represents a higher level of sensitivity to atractyloside than reported for other plants.     

OXIDATIVE PHOSPHORYLATION IN MITOCHONDRIA FROM LIVERS SHOWING CLOUDY SWELLING

Using succinate and α-ketoglutarate as substrates, oxidative phosphorylation has been measured in mitochondria isolated from livers showing cloudy swelling. This cellular change was obtained by injecting rats with S. typhi murium toxin and guinea pigs with diphtheria toxin. It has been found that phosphorylation associated with the oxidation of either of these substrates was partially inhibited in mitochondria from livers showing cloudy swelling, while the oxygen consumption was unchanged. Thus, the P:O ratios for both succinate and α-ketoglutarate were lower in mitochondria from treated animals than they were in normal mitochondria. Dephosphorylation of ATP was not significantly modified in mitochondria from livers showing cloudy swelling as compared with normal controls. No dephosphorylation of AMP and G-6-P was observed either in normal mitochondria or in mitochondria from treated animals.     

CORTISONE-INDUCED ALTERATIONS IN MITOCHONDRIAL FUNCTION AND STRUCTURE

The effects of cortisone treatment on oxygen consumption, oxidative phosphorylation, and fine structure of rat liver mitochondria have been studied. Male rats weighing 125 g were treated for 6 days with 5 mg of cortisone acetate or isotonic saline. On the 7th day, sections of liver were excised and processed for light and electron microscopy. Mitochondrial respiration and oxidative phosphorylation were studied with mitochondria isolated from these livers. Cortisone treatment is responsible for a 14–40% decrease in the amount of oxygen consumed per mg of mitochondrial protein when succinate, α-ketoglutarate, or β-hydroxybutyrate are used as substrates, or with ascorbate and N,N,N¹,N¹-tetramethyl p-phenylenediamine as electron donors. In addition, oxidative phosphorylation is uncoupled with a lowering of the P:O ratios. Randomly selected liver cells have been analyzed by quantitative morphometric techniques. The average mitochondrial volume is increased fourfold in the peripheral and midzonal regions with a commensurate decrease in the number of mitochondria per cell. These alterations are present throughout the hepatic lobule, but are most marked in midzonal cells. The total mitochondrial volume per cell and the per cent of the total cytoplasmic volume occupied by mitochondria remains relatively unaltered, as does the total amount of cristae surface per cell. While the mitochondria are enlarged, they are not "swollen." The relationships between the steroid hormone treatment and the alterations in mitochondrial function and structure are discussed.     

Calcium Uptake and Associated Adenosine Triphosphatase Activity of Isolated Platelet Membranes

A platelet subcellular fraction, sedimenting between 14,000 and 40,000 g and consisting primarily of membrane vesicles, accumulates up to 200–400 nmoles calcium/mg protein in the presence of ATP and oxalate. Steady-state levels of calcium accumulation are attained in 40–60 min. Calcium uptake requires adenosine triphosphate (ATP), is enhanced by oxalate, and is accompanied by the release of inorganic phosphate. Calcium accumulation and phosphate release require magnesium and are inhibited by Salyrgan (10 µM) and adenosine diphosphate (ADP) (1 mM), but not by ouabain (0.1 mM). The ATPase activity is stimulated by low concentrations of calcium (5–10 µM) and is inhibited by 2 mM EGTA. Electron microscopic histochemistry using lead nitrate to precipitate released phosphate results in lead precipitates localized primarily at the inner surface of membrane vesicles. These results provide evidence for a membrane ATPase that is stimulated by low concentrations of calcium and may be involved in the transport of calcium across the membrane. It is postulated that the observed calcium uptake activity is an in vitro manifestation of a calcium extrusion pump in the intact platelet.     

Energy-independent protection of the oxidative phosphorylation capacity of mitochondria against anoxic damage by ATP and its non-metabolizable analogs

Preservation of the oxidative phosphorylation capacity of mitochondria by addition of ATP under anaerobic conditions was analyzed by use of non-metabolizable adenine nucleotide analogs. The capacity was well preserved in the presence of ATP and did not require the hydrolysis of ATP, since ATP analogs, such as beta, gamma-methylene adenosine triphosphate (AMPPCP), alpha, beta-methylene adenosine triphosphate (AMPCPP), and adenylyl imidodiphosphate (AMPPNP), were as effective as ATP. These analogs were incorporated into mitochondria through ATP/ADP translocase to maintain the original level of total adenine nucleotides in the mitochondria. ADP apparently had the same effect as ATP, but its effect was shown to be due to ATP generated from it by adenylate kinase in mitochondria. An analog of ADP, alpha, beta-methylene adenosine diphosphate (AMPCP), which was found to be a substrate of the translocase but not of adenylate kinase, could not replace ADP or ATP. From these results, it was concluded that the oxidative phosphorylation capacity of mitochondria was maintained by ATP, but not ADP, through a process not requiring energy.     

MICROTUBULES IN RELATION TO THE MOTILITY OF A SPERM SYNCYTIUM IN AN ARMORED SCALE INSECT

Male scale insects of the species Parlatoria oleae Colvée (Homoptera: Coccoidea) produce motile sperm bundles. The bundle is a syncytium consisting of 10 to 20 closely packed, filamentous spermatozoa, which share a common cytoplasm and are enclosed in a common membrane. The individual spermatozoon is not surrounded by a plasma membrane, but is delimited by a scroll-like sheath composed of 45 to 50 microtubules. The microtubules run parallel to the long axis of the spermatozoon and are arranged in a spiral pattern as seen in transection. The outside diameter measures approximately 140 to 220 A and the inside diameter, 70 to 100 A. The spermatozoon is about 300 µ long and tapers gradually from a diameter of approximately 0.3 µ anteriorly to 0.1 µ posteriorly. The anterior half (150 µ) has a threadlike core of chromatin about 0.07 µ in diameter. A homogeneous cytoplasm surrounds the nuclear core and fills the posterior half of the spermatozoon. Neither osmium tetroxide nor glutaraldehyde fixation revealed the presence of a nuclear envelope, acrosomal membranes, mitochondria, flagellum, or centrioles. In spite of the apparent lack of orthodox cell organelles, the spermatozoon is actively motile upon release from the bundle. It exhibits capactiy for motility throughout its entire length. Since the sheath of microtubules is the only structure which extends the full length of the spermatozoon, it probably plays a significant role in spermatozoan motility.     

Effects of 2-Butylmalonate, 2-Phenylsuccinate, Benzylmalonate, and p-Iodobenzylmalonate on the Oxidation of Substrates by Mung Bean Mitochondria

The effect of inhibitors of carboxylic acid anion transport on the oxidation of substrates by mung bean (Phaseolus aureus) mitochondria was investigated. The oxidation of malate in the presence of either glutamate or cysteine sulfinate was inhibited by 2-butylmalonate, 2-phenylsuccinate, benzylmalonate, and p-iodobenzylmalonate in both intact and broken mitochondria. The oxidation of succinate, on the other hand, was inhibited in intact but not in broken mitochondria. The oxidation of reduced nicotinamide adenine dinucleotide was inhibited only by p-iodobenzylmalonate. This inhibition occurred only in coupled mitochondria and could be reversed by the addition of adenosine diphosphate.