Degradation of mitochondria by cytosolic factors in reticulocytes.

The degradation process of mitochondria in rabbit reticulocytes proceeds predominately directly in the cytosol rather than in secondary lysosomes as judged by electronmicroscopy. At least five cytosolic protein factors are present in reticulocytes, which could be related to the degradation of mitochondria: the two inhibitory proteins of the respiratory chain RF and RC and three enzymes which cause a lysis of mitochondria in vitro (lipoxygenase, proteinase, phospholipase A). The properties of these factors are the subject of this paper. A hypothetic scheme of the degradation of mitochondria in reticulocytes is proposed. The degradation of mitochondria in reticulocytes is viewed as a complex interplay of various cytosolic factors and the functional state of the mitochondrial membranes. The lipoxygenase damages the membranes and triggers the penetration of the respiratory inhibitors. In this manner, a catastrophic cycle is initiated which leads to the complete breakdown of the mitochondria.     

Clocking out: modeling phage-induced lysis of Escherichia coli

Phage lambda lyses the host Escherichia coli at a precisely scheduled time after induction. Lysis timing is determined by the action of phage holins, which are small proteins that induce hole formation in the bacterium's cytoplasmic membrane. We present a two-stage nucleation model of lysis timing, with the nucleation of condensed holin rafts on the inner membrane followed by the nucleation of a hole within those rafts. The nucleation of holin rafts accounts for most of the delay of lysis after induction. Our simulations of this model recover the accurate lysis timing seen experimentally and show that the timing accuracy is optimal. An enhanced holin-holin interaction is needed in our model to recover experimental lysis delays after the application of membrane poison, and such early triggering of lysis is possible only after the inner membrane is supersaturated with holin. Antiholin reduces the delay between membrane depolarization and lysis and leads to an earlier time after which triggered lysis is possible.     

Regulation of nonphosphorylating electron transport pathways in soybean cotyledon mitochondria and its implications for fat metabolism

The respiration of mitochondria isolated from germinating soybean cotyledons was strongly resistant to antimycin and KCN. This oxygen uptake was not related to lipoxygenase which was not detectable in purified mitochondria. The antimycin-resistant rate of O₂ uptake was greatest with succinate as substrate and least with exogenous NADH. Succinate was the only single substrate whose oxidation was inhibited by salicyl hydroxamic acid alone, indicating engagement of the alternative oxidase. Concurrent oxidation of two or three substrates led to greater involvement of the alternative oxidase. Despite substantial rotenone-resistant O₂ uptake with NAD-linked substrates, respiratory control was observed in the presence of antimycin, indicating restriction of electron flow through complex I. Addition of succinate to mitochondria oxidizing NAD-linked substrates in state four stimulated O₂ uptake substantially, largely by engaging the alternative oxidase. We suggest that these properties of soybean cotyledon mitochondria would enable succinate received from the glyoxysome during lipid metabolism to be rapidly oxidized, even under a high cytosolic energy charge.     

Variations of matrix electrondensity in brain mitochondria.

The configuration of brain mitochondria was compared in situ, after aldehyde perfusion and/or osmium immersion fixation and in isolated fractions of different functional performance. After combined aldehyde perfusion osmium immersion fixation in situ, mitochondria were condensed having a dark matrix. Fractions capable of controlled respiration also consisted of condensed mitochondria. On the contrary, expanded mitochondria with light matrix were brought about by immersion fixation. Fractions consisting predominantly of light mitochondria displayed no controlled respiration. Light matrix and expanded form are therefore regarded as a functionally impaired state of brain mitochondria. The condensed form is thought to be a landmark of good fixation.     

Natural killer cell-mediated lysis involves an hydroxyl radical-dependent step

The role of oxygen radicals in lysis of K562 target cells by human natural killer (NK) cells was determined by addition of scavengers of these free radicals. Lysis was greatly reduced under hypoxic conditions. Superoxide dismutase and cytochrome c, scavengers of superoxide anions, and catalase and scavengers of hypochlorite had no effect on lysis. Of 15 hydroxyl radical scavengers tested, 13 inhibited lysis. These were not toxic, because cell morphology and spontaneous chromium release were not affected and preculture with scavengers was not inhibitory. These scavengers differed widely in structure, but degree of inhibition of lysis correlated with their rate constants (k) for reaction with hydroxyl radical (k vs log inhibitor concentration required to decrease lysis by 50%: r = -0.9202, p less than 0.001), showing that inhibition was due to inactivation of the hydroxyl radical. Target cell binding was not reduced at concentrations that inhibited lysis. Inhibitors of the lipoxygenase pathway also decreased lysis, suggesting this pathway to be the source of hydroxyl radicals. In view of the reported requirements for hydroxyl radical-mediated lipid peroxidation for optimal secretory activity in a number of cell types, it appears that the generation of hydroxyl radicals by NK cells is required for delivery of cytotoxic factors.     

Erythrocyte bisulfite transport

The wide range of transport rates for anions of differing chemical structure by the human erythrocyte anion transport protein (Band 3 protein) suggests that this protein is highly selective for anions that chemically resemble its natural substrate bicarbonate. To test this hypothesis, the influx of bisulfite (HSO3-), a bicarbonate analog, was compared to influxes of chloride, sulfate, and bicarbonate, as measured by the technique of colloid osmotic lysis in isotonic ammonium salt solution. The lysis time induced in chloride solution (much greater than 10 min) was markedly accelerated to 0.6 min by the addition of small amounts (5 mM) of bicarbonate, an effect characteristic of colloid osmotic lysis induced by the anion transport pathway. Lysis in bicarbonate solution was extremely rapid (0.09 min), and was markedly inhibited by acetazolamide (2.9 min). Lysis in bisulfite solution occurred spontaneously (2.2 min) but was markedly accelerated to a time similar to that of chloride (0.56 min) by addition of 5 mM bicarbonate. In contrast, sulfate induced lysis was extremely slow (less than 10% lysis at 40 min in the presence of bicarbonate). Preincubation of erythrocytes with SITS, an inhibitor of anion exchange, prevented lysis by chloride, but had no effect on lysis by bicarbonate, indicating that lysis by bicarbonate was predominantly through diffusion and not anion transport. SITS treatment of erythrocytes eliminated the catalytic effect of bicarbonate during lysis by bisulfite, indicating that anion transport of bisulfite and diffusion of the conjugate acid in the form of SO2 both contribute to the total membrane flux. When the contribution of diffusion is taken into account, the rate of bisulfite influx through the anion exchange pathway is at least 100-fold faster than that for sulfate.     

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.     

Lysis of Vibrio succinogenes by ethylenediamine-tetraacetic acid or lysozyme

Wolin, M. J. (University of Illinois, Urbana). Lysis of Vibrio succinogenes by ethylenediaminetetraacetic acid or lysozyme. J. Bacteriol. 91:1781-1786. 1966.-Cell suspensions of Vibrio succinogenes are lysed by ethylenediaminetetraacetic acid (EDTA) or lysozyme. Lysis occurs at alkaline pH and is prevented by 0.15 m NaCl or KCl or 0.3 m sucrose. The addition of 10(-3)m Mg(++), 10(-3)m spermine, or 10(-2)m Ca(++) prevents lysozyme lysis, and 10(-4)m spermine prevents EDTA lysis. EDTA lysis leads to the formation of a cell ghost, and lysozyme lysis leads to the formation of an empty round body. Freezing and thawing of cells permits lysozyme attack which is not prevented by the protective agents mentioned above. Much of the cell protein, and almost all of the nucleic acids, are released from the cells during EDTA lysis. Treatment of frozen-thawed cells with lysozyme at neutral pH does not cause release of more than 50% of the cell protein and 60% of the nucleic acids of the cells.     

Fe2+-Induced Lysis and Lipid Peroxidation of Chromaffin Granules

Chromaffin granules, the catecholaminergic storage granules from adrenal chromaffin cells, lysed in 10(-9)-10(-7) M Fe2+. Lysis was accompanied by the production of malondialdehyde which results from lipid peroxidation. Both chromaffin granule lysis and malondialdehyde production were inhibited by the free radical trapping agent butylated hydroxytoluene but not by catalase and/or superoxide dismutase. The results suggest that lysis resulted from a direct transfer of electrons from Fe2+ to a component of the chromaffin granule membrane without the participation of either superoxide or hydrogen peroxide and may have resulted from lipid peroxidation. In some experiments, ascorbate alone induced chromaffin granule lysis which was inhibited by EDTA, EGTA, or deferoxamine. The lysis was probably caused by trace amounts of reducible polyvalent cation. Lysis sometimes occurred when Ca2+ was added with EGTA (10 microM free Ca2+ concentration) and was consistently observed together with malondialdehyde production in the presence of Ca2+, EGTA, and 10 microM Fe2+ (total concentration). The apparent Ca2+ dependency for chromaffin granule lysis and malondialdehyde production was probably caused by a trace reducible polyvalent ion displaced by Ca2+ from EGTA and not by a Ca2+-dependent reaction involving the chromaffin granule.     

Interaction between Mitochondrial Cytochromes and Linoleic Acid Hydroperoxide: POSSIBLE CONFUSION WITH LIPOXYGENASE AND ALTERNATIVE PATHWAY

O₂ uptake by tissue extracts in the presence of linoleic acid is generally ascribed to lipoxygenase. Such an O₂ uptake can be observed not only with mitochondria of Solanum tuberosum L. and Arum maculatum L. and pure lipoxygenase but also with cytochrome c. However, the rate of oxidation is highly dependent on the procedure used to prepare the solutions of linoleic acid. Unless special care is taken to prevent contact between linoleic acid and O₂, it appears that linoleic acid hydroperoxide is readily formed. This derivative can be readily oxidized by mitochondria or cytochrome c. On the other hand, the use of a rapid and specific enzymic procedure to estimate the disappearance of linoleic acid demonstrates that linoleic acid itself is not consumed at any appreciable rate by mitochondria or cytochrome c, the true substrate being linoleic acid hydroperoxide. During the reaction, the heme nucleus of added cytochrome c or of mitochondrial cytochromes undergoes deep alterations. Therefore, caution should be exerted when equating an O₂ uptake observed in the presence of linoleic acid to a lipoxygenase activity. The same holds true for the similarity of reaction towards specific inhibitors between lipoxygenase and the cyanide-insensitive pathway oxidase.     

Functional characteristics of light and dark cells]

Comparison of peculiarties attending the structure of mitochondria in the dark and light cells with the results of studying the respiration of condensed, intermediate and orthodoxic mitochondria showed that dark cells containing condensed mitochondria were at the state of rest or specific activity associated with a high level of bioenergetic and biosynthetic processes. Light cells with a preponderant content of intermediate mitochondria are characterized by the state of active specific activity accompanied by a high energy potential, intensified energy expenditure and a reduction of the level of biosynthetic processes. Light cells with a prevalence of orthodoxic mitochondria are characterized by a low level of energy provision and a predominance of the processes of decomposition of structures over their resynthesis.     

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.     

Leukemia cell lysis by sulphydryl reagents and thiols.

Iodoacetate and other sulfhydryl reagents as well as cysteine and other thiols caused lysis of the mouse leukemic lymphoblasts L5178Y, L1210, and P388 in culture. Lysis by either iodoacetate or cysteine was preceded by ATP depletion. However ATP depletion was not a sufficient explanation for lysis since deoxyglucose depleted ATP to the same extent as did iodoacetate but did not cause lysis. It was concluded that sulfhydryl-disulfide equilibrium was essential to the maintenance of cellular integrity and ATP concentration.     

An analysis of the contribution of the preparatory technique to the appearance of condensed and orthodox conformations of liver mitochondria

The structure and volume of isolated mitochondria embedded for electron microscopy during different respiratory states were analyzed in thin sections. Three different embedding methods were compared; osmium tetroxide fixation/acetone dehydration, glutaraldehyde fixation/acetone dehydration, and glutaraldehyde fixation-osmium tetroxide postfixation/acetone dehydration. Analysis of fresh mitochondria, isolated in a sucrose medium, revealed the presence of a homogeneous population with respect to structure when any of the three methods were applied. After fixation with osmium alone, or in combination with glutaraldehyde, nearly 100% of the mitochondria were in a "condensed" conformation. Mitochondria fixed with glutaraldehyde alone resulted in a population of mitochondria that had large spaces separating the two membranes of the cristae which corresponds to the condensed conformation as observed after osmium fixation. Transfer of the mitochondria to the incubation medium led to the appearance of two classes of mitochondria with respect to size. One class had a volume close to that observed when suspended in sucrose, and another class was present that was 30-45% larger. In osmium fixed or in double-fixed preparations, these small and large classes corresponded to "condensed" and "orthodox" forms of mitochondria respectively. When glutaraldehyde was used alone as the fixative, the two size classes were also present. However, the mitochondria were homogeneous with respect to structure. In these mitochondria, the width of the space that separated the cristae membranes had become reduced when compared to mitochondria suspended in sucrose. The two size classes were also present in samples of mitochondria prepared during both states 3 and 4. State 4 conditions did not lead to any significant increase of the number of condensed mitochondria. In state 3 preparations, 65-70% of the population were condensed. The condensed and orthodox forms could be related to normal and swollen forms of mitochondria. Conditions that led to a swelling also led to an increase in the number of orthodox mitochondria in osmium-fixed material. The different appearance of the mitochondria is explained by the different conditions for fixation of the mitochondria that exist when nonswollen and swollen mitochondria are fixed. This difference is particularly crucial in the case of osmium tetroxide due to the unique way this fixative, among generally used fixatives, denatures proteins.     

Structural changes during lysis of a psychorophilic marine bacterium

The marine psychrophile, a red, gram-negative motile rod with a single polar flagellum, is stable when suspended in 0.1 m Mg(2+) plus 0.5 m NaCl at 0 C and neutral pH but lyses if the salt composition of the medium is changed, the temperature raised above 20 C, or the pH lowered. Lysis is accompanied by a fall in turbidity, a release of ultraviolet-absorbing substances, and a loss of deoxyribonucleic acid and ribonucleic acid. Ultrastructural changes accompanying lysis were studied. Thin sections of cells fixed while intact showed a triple-layered cell wall and cytoplasmic membrane, each 6.0 to 7.5 nm thick. Mesosomes were also observed. Either Na(+) or Mg(2+) could maintain wall integrity, whereas Mg(2+) was needed for membrane integrity. In distilled water, lysis was very extensive, and much material was released as wall fragments and as vesicles which probably came from the wall and cytoplasmic membrane. Lysis at 37 C resulted in degradation of the wall and liberation of wall fragments. The cell membrane was rarely observed as a triple-layered structure in such temperature-lysed cells. After lysis at pH 5.0, the cell wall was distorted, and only a suggestion of the cell membrane remained. Replicas showed that this organism had a matted surface which was distorted under different conditions of lysis.     

A novel technique for gentle lysis of eucaryotic cells Isolation of plasma membranes from Dictyostelium discoideum

A new type of lysis technique for eucaryotic cells was used for the isolation of highly purified plasma membranes from Dictyostelium discoideum. Suspensions of amoebae (10 μm diameter) were lysed by forced passage through Nuclepore filters with pores of 5 μm diameter. Virtually complete lysis was effected with minimal fragmentation of lysosomes and mitochondria. By subsequent differential and isopycnic centrifugation, 25–35-fold purified plasma membranes were isolated in 35–50% yield for cells from vegetative through tip formation stages of development. Lysis, yield and purity were enhanced by use of slightly alkaline conditions. Contamination with other organelles and with soluble proteins was found to be minimal. At each developmental stage, the plasma membranes generated three closely-spaced, equally pure bands in a sucrose density gradient. Two-dimensional electrophoretic analysis of the individual bands showed that they were very similar to each other, indicating that the density differences are not due to gross differences in protein composition.     

Prm1 prevents contact-dependent lysis of yeast mating pairs

Membrane fusion requires localized destabilization of two phospholipid bilayers, but unrestrained membrane destabilization could result in lysis. prm1 mutant yeast cells have a defect at the plasma membrane fusion stage of mating that typically results in the accumulation of prezygotes that have fingers of membrane-bound cytoplasm projecting from one cell of each pair into its mating partner in the direction of the osmotic gradient between the cells. However, some prm1 mating pairs fuse successfully whereas the two cells in other prm1 mating pairs simultaneously lyse. Lysis only occurs if both mating partners are prm1 mutants. Osmotic stabilization does not protect prm1 mating pairs from lysis, indicating that lysis is not caused by a cell wall defect. prm1 mating pairs without functional mitochondria still lyse, ruling out programmed cell death. No excess lysis was found after pheromone treatment of haploid prm1 cells, and lysis did not occur in mating pairs when prm1 was combined with the fus1 and fus2 mutations to block cell wall remodeling. Furthermore, short (<1 microm) cytoplasmic microfingers indicating the completion of cell wall remodeling appeared immediately before lysis. In combination, these results demonstrate that plasma membrane contact is a prerequisite for lysis. Cytoplasmic microfingers are unlikely to cause lysis since most prm1 mating pairs with microfingers do not lyse, and microfingers were also detected before fusion in some wild-type mating pairs. The lysis of prm1 mutant mating pairs suggests that the Prm1 protein stabilizes the membrane fusion event of yeast mating.     

Characterization of the Highly Autolytic Lactococcus lactis subsp. cremoris Strains CO and 2250

Two highly autolytic Lactococcus lactis subsp. cremoris strains (CO and 2250) were selected and analyzed for their autolytic properties. Both strains showed maximum lysis when grown in M17 broth containing a limiting concentration of glucose (0.4 to 0.5%) as the carbohydrate source. Lysis did not vary greatly with pH or temperature but was reduced when strains were grown on lactose or galactose. Growth in M17 containing excess glucose (1%) prevented autolysis, although rapid lysis of L. lactis subsp. cremoris CO did occur in the presence of 1% glucose if sodium fluoride (an inhibitor of glycolysis) was added to the medium. Maximum cell lysis in a buffer system was observed early in the stationary phase, and for CO, two pH optima were observed for log-phase and stationary-phase cells (6.5 and 8.5, respectively). Autolysins were extracted from the cell wall fraction of each strain by using either 4% sodium dodecyl sulfate (SDS), 6 M guanidine hydrochloride, or 4 M lithium chloride, and their activities were analyzed by renaturing SDS-polyacrylamide gel electrophoresis on gels containing Micrococcus luteus or L. lactis subsp. cremoris CO cells as the substrate. More than one lytic band was observed on each substrate, with the major band having an apparent molecular mass of 48 kDa for CO. Each lytic band was present throughout growth and lysis. These results suggest that at least two different autolytic enzymes are present in the autolytic L. lactis subsp. cremoris strains. The presence of the lactococcal cell wall hydrolase gene, acmA (G. Buist, J. Kok, K. J. Leenhouts, M. Dabrowska, G. Venema, and A. J. Haandrikman, J. Bacteriol. 177:1554-1563, 1995), in strains 2250 and CO was confirmed by Southern hybridization. Analysis of an acmA deletion mutant of 2250 confirmed that the gene was involved in cell separation and had a role in cell lysis.     

Consequences of self-presentation of peptide antigen by cytolytic T lymphocytes

We have used H-2Db-restricted CTL clones specific for peptide 365 to 380 of the influenza nucleoprotein to seek evidence for interaction between the TCR and peptide Ag. Preincubation of these CTL with peptide 365 to 380 resulted in inhibition of target cell lysis. In addition, CTL lysed allogeneic targets in the presence of soluble peptide Ag. Investigation of the basis of these two phenomena revealed a requirement for expression of H-2Db molecules by the effector cells. Either preincubation with anti-Db mAb or the use of chimera-derived H-2d CTL specific for Db plus peptide ablated both peptide-dependent inhibition and lysis of allogeneic cells, suggesting these activities are a consequence of self-presentation of peptide Ag by CTL. Lysis of allogeneic cells appears to represent bystander lysis by CTL in response to recognition of peptide on other effector cells. Lysis inhibition is attributable to a highly potent form of cold target inhibition in which CTL serve as their own cold targets.     

EFFECTS OF OSMOLAR CHANGES ON ISOLATED MITOCHONDRIA OF BRAIN AND LIVER

Abstract— The effects of altered osmolarity on respiration and fine structure were compared in isolated rat cerebral versus liver mitochondria.
Polarographic study of cerebral mitochondria in hypo-osmolar media showed inhibition of State 3 (ADP-dependent) respiration which was not reversed by dinitrophenol. In hyperosmolar media, State 3 respiration was transiently inhibited and State 4 (ADP-independent) respiration increased with the NAD-linked substrate pair, glutamate and malate. With succinate as substrate, respiration was not affected by moderate hyperosmolarity. In the most hyperosmolar medium, State 3 respiration was inhibited with both substrates.
In contrast to the results with cerebral mitochondria, State 4 respiration was increased in hypo-osmolar media and State 3 respiration was persistently inhibited in hyperosmolar media in liver mitochondria with both substrates.
In both cerebral and liver mitochondria, cytochrome c oxidase (EC 1.9.3.1.) activity was mildly inhibited in hypo-osmolar media and increased in hyperosmolar media.
Electron microscopy showed that liver mitochondria were swollen in hypo-osmolar media and condensed in hyperosmolar media. Cerebral mitochondria showed mild rarefaction in hypo-osmolar media and, in hyperosmolar media, more than half the mitochondria showed either no or minimal changes in fine structure.
Our results suggest that there are differences in metabolic control and structure between mitochondria from different cell types, which may be important in the cellular metabolic response to pathologic changes in water or osmolarity.