ENZYMATIC PROPERTIES OF THE INNER AND OUTER MEMBRANES OF RAT LIVER MITOCHONDRIA

Treatment of rat liver mitochondria with digitonin followed by differential centrifugation was used to resolve the intramitochondrial localization of both soluble and particulate enzymes. Rat liver mitochondria were separated into three fractions: inner membrane plus matrix, outer membrane, and a soluble fraction containing enzymes localized between the membranes plus some solublized outer membrane. Monoamine oxidase, kynurenine hydroxylase, and rotenone-insensitive NADH-cytochrome c reductase were found primarily in the outer membrane fraction. Succinate-cytochrome c reductase, succinate dehydrogenase, cytochrome oxidase, β-hydroxybutyrate dehydrogenase, α-ketoglutarate dehydrogenase, lipoamide dehydrogenase, NAD- and NADH-isocitrate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase, and ornithine transcarbamoylase were found in the inner membrane-matrix fraction. Nucleoside diphosphokinase was found in both the outer membrane and soluble fractions; this suggests a dual localization. Adenylate kinase was found entirely in the soluble fraction and was released at a lower digitonin concentration than was the outer membrane; this suggests that this enzyme is localized between the two membranes. The inner membrane-matrix fraction was separated into inner membrane and matrix by treatment with the nonionic detergent Lubrol, and this separation was used as a basis for calculating the relative protein content of the mitochondrial components. The inner membrane-matrix fraction retained a high degree of morphological and biochemical integrity and exhibited a high respiratory rate and respiratory control when assayed in a sucrose-mannitol medium containing EDTA.     

THE SURFACE CHARGE OF RAT LIVER MITOCHONDRIA AND THEIR MEMBRANES : Clarification of Some Controversies Concerning Mitochondrial Structure

The surface charge of intact mitochondria and submitochondrial particles was examined by the technique of preparative free flow electrophoresis. When submitochondrial preparations obtained by a swelling-contraction procedure were examined with this technique, two fractions were observed. One of these fractions exhibited the same electrophoretic properties as intact mitochondria, which indicated that it was derived from the outer limiting membrane of the mitochondrion. This fraction was found to contain the enzymes monoamine oxidase and rotenone-insensitive NADH-cytochrome c reductase which have been reported to be localized in the outer mitochondrial membrane. The other fraction exhibited an electrophoretic mobility which was different from that of intact mitochondria, and this fraction contained enzymes characteristic of the inner membrane-matrix fraction such as soluble and particulate enzymes of the Krebs cycle. Microsomes exhibited an electrophoretic mobility which was almost identical with that of the outer mitochondrial membrane. In addition to resolving the localization of enzymes in mitochondrial membranes, these data indicate that the outer limiting membrane of the mitochondrion is the sole determinant of the surface charge of mitochondria.     

Preparation of an inner membrane-matrix fraction from yeast yeast mitochondria.

Treatment of yeast mitochondria with digitonin was used in order to prepare an inner membrane-matrix fraction preserving its permeability properties. The incubation time of mitochondria with digitonin was an essential parameter for the selective solubilization of the outer membrane. The incubation of mitochondria for l min at different concentrations of digitonin led to a three-step release of mitochondrial enzymes: (a) at low concentrations of digitonin, adenylate kinase was released; (b) higher concentrations were required to solubilize kynurenine hydroxylase, an outer membrane marker; (c) inner membrane markers (succinate dehydrogenase and oligomycin-sensitive adenosine triphosphatase) and matrix markers (fumarase and isocitrate dehydrogenase) were significantly released at concentrations of digitonin higher than 0.4 mg/mg of protein. The electron microscopic aspects of yeast mitoplasts (inner membrane-matrix fraction obtained by treatment with 0.4 mg of digitonin) showed an orthodox and a twisted configuration. These new organelles retained respiratory control when assayed with ethanol as the substrate. Their selective permeability properties were preserved as shown by isoosmotic swelling in potassium or ammonium salt solutions.     

The orientation of phosphate-dependent glutaminase on the inner membrane of rat renal mitochondria

Phosphate-dependent glutaminase is associated with the inner membrane of rat renal mitochondria. The orientation of this enzyme was characterized by comparing its sensitivity in isolated mitochondria and in mitoplasts to two membrane impermeable inhibitors. Mitoplasts were prepared by repeated swelling of mitochondria in a hypotonic phosphate solution. This procedure released greater than 70% of the adenylate kinase from the intermembrane space, but less than 10 and 25% of the marker activities characteristic of the inner membrane and matrix compartments, respectively. The addition of 20 microM p-chloromercuriphenylsulfonate (pCMPS) caused a rapid inactivation of the purified glutaminase. In contrast, the glutaminase contained in isolated mitochondria and mitoplasts was only slightly affected by the addition of up to 2 mM pCMPS. Similarly, the activity in mitochondria and mitoplasts was not inhibited by the addition of an excess of inactivating Fab antibodies. However, a similar extent of inactivation occurred when either membrane fraction was incubated with concentrations of octylglucoside greater than 0.35%. Mitochondria were also treated with increasing concentrations of digitonin. At 0.4 mg digitonin/mg protein, all of the adenylate kinase was released but the glutaminase activity was either slightly inhibited or unaffected by the addition of pCMPS or the Fab antibodies, respectively. These studies establish that the glutaminase is localized on the inner surface of the inner membrane. Therefore, mitochondrial catabolism of glutamine must occur only within the matrix compartment.     

Separation of outer and inner membranes of mitochondria from the brown adipose tissue of infant rats.

Digitonin treatment and the swelling-shrinkage-sonication procedure as used to separate mitochondria membranes were applied to mitochondria from the brown adipose tissue (BAT) of infant rats. Digitonin at a concentration of 0.15 mg/mg mitochondrial protein produced disruption of the outer membrane of BAT mitochondria and a complete release of adenylate kinase. However, fragments of the outer membrane remained firmly attached to the inner membrane-matrix particles (mitoplasts) and sedimented at 10 000 g, as indicated by the activity of monoamine oxidase in the pellet. Only at 0.5 mg digitonin/mg protein did outer membrane become almost entirely separated. Oxidation of external cytochrome c by mitoplasts was only 50% of the total cytochrome oxidase at 0.5 mg digitonin/mg protein, indicating an incomplete exposure of the inner membrane to the external medium. Ultrastructural studies revealed that a large proportion of mitoplasts retained the orthodox configuration under these conditions. Outer membrane fragments obtained by the swelling-shrinkage-sonication procedure were of buoyant density corresponding to 20–30% (weight/vol) sucrose. After a 10 sec sonication of mitochondria, a relatively pure outer membrane fraction could be obtained with a yield not exceeding 20%. Longer sonication increased the yield, but also increased the degree of contamination by inner membrane fragments. Optimum conditions for the separation of outer and inner membranes from brown adipose tissue mitochondria are described.     

Flow Cytometric Analysis of Rhodamine 123 Fluorescence during Modulation of the Membrane Potential in Plant Mitochondria

The fluorescent dye rhodamine 123, which selectively accumulates in mitochondria based on the membrane potential, was used with flow cytometry to evaluate variations in activity of mitochondria isolated from plant tissues. In the presence of succinate and ATP, potato (Solanum tuberosum L.) tuber mitochondrial activity was affected by metabolic inhibitors and compounds that modify the membrane potential. The more uniform the mitochondrial population, the higher the observed membrane potential. The reactive population corresponds to the proportion of intact mitochondria (94-97%) defined by classic methods. Changes in the light-scattering properties are more related to internal modifications affecting the inner membrane-matrix system of the mitochondria during metabolic modulation than to specific volume change or outer membrane surface modifications. We tested our approach using an Arum maculatum preparation that contains three different types of mitochondria and demonstrated the validity of the light-scatter measurements to distinguish the α, β, and [ill] mitochondria and to measure their ability to built up a membrane potential in the presence of succinate. These results demonstrate clearly that flow cytometric techniques using rhodamine 123 can be employed to study the activity in isolated plant mitochondria.     

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.     

ULTRASTRUCTURE OF ISOLATED KIDNEY MITOCHONDRIA TREATED WITH PHLORIZIN AND ATP

Direct electron microscopic evidence is reported of the ultrastructure of mitochondrial membranes and compartments in mitochondria isolated in 0.5 M sucrose from the rat kidney cortex and the experimental changes they undergo with phlorizin and ATP treatment. A heterogeneous population of mitochondria is recognized under control conditions. The mitochondria appear to be of 3 main types, normal, swollen, and contracted. Under phlorizin treatment, most of the mitochondria swell in less than 15 minutes, apparently at the expense of the matrix. Treatment with ATP, on the other hand, produces, during the same time, a marked contraction of the isolated mitochondria, with many refoldings of the inner membrane and marked increase in the electron opacity of the matrix. It is concluded from these observations that mitochondrial swelling and contraction should be related mainly to the matrix content.     

ENZYME LOCALIZATION IN THE ANAEROBIC MITOCHONDRIA OF ASCARIS LUMBRICOIDES

Mitochondria from the muscle of the parasitic nematode Ascaris lumbricoides var. suum function anaerobically in electron transport-associated phosphorylations under physiological conditions. These helminth organelles have been fractionated into inner and outer membrane, matrix, and intermembrane space fractions. The distributions of enzyme systems were determined and compared with corresponding distributions reported in mammalian mitochondria. Succinate and pyruvate dehydrogenases as well as NADH oxidase, Mg⁺⁺-dependent ATPase, adenylate kinase, citrate synthase, and cytochrome c reductases were determined to be distributed as in mammalian mitochondria. In contrast with the mammalian systems, fumarase and NAD-linked "malic" enzyme were isolated primarily from the intermembrane space fraction of the worm mitochondria. These enzymes are required for the anaerobic energy-generating system in Ascaris and would be expected to give rise to NADH in the intermembrane space. The need for and possible mechanism of a proton translocation system to obtain energy generation is suggested.     

An electron microscopic study of structural changes during the large amplitude swelling and contraction of isolated beef-heart mitochondria

Summary The structural changes of isolated beef-heart mitochondria undergoing hypotonic swelling and ATP-induced contraction are described. During swelling the mitochondria take up water, the matrix space enlarges, the inner membrane becomes greatly extended with material which is apparently derived from the cristae (the rest of which fragment) and the outer membrane is ruptured. On addition of ATP, Mn⁺⁺ and Ca⁺⁺ water is extruded as the extended inner membrane contracts around the remnants of the cristae to give compact bundles of vesicles which bear little resemblance to the original organized structure.On leave of absence from Agricultural Research Council Institute of Animal Physiology, Babraham, Cambridge, England.     

Ketogenesis in vertebrate livers.

1. The hypothesis is advanced that a gluconeogenic organ such as the liver would evolve to oxidise fatty acids as its source of ATP for gluconeogenesis. It is also argued that such an organ might, in the light of current knowledge, be expected to be ketogenic. The animals investigated were lamprey, rainbow trout, eel, toad, axolotl, lizard and rat. 2. The respiratory quotients of liver slices from all animals was close to 0.74. Ketone bodies were produced from butyrate by all livers excepting the lamprey and ketone bodies were present in all blood samples examined. 3. There was no convincing evidence that direct deacylation of acetoacetyl CoA was important in any liver. HMGCoA synthase activity could not be found in the livers of the lamprey and eel. This enzyme was present in livers of the other animals. There was a large amount of acetoacetyl CoA-succinate transferase in the livers of the rainbow trout and eel, but only small amounts in the higher animals. 4. It is suggested that, initially the transferase was the important ketogenic pathway and the HMGCoA pathway evolved later.     

Yeast mitochondrial outer membrane specifically binds cytoplasmically-synthesized precursors of mitochondrial proteins

The precursor of cytochrome b₂ (a cytoplasmically-synthesized mitochondrial protein) binds to isolated mitochondria or to isolated outer membrane vesicles. Binding does not require an energized inner membrane, is diminished by trypsin treatment of the membranes and is not observed with the partially processed (intermediate) form of the cytochrome b₂ precursor or with non-mitochondrial proteins. Upon energization of the mitochondria, the bound precursor is imported and cleaved to the mature form. Similar results were obtained with the precursor of citrate synthase. This receptor-like binding activity was present in isolated outer, but not inner membrane. It was solubilized from outer membrane with non-ionic detergent and reconstituted into liposomes.     

Treatment of rats with glucagon or mannoheptulose increases mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity and decreases succinyl-CoA content in liver.

1. The activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (EC 4.1.3.5) in extracts of rapidly frozen rat livers was doubled in animals treated in various ways to increase ketogenic flux. 2. Some 90% of the activity measured was mitochondrial, and changes in mitochondrial activity dominated changes in total enzyme activity. 3. The elevated HMG-CoA synthase activities persisted throughout the isolation of liver mitochondria. 4. Intramitochondrial succinyl-CoA content was lower in whole liver homogenates and in mitochondria isolated from animals treated with glucagon or mannoheptulose. 5. HMG-CoA synthase activity in mitochondria from both ox and rat liver was negatively correlated with intramitochondrial succinyl-CoA levels when these were manipulated artificially. Under these conditions, the differences between mitochondria from control and hormone-treated rats were abolished. 6. These findings show that glucagon can decrease intramitochondrial succinyl-CoA concentration, and that this in turn can regulate mitochondrial HMG-CoA synthase. They support the hypothesis that the formation of ketone bodies from acetyl-CoA may be regulated by the extent of succinylation of mitochondrial HMG-CoA synthase.     

Ultrastructural studies of beef heart mitochondria. 3. The inequality of gross morphological change and oxidative phosphorylation

The relationships between membranes and intramembrane compartments of isolated heart mitochondria are inadequately defined to express the induced morphological changes associated with the structural organization. The inner membrane and matrix are the major structural entities which undergo transformation upon alteration of metabolism or incubation conditions. To better express these morphological changes within a mitochondrion, two inner membranes plus enclosed matrix are defined as an inmerix (plural inmerices). Three general morphological forms of mitochondria can be distinguished by the size and shape of inmerices. These are distended, condensed, and coalesced inmerixal configurations. Hypotonic conditions and P_i in isotonic sucrose generate distended configurations. This P_i distention is apparently dependent on utilization of energy. It does not occur under anaerobic conditions. Oxidizable substrates generate condensed configurations. ADP and dADP generate coalesced configurations and stop formation of condensed and distended inmerixal configurations in the absence of inhibitors. ADP coalescence is apparently not dependent on an energy input. It occurs under aerobic and anaerobic conditions, and in isotonic and hypotonic media. Atractyloside completely inhibits the effects of ADP on inmerixal membranes whereas oligomycin does not. Distention by P_i is unaffected by the two inhibitors. Distended inmerices, without added P_i (12 mM and 62 mM sucrose), are coalesced by ADP. These studies indicate that coalescence of inmerixal membranes probably reflects the consequences of specific stoichiometric binding or translocation of adenine nucleotides.     

Localization of catalytic and regulatory subunits of cyclic AMP-dependent protein kinases in mitochondria from various rat tissues.

Observation and quantification of the catalytic subunit C of cyclic AMP-dependent protein kinases by immuno-gold electron microscopy suggested a high concentration of cyclic AMP-dependent protein kinases in mitochondria from liver, kidney, heart and skeletal muscle, pancreas, parotid gland and brain cells. The position of gold particles pointed to a localization in the inner membrane/matrix space. A similar distribution was obtained by immunolocalization of the cyclic AMP-dependent protein kinase regulatory subunits RI and RII in liver, pancreas and heart cells. The results indicated the presence of both the type I and the type II cyclic AMP-dependent protein kinases in mitochondria of hepatocytes, and the preferential occurrence of the type I protein kinase in mitochondria from exocrine pancreas and heart muscle. The immunocytochemical results were confirmed by immunochemical determination of cyclic AMP-dependent protein kinase subunits in fractionated tissues. Determinations by e.l.i.s.a. of the C-subunit in parotid gland cell fractions indicated about a 4-fold higher concentration of C-subunit in the mitochondria than in a crude 1200 g supernatant. Immunoblot analysis of subfractions from liver mitochondria supported the localization in situ of cyclic AMP-dependent protein kinases in the inner membrane/matrix space and suggested that the type I enzyme is anchored by its regulatory subunit to the inner membrane. In accordance with the immunoblot data, the specific activity of cyclic AMP-dependent protein kinase measured in the matrix fraction was about twice that measured in whole mitochondria. These findings indicate the importance of cyclic AMP-dependent protein kinases in the regulation of mitochondrial functions.     

Resolution of rat mitochondrial matrix proteins by two-dimensional polyacrylamide gel electrophoresis.

The mitochondrial matrix subfractions from rat liver, kidney cortex, brain, heart, and skeletal muscle were isolated and their protein components were resolved by two-dimensional polyacrylamide gel electrophoresis, revealing between 120 and 150 components for each matrix subfraction. Excellent resolution was obtained utilizing a pH 5 to 8 gradient in the first dimension and in 8 to 13% exponential acrylamide gradient in the second dimension, increasing the number of mitochondrial matrix proteins observed 3-fold over one-dimensional systems. Protein components tentatively identified by co-migration with pure enzymes and by known tissue distributions are carbamoyl-phosphate synthetase (EC 2.7.2.5), ornithine transcarbamylase (EC 2.1.3.3), glutamate dehydrogenase (EC 1.4.1.3), pyruvate carboxylase (EC 6.4.1.1), citrate synthase (EC 4.1.3.7), fumarase (EC 4.2.1.2), aconitase (EC 4.2.1.3), alpha-ketoglutarate dehydrogenase (EC 1.2.4.2), dihydrolipoyl transsuccinylase (EC 2.3.1.12), lipoamide dehydrogenase (EC 1.6.4.3), glutamate-aspartate aminotransferase (EC 2.6.1.1), and the two subunits of pyruvate dehydrogenase (EC 1.2.4.1). Protein components unambiguously identified by peptide mapping are citrate synthase, aconitase, and pyruvate carboxylase. The inner membrane subfraction from rat liver mitochondria was also resolved two dimensionally; the alpha and beta subunits of ATPase (F1) (EC 3.6.1.3) were identified by peptide mapping.     

Regulation of mitochondrial membrane protein turnover by thyroid hormone(s)

Effects of thyroidectomy on turnover rate of proteins of rat liver mitochondria, mitochondrial membranes and microsomes were examined. Thyroidectomy resulted in a significant increase in the half-lives of whole mitochondria and inner membrane-matrix, the effect being less pronounced on the half-lives of outer mitochondrial membrane and microsomes.     

Alanine aminotransferase and some other enzymes in different populations of free brain cortex mitochondria

The activity of certain key enzymes involved in glutamic acid metabolism was studied in purified brain mitochondria and in mitochondrial subfractions separated in a discontinuous 1.2--1.6 mol/l sucrose gradient. Alanine aminotransferase and glutamate dehydrogenase were found to be matrix enzymes and aspartate aminotransferase to be associated with the inner mitochondrial membranes. After the purified mitochondria had been separated into 5 subfractions, aspartate aminotransferase and NAD+-dependent isocitrate dehydrogenase were found to be bound to the lighter mitochondrial subfractions settling at the 1.4--1.5 mol/l sucrose boundary while alanine aminotransferase, 4-aminobutyrate transaminase and glutamate dehydrogenase were associated with the heavier subfractions settling below 2.4 mol/l sucrose. The highest specific activity of the given enzymes was found in the subfraction settling at the 1.4--1.5 mol/l sucrose boundary, the only exception being alanine aminotransferase activity, whose maximum was found in the subfractions settling in 1.5 and 1.6 mol/l sucrose. It was concluded that alanine aminotransferase, in conjunction with glutamate dehydrogenase, is linked to NH3 binding and to the oxidation of reduced adenine nucleotides; in addition, alanine aminotransferase is presumed to have the function of transporting glutamate from the mitochondria to the extramitochondrial space.     

SEPARATION OF MITOCHONDRIAL MEMBRANES OF NEUROSPORA CRASSA : II. Submitochondrial Localization of the Isoleucine-Valine Biosynthetic Pathway

Separation of Neurospora mitochondrial outer membranes from the inner membrane/matrix fraction was effected by digitonin treatment and discontinuous density gradient centrifugation. The solubilization of four isoleucine-valine biosynthetic enzymes was studied as a function of digitonin concentration and time of incubation in the detergent. The kinetics of the appearance of valine biosynthetic function in fractions outside of the inner membrane/matrix fraction, coupled with enzyme solubilization patterns similar to that for the matrix marker, mitochondrial malate dehydrogenase, indicate that the four isoleucine-valine pathway enzymes are localized in the mitochondrial matrix.     

Electron paramagnetic resonance characterization of membrane bound iron-sulfur clusters and aconitase in plant mitochondria

Electron paramagnetic resonance (EPR) characteristics of the iron-sulfur clusters of potato tuber mitochondria have been examined in various subfractions of the mitochondria. We confirm that EPR signals comparable to those of the iron-sulfur proteins of mammalian mitochondria respiratory complexes are also present in plant mitochondria. Two distinct iron-sulfur centers paramagnetic in the oxidized state exhibit signals which differ in their detailed line shape and field position. One of these which is present in the inner membrane corresponds to center S.3. The EPR spectrum of the soluble fraction revealed the presence of another center with a low field maximum at g = 2.03 and is associated with aconitase. The EPR signal observed in the mitochondrial matrix from potato tuber and characteristic of 3Fe cluster is significantly changed in shape after addition of citrate and differs clearly from the spectrum of pig heart mitochondrial aconitase. The aconitase in plant mitochondria differs from that of mammalian mitochondria by several features.