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1.
H. A. I. Newman Stanley E. Gordesky Charles Hoppel Cecil Cooper 《The Biochemical journal》1968,107(3):381-385
1. The amount and types of phospholipid and the fatty acid composition of the various phospholipids were examined in intact rat liver mitochondria, in mitochondria devoid of their outer membrane (preparation A) and in very small pieces derived from the disruption of the inner-membrane complexes (preparation B). The latter two preparations were obtained by digitonin treatment and carry out oxidative phosphorylation. 2. The ratio mug.atoms of phospholipid P/mg. of protein was 0.163 for intact mitochondria, decreased to 0.118 on removal of the outer membrane and increased markedly to 0.292 on disruption of the inner-membrane complex. 3. Examination of the various types of phospholipid present showed that the molar proportions cardiolipin:phosphatidylcholine:phosphatidylethanolamine were approx. 1:6:6 for intact mitochondria and 1:3:3 for preparations A and B. 4. There was a correlation between the recovery of cardiolipin and adenosine triphosphatase activity in the conversion of intact mitochondria into preparations A and B. 5. The fatty acid contents of the various types of phospholipid purified by thin-layer chromatography were identical in all three preparations. Our results show a considerably higher content of arachidonic acid and lower content of oleic acid for phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol than have previously been reported for mitochondrial phospholipids. 相似文献
2.
ENZYMATIC PROPERTIES OF THE INNER AND OUTER MEMBRANES OF RAT LIVER MITOCHONDRIA 总被引:125,自引:29,他引:96
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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. 相似文献
3.
Regulation of mitochondrial hexokinase in cultured HT 29 human cancer cells. An ultrastructural and biochemical study 总被引:2,自引:0,他引:2
C Denis-Pouxviel I Riesinger C Bühler D Brdiczka J C Murat 《Biochimica et biophysica acta》1987,902(3):335-348
The involvement of the mitochondrial bound hexokinase in aerobic glycolysis was investigated in two subpopulations of the HT 29 human colon cancer cell line: a poorly differentiated one with high aerobic lactate production (referred as undifferentiated or standard cells), and an enterocyte-like differentiated one with lower lactate production (referred as differentiated or Glc- cells). After mild digitonin treatment, 85% of the total cellular hexokinase activity remained in the particulate fraction in both cell types. In both cases mitochondria appeared to be tightly coupled but the Glc- cells exhibited a significantly higher oxidation rate in the presence of glucose. Electron microscopy of freeze-fractured cells revealed the absence of contacts between the two limiting mitochondrial membranes in the highly glycolytic standard cells, whereas the contacts were present in the Glc- cells. Furthermore, we investigated the functional relationship between bound hexokinase (as hexokinase-porin complex) and the inner compartment of mitochondria isolated from standard and Glc- HT 29 cells. In contrast to the differentiated cells the hexokinase in undifferentiated standard cells was not functionally coupled to the oxidative phosphorylation. This suggests that the high rate of lactate formation in neoplastic cells is not caused by an increase of particulate hexokinase activity but rather by a disregulation of the hexokinase-porin complex caused by the absence of contact sites between the two mitochondrial membranes. In agreement with this interpretation, the hexokinase-porin complex could be completely removed by digitonin treatment in standard HT 29 cells, while this was not possible in mitochondria from Glc- cells. 相似文献
4.
A major energy source in brain is glucose, which is committed to metabolism by hexokinase (Type I isozyme), an enzyme usually considered to be bound to the outer mitochondrial membrane. In this study, the subcellular location of hexokinase in brain has been rigorously investigated. Mitochondrial fractions containing hexokinase (greater than 500 milliunits/mg protein) were prepared by two different procedures, and then subjected to density gradient centrifugation before and after loading with barium phosphate, a technique designed to increase the density of the mitochondria. The gradient distribution patterns of both unloaded and loaded preparations show that brain hexokinase does not distribute exclusively with mitochondrial marker enzymes. This is particularly evident in the loaded preparations where there is a clear distinction between the peak activities of hexokinase and mitochondrial markers. The same observation was made when the mitochondrial fraction of either untreated or barium phosphate-loaded mitochondria was subjected to titration with digitonin. In fact, at concentrations of digitonin, which almost completely solubilize marker enzymes for both the inner and outer mitochondrial membranes, a significant fraction of the total hexokinase remains particulate bound. Electron microscopy confirmed that particulate material is still present under these conditions. Significantly, hexokinase is released from particulate material only at high concentrations of digitonin which solubilize the associated microsomal marker NADPH-cytochrome c reductase. Glucose 6-phosphate, which is known to release hexokinase from the brain "mitochondrial fraction" also releases hexokinase from this unidentified particulate component. These results on brain, a normal glucose utilizing tissue, differ from those obtained previously on highly glycolytic tumor cells where identical subfractionation procedures revealed a strictly outer mitochondrial membrane location for particulate hexokinase (Parry, D. M., and Pedersen, P. L. (1983) J. Biol. Chem. 258, 10904-10912). It is concluded that in brain, hexokinase has a greater propensity to localize at nonmitochondrial receptor sites than to those known to be associated with the outer mitochondrial membrane. 相似文献
5.
The localization of some coenzyme A-dependant enzymes in rat liver mitochondria 总被引:5,自引:1,他引:4
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1. CoA, acetyl-CoA, l-carnitine and acetyl-l-carnitine when added to rat liver mitochondria equilibrate with approximately two-thirds of the total intramitochondrial water. The mitochondrial space calculated to be freely permeable to these solutes was identical with that obtained for sucrose. 2. Acetyl-CoA is rapidly deacylated by rat liver mitochondria at 0 degrees C, and special precautions are required to measure its mitochondrial permeation. 3. Rat liver mitochondria were separated into fractions that correspond to the inner membrane, the outer membrane, and the soluble proteins of the matrix and intermembrane compartment. Soluble enzymes considered to be located in the matrix were citrate synthase (EC 4.1.3.7), palmitoyl-CoA dehydrogenase (EC 1.3.2.2), electron-transferring flavoprotein, medium-chain-length ATP-specific fatty acyl-CoA synthetase (EC 6.2.1.2), l-3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35) and 3-keto-acyl-CoA thiolase (EC 2.3.1.16). Carnitine palmitoyltransferase (EC 2.3.1.-) is largely associated with the inner-membrane fraction. A long-chain-length ATP-specific fatty acyl-CoA synthetase (EC 6.2.1.3) is associated with the outer-membrane fraction. 相似文献
6.
Study on the localization of proteases of mitochondrial origin 总被引:1,自引:0,他引:1
A marked proteolytic activity against casein can be demonstrated in rat liver mitochondria. The proteases degrading casein appear distributed between a sedimentable fraction (Po) and a soluble extract (So). Part of the soluble fraction activity, which may be recovered in the mitochondrial intermembrane space, results from a contamination by lysosomal proteases and can be eliminated by previously washing the mitochondria with digitonin. The pre-exposure to digitonin causes an enhancement of the caseinolytic activity associated with the membrane fragments, proving that this activity is not due to lysosomal enzymes. When rats have been injected in vivo with the compound 48/80 which, by degranulating the mast cells prevents contamination of the mitochondrial preparations by mast cell proteases, the membrane fraction (Po) retains a caseinolytic activity of the order of 80 per cent of the control preparations. A similar value of activity is observed in the membranes of brain mitochondria, isolated by a method which removes the rare mast cells they may contain. This shows that the greater part of the caseinolytic activity associated with the rat liver membranes does not originate from mast cell granules. Liver mitochondria pre-exposed to digitonin to eliminate lysosomal contaminants, have been subfractionated into matrix, intermembrane space, inner and outer membrane. Each of the fractions exhibits a caseinolytic activity, but the largest part is localized in the inner compartments of mitochondria: the matrix and the inner membrane. The optimal pH and the sensitivity to inhibitors of the proteases in the different compartments indicate that we are dealing with distinct enzymes. 相似文献
7.
THE SURFACE CHARGE OF RAT LIVER MITOCHONDRIA AND THEIR MEMBRANES : Clarification of Some Controversies Concerning Mitochondrial Structure 总被引:5,自引:3,他引:2
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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. 相似文献
8.
V A Saks A V Kuznetsov V V Kupriyanov M V Miceli W E Jacobus 《The Journal of biological chemistry》1985,260(12):7757-7764
To define more clearly the interactions between mitochondrial creatine kinase and the adenine nucleotide translocase, the outer membrane of rat heart mitochondria was removed by digitonin, producing an inner membrane-matrix (mitoplast) preparation. This mitoplast fracton was well-coupled and contained a high specific activity of mitochondrial creatine kinase. Outer membrane permeabilization was documented by the loss of adenylate kinase, a soluble intermembrane enzyme, and by direct antibody inhibition of mitochondrial creatine kinase activity. With this preparation, we documented four important aspects of functional coupling. Kinetic studies showed that oxidative phosphorylation decreased the value of the ternary enzyme-substrate complex dissociation constant for MgATP from 140 to 16 microM. Two approaches were used to document the adenine nucleotide translocase specificity for ADP generated by mitochondrial creatine kinase. Exogenous pyruvate kinase (20 IU/ml) could not readily phosphorylate ADP produced by creatine kinase, since added pyruvate kinase did not markedly inhibit creatine + ATP-stimulated respiration. Additionally, when ADP was produced by mitochondrial creatine kinase, the inhibition of the translocase required 2 nmol of atractyloside/mg of mitoplast protein, while only 1 nmol/mg was necessary when exogenous ADP was added. Finally, the mass action ratio of the mitochondrial creatine kinase reaction exceeded the apparent equilibrium constant when ATP was supplied to the creatine kinase reaction by oxidative phosphorylation. Overall, these results are consistent with much data from intact rat heart mitochondria, and suggest that the outer membrane plays a minor role in the compartmentation of adenine nucleotides. Furthermore, since the removal of the outer membrane does not alter the unique coupling between oxidative phosphorylation and mitochondrial creatine kinase, we suggest that this cooperation is the result of protein-protein proximity at the inner membrane surface. 相似文献
9.
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. 相似文献
10.
Analytical study of microsomes and isolated subcellular membranes from rat liver VIII. Subfractionation of preparations enriched with plasma membranes, outer mitochondrial membranes, or Golgi complex membranes 总被引:8,自引:6,他引:2
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《The Journal of cell biology》1981,89(3):456-474
Preparations enriched with plasmalemmal, outer mitochondrial, or Golgi complex membranes from rat liver were subfractionated by isopycnic centrifugation, without or after treatment with digitonin, to establish the subcellular distribution of a variety of enzymes. The typical plasmalemmal enzymes 5'-nucleotidase, alkaline phosphodiesterase I, and alkaline phosphatase were markedly shifted by digitonin toward higher densities in all three preparations. Three glycosyltransferases, highly purified in the Golgi fraction, were moderately shifted by digitonin in both this Golgi complex preparation and the microsomal fraction. The outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the out mitochondrial membrane preparation, in agreement wit its behavior in microsomes. With the exception of NADH cytochrome c reductase (which was concentrated in the outer mitochondrial membrane preparation), typical microsomal enzymes (glucose-6-phosphatase, esterase, and NADPH cytochrome c reductase) displayed low specific activities in the three preparations; except for part of the glucose-6-phosphatase activity in the plasma membrane preparation, their density distributions were insensitive to digitonin, as they were in microsomes. The influence of digitonin on equilibrium densities was correlated with its morphological effects. Digitonin induced pseudofenestrations in plasma membranes. In Golgi and outer mitochondrial membrane preparations, a few similarly altered membranes were detected in subfractions enriched with 5'-nucleotidase and alkaline phosphodiesterase I. The alterations of Golgi membranes were less obvious and seemingly restricted to some elements in the Golgi preparation. No morphological modification was detected in digitonin-treated outer mitochondrial membranes. These results indicate that each enzyme is associated with the same membrane entity in all membrane preparations and support the view that there is little overlap in the enzymatic equipment of the various types of cytomembranes. 相似文献
11.
D. Brdiczka W. Krebs P. Kloock 《Biochimica et Biophysica Acta (BBA)/General Subjects》1973,297(2):203-212
A method is described which makes it possible to determine whether a given enzyme is located on the outer surface of a mitochondrial membrane, or wether it is localized within a mithondrial compartment. The method combines the use of proteases with digitonin. Depending on the concentration of digitonin, enzymes behind the microsomal membranes and the outer mitochondrial membrane may be successively exposed to the action of proteases. Thus, enzymes located within microsomes contaminating the mitochondrial fraction may be easily distinguished from true intramitochondrial enzymes.Applying this technique to the mitochondrial fraction of rat liver, it is shown that lactate dehydrogenase (l-lactate: NAD+ oxidoreductase, EC 1.1.1.27) is located on the outer surface of the mitochondria and within microsomal contaminants. 相似文献
12.
Krause F Reifschneider NH Vocke D Seelert H Rexroth S Dencher NA 《The Journal of biological chemistry》2004,279(46):48369-48375
Higher plant mitochondria have many unique features compared with their animal and fungal counterparts. This is to a large extent related to the close functional interdependence of mitochondria and chloroplasts, in which the two ATP-generating processes of oxidative phosphorylation and photosynthesis, respectively, take place. We show that digitonin treatment of mitochondria contaminated with chloroplasts from spinach (Spinacia oleracea) green leaves at two different buffer conditions, performed to solubilize oxidative phosphorylation supercomplexes, selectively extracts the mitochondrial membrane protein complexes and only low amounts of stroma thylakoid membrane proteins. By analysis of digitonin extracts from partially purified mitochondria of green leaves from spinach using blue and colorless native electrophoresis, we demonstrate for the first time that in green plant tissue a substantial proportion of the respiratory complex IV is assembled with complexes I and III into "respirasome"-like supercomplexes, previously observed in mammalian, fungal, and non-green plant mitochondria only. Thus, fundamental features of the supramolecular organization of the standard respiratory complexes I, III, and IV as a respirasome are conserved in all higher eukaryotes. Because the plant respiratory chain is highly branched possessing additional alternative enzymes, the functional implications of the occurrence of respiratory supercomplexes in plant mitochondria are discussed. 相似文献
13.
Polarographic assay and intracellular distribution of superoxide dismutase in rat liver. 总被引:10,自引:6,他引:4
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D D Tyler 《The Biochemical journal》1975,147(3):493-504
1. A polarographic assay of superoxide (O2--) dismutase (EC 1.15.1.1) activity is described, in which the ability of the enzyme to inhibit O2---dependent sulphite oxidation, initiated by xanthine oxidase activity, is measured. The assay was used in a study of the intracellular distribution of superoxide dismutase in rat liver. Both cyanide-sensitive cupro-zinc dismutase (92% of the total activity) and cyanide-insensitive mangano-dismutase (8%) were measured. 2. Rat liver homogenates contained both particulate (16%y and soluble (84%) dismutase activity. The particulate activity contained both types of dismutase, whereas nearly all the soluble dismutase was a cupro-zinc enzymes. The distribution pattern of mangano-dismutase was similar to that of cytochrome oxidase and glutamate dehydrogenase, indicating that the enzyme was probably present exclusively in the mitochondria. 3. Superoxide dismutase activity in the heavy-mitochondrial (M) fraction was latent and was activated severalfold and largely solubilized by sonication. Treatment of the M fraction with digitonin or a hypo-osmotic suspending medium indicated that most of the cupro-zinc dismutase was located in the mitochondrial intermembrane space, whereas the mangano-enzyme was located in the inner-membrane and matrix space. 4. A small amount of dismutase activity appeared to be present in the nuclei and microsomal fraction, but little or no activity in the lysosomes or peroxisomes. 5. The results are discussed in relation to the intracellular location of known O2---generating enzymes, the possible role of superoxide dismutase activity in intracellular H2O2 formation, and to current views on the physiological function of the enzyme. 相似文献
14.
Summary The removal of the outer mitochondrial membrane and hence of constituents of the intermembrane space in rat-liver mitochondria using digitonin showed that phosphate-dependent glutaminase, alanine and aspartate aminotransferase were localized in the mitoplasts. Further fractionation of mitoplasts following their sonication resulted in 90% of glutaminase, 98% of alanine aminotransferase and 48% of aspartate aminotransferase being recovered in the soluble fraction while the remainder of each enzyme was recovered in the sonicated vesicles fraction. These results indicated that glutaminase and alanine aminotransferase were soluble matrix enzymes, the little of each enzyme recovered in the sonicated vesicles fraction being probably due to entrapment in the vesicles. Aspartate aminotransferase had dual localization, in the inner membrane and matrix with the high specific activity in sonicated vesicles confirming its association with the membrane. Activation experiments suggested that the membrane-bound enzyme was localized on the inner side of the inner mitochondrial membrane. 相似文献
15.
Mitochondrial boundary membrane contact sites in brain: points of hexokinase and creatine kinase location, and control of Ca2+ transport 总被引:1,自引:0,他引:1
M Kottke V Adam I Riesinger G Bremm W Bosch D Brdiczka G Sandri E Panfili 《Biochimica et biophysica acta》1988,935(1):87-102
The location of hexokinase at the surface of brain mitochondria was investigated by electron microscopy using immuno-gold labelling techniques. The enzyme was located where the two mitochondrial limiting membranes were opposed and contact sites were possible. Disruption of the outer membrane by digitonin did not remove bound hexokinase and creatine kinase from brain mitochondria, although the activity of outer membrane markers and adenylate kinase decreased, suggesting a preferential location of both enzymes in the contact sites. In agreement with that, a membrane fraction was isolated from osmotically lysed rat brain mitochondria in which hexokinase and creatine kinase were concentrated. The density of this kinase-rich fraction was specifically increased by immuno-gold labelling of hexokinase, allowing a further purification by density gradient centrifugation. The fraction was composed of inner and outer limiting membrane components as shown by the specific marker enzymes, succinate dehydrogenase and NADH-cytochrome-c-oxidase (rotenone insensitive). As reported earlier for the enriched contact site fraction of liver mitochondria the fraction from brain mitochondria contained a high activity of glutathione transferase and a low cholesterol concentration. Moreover, the contacts showed a higher Ca2+ binding capacity in comparison to outer and inner membrane fractions. This finding may have regulatory implications because glucose phosphorylation via hexokinase activated the active Ca2+ uptake system and inhibited the passive efflux, resulting in an increase of intramitochondrial Ca2+. 相似文献
16.
Bornhövd C Vogel F Neupert W Reichert AS 《The Journal of biological chemistry》2006,281(20):13990-13998
The F1F0-ATP synthase in mitochondria, in addition to its function in energy transduction, has a structural role in determining cristae morphology. This depends on its ability to form dimeric and higher oligomeric supracomplexes. Here we show that mutants of the dimer-specific subunits e and g, which destabilize dimeric and oligomeric F1F0-ATP synthase supracomplexes, have a decreased mitochondrial membrane potential delta psi. The degree of destabilization correlated with the reduction of the membrane potential. The enzymatic activities of F1F0-ATP synthase and cytochrome c oxidase, maximal respiration rate, coupling of oxidative phosphorylation, and tubular mitochondrial morphology were not affected or only to a minor extent. In mutants lacking one or two coiled-coil domains of subunit e, the reduction of the mitochondrial membrane potential was not due to loss of mitochondrial DNA, a reduced capacity of oxidative phosphorylation, or to altered cristae morphology. We propose a role for the supracomplexes of the F1F0-ATP synthase in organizing microdomains within the inner membrane, ensuring optimal bioenergetic competence of mitochondria. 相似文献
17.
The bulk of the enzyme L-alanine: 4,5-dioxovalerate transaminase, which catalyses the transamination reaction between L-alanine and 4,5-dioxovalerate to synthesize delta-aminolevulinic acid was predominantly recovered in the mitochondrial matrix. Sub-fractionation procedure of the mitochondria involved the use of digitonin and lubrol followed by differential centrifugation to separate soluble and particulate enzymes. Lubrol did not inhibit this enzyme. Presence of this enzyme in the mitochondrial matrix was further confirmed by western blot analysis. The results support the conclusion that L-alanine: 4,5-dioxovalerate transaminase is localized and functions in the mitochondrial matrix. 相似文献
18.
The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane 总被引:32,自引:0,他引:32
R R Anholt P L Pedersen E B De Souza S H Snyder 《The Journal of biological chemistry》1986,261(2):576-583
We have investigated the subcellular localization of the peripheral-type benzodiazepine receptor in rat adrenal gland using the high affinity ligand 3H-labeled 1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide ([3H]PK11195). The autoradiographic pattern of [3H]PK11195 binding sites in tissue sections of adrenal gland is similar to the histochemical distribution of the mitochondrial marker enzymes, cytochrome oxidase and monoamine oxidase, which are present in high concentrations only in the cortex. Subcellular fractionation studies of homogenates of adrenal gland indicate that the recovery and enrichment of [3H]PK11195 binding sites in the nuclear, mitochondrial, microsomal, and soluble fractions correlate closely with cytochrome oxidase activity, but not with markers for the nuclei, lysosomes, peroxysomes, endoplasmic reticulum, plasma membrane, or cytoplasm, indicating an association of the peripheral-type benzodiazepine receptor with the mitochondrial compartment. Titration of isolated mitochondria with digitonin results in the simultaneous release of the peripheral-type benzodiazepine receptor and of monoamine oxidase, but not cytochrome oxidase, indicating association of the peripheral-type benzodiazepine receptor with the mitochondrial outer membrane. Scatchard analysis and drug displacement studies of the binding of [3H] PK11195 to intact mitochondria and to the outer membrane-enriched digitonin extract further confirm the localization of the peripheral-type benzodiazepine receptor to the mitochondrial outer membrane. 相似文献
19.
SEPARATION OF MITOCHONDRIAL MEMBRANES OF NEUROSPORA CRASSA : II. Submitochondrial Localization of the Isoleucine-Valine Biosynthetic Pathway 总被引:4,自引:0,他引:4
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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. 相似文献
20.
The structural changes accompanying digitonin-induced release of enzymes and metabolites from isolated hepatocytes have been studied by scanning and transmission electron microscopy. In the initial phase, characterized by total release of the cytosolic marker enzyme, lactate dehydrogenase, the plasma membrane was immediately damaged, rapidly followed by extensive damage to the endoplasmic reticulum. The shape of the cell, however, was maintained, and the mitochondria and nucleus remained tightly held together by the cytoskeleton. Mitochondria remained intact initially, whereas the cytosol became less electron dense and the nuclear chromatin was more dispersed. An intermediate phase was characterized by total release of adenylate kinase and most of the glucose-6-phosphatase, marker enzymes for the mitochondrial intermembrane space and the endoplasmic reticulum, respectively. The outer mitochondrial membrane was ruptured, but mitochondria maintained their normal matrix electron density. In the final phase, characterized by the beginning of citrate synthase release from the mitochondrial matrix space, the mitochondria became swollen, and only the nucleus, inner and outer mitochondrial membranes, and the cytoskeleton could be clearly distinguished. Although the plasma membrane could not be readily discerned in electron micrographs after the initial phase, the plasma membrane marker enzyme 5'-nucleotidase remained associated with digitonin-treated hepatocytes. Acetyl-CoA carboxylase was released much more slowly than lactate dehydrogenase, indicating some severe restriction on its release. The release of acetyl-CoA carboxylase closely paralleled the release of glucose-6-phosphatase. The controlled exposure of hepatocytes to digitonin, therefore, leads to the sequential release of soluble, compartmentalized cellular components and some membrane-bound components, but the mitochondrial membrane, cytoskeleton and the nucleoskeleton survive even long-term digitonin treatment. 相似文献