Stimulation of mitoplast respiration by mitochondrial porin

Mitochondrial porin (2 ng/ml) being added to the rat liver mitoplasts considerably stimulates the respiration in the third and uncoupled states. As the same effect was observed previously with the addition of outer membrane fraction to the mitoplast suspension, it is concluded that mitochondrial porin participates in regulation of the mitochondria respiration and, probably, is the natural activator of the ADP/ATP carrier function.     

Protein kinase activity and endogenous phosphorylation in subfractions of rat liver mitochondria

Rat liver mitochondria were subfractionated into outer membrane, intermembrane and mitoplast (inner membrane and matrix) fractions. Of the recovered protein kinase activity, 80–90% was found in the intermembrane fraction, while the rest was associated with mitoplast. The intermembrane prostimulated kinase was stimulated by cyclic AMP, while the mitoplast enzyme was stimulated by the nucleotide only after treatment with Triton X-100. Extracted protein kinase resolved into three peaks on DEAE-cellulose chromatography. All three peaks were present both in the intermembrane fraction and in mitoplast. One peak corresponded to the catalytic subunit of cyclic AMP-dependent protein kinase, one was a cyclic AMP-independent enzyme, and the third was the cyclic AMP-dependent type II enzyme. The endogenous incorporation of phosphate was particularly high in the outer mitochondrial membrane, and occurred also in the mitoplast fraction. The incorporation in mitoplasts was to a double band of Mr 47 500, and in outer membranes to apparently heterogeneous material of comparatively low molecular weight.     

Protein kinase activity and endogenous phosphorylation in subfractions of rat liver mitochondria

Rat liver mitochondria were subfractionated into outer membrane, intermembrane and mitoplast (inner membrane and matrix) fractions. Of the recovered protein kinase activity, 80-90% was found in the intermembrane fraction, while the rest was associated with mitoplasts. The intermembrane protein kinase was stimulated by cyclic AMP, while the mitoplast enzyme was stimulated by the nucleotide only after treatment with Triton X-100. Extracted protein kinase resolved into three peaks on DEAE-cellulose chromatography. All three peaks were present both in the intermembrane fraction and in mitoplasts. One peak corresponded to the catalytic subunit of cyclic AMP-dependent protein kinases, one was a cyclic AMP-independent enzyme, and the third was the cyclic AMP-dependent type II enzyme. The endogenous incorporation of phosphate was particularly high in the outer mitochondrial membrane, and occurred also in the mitoplast fraction. The incorporation in mitoplasts was to a double band of Mr 47 500, and in outer membranes to apparently heterogeneous material of comparatively low molecular weight.     

Creatine kinase of rat heart mitochondria. The demonstration of functional coupling to oxidative phosphorylation in an inner membrane-matrix preparation

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.     

Evidence for ΔpH surface component (ΔpH) of proton motive force in ATP synthesis of mitochondria

Background

One of the central debates in membrane bioenergetics is whether proton-dependent energy coupling mechanisms are mediated exclusively by protonic transmembrane electrochemical potentials, as delocalized pmf, ΔµH⁺, or by more localized membrane surface proton pathways, as interfacial pmf, ΔµH^S.

Methods

We measure ?pH^S in rat liver mitoplasts energized by respiration or ATP hydrolysis by inserting pH sensitive fluorescein-phosphatidyl-ethanolamine(F-PE) into mitoplast surface.

Results

In the presence of rotenone and Ap5A, succinate oxidation induces a bi-phasic interfacial protonation on the mitoplast membranes, a fast phase followed by a slow one, and an interfacial pH decrease of 0.5 to 0.9 pH units of mitoplast with no simultaneous pH changes in the bulk. Antimycin A, other inhibitors or uncouplers of mitochondrial respiration prevent the decrease of mitoplast ?pH^S, supporting that ΔµH^S is dependent and controlled by energization of mitoplast membranes. A quantitative assay of ATP synthesis coupled with ?pH^S of mitoplasts oxidizing succinate with malonate titration shows a parallel correlation between ATP synthesis, State 4 respiration and ?pH^S, but not with ?Ψ_E.

General Significance

Our data substantiate ?pH^S as the primary energy source of pmf for mitochondrial ATP synthesis. Evidence and discussion concerning the relative importance and interplay of ?pH^S and ?Ψ_E in mitochondrial bioenergetics are also presented.     

Influence of thyroid status on the electrophoretic pattern of rat liver mitochondrial proteins]

Liver mitochondria were isolated from normal and thyroidectomized rats and their protein components analyzed by polyacrylamide gel electrophoresis. In whole mitochondria 35 protein fractions with MW ranging from 10,000 to 135,000 were characterized. In the absence of thyroid hormone secretion, the amount of a MW 54,000 fraction was always decreased. Injection of small doses of 3,5,3'-triiodo-L-thyronine to the thyroidectomized animal restored the quantity of that protein fraction to normal. Isolated outer mitochondrial membranes showed the presence of 20 protein fractions. These fractions revealed no change after thyroidectomy. The mitoplast, which contained 35 fractions, exhibited a decrease of the MW 54,000 component in thyroidectomized rats. The mitoplast was separated into several fractions. Water soluble matrix proteins presented molecular weights ranging between 40,000 and 55,000. Proteins, which were slightly bound to the inner mitochondrial membrane and could be extracted by KCl, presented molecular weights between 25,000 and 45,000. Structural proteins showed a principal specific component of MW equals 23,000. Electrophoretic patterns obtained with these submitochondrial fractions were similar in normal and thyroidectomized animals. The mitoplast fraction which contained the insoluble cytochromes (a, a3, b, c1) was isolated ; its principal constituent, of MW 54,000 was significantly decreased after thyroidectomy. Thus, the lack of thyroid hormone secretion lowered the level of a protein constituent bound to the inner membrane of liver mitochondria. The synthesis of this constituent could be controlled by mitochondrial nucleic acids.     

Studies on the nature of adenosine diphosphatase activity from rat liver mitochondria

Adenosine diphosphatase (ADPase) activity was studied in rat liver with [beta-32P]ADP as a substrate. Mitochondria and outer mitochondrial membrane fractions were isolated and assayed for ADPase and various marker enzymes. ADPase activity was strikingly reduced when the outer membranes were removed from the mitochondria whether by digitonin treatment or osmotic shock. Addition of the inter-membrane space subfraction to the purified outer membranes resulted in enhanced ADPase activity. Addition of the inter-mitochondrial membrane enzyme adenylate kinase to outer membranes also produced a large stimulation of activity. The ADPase activity could also be reconstituted in vitro with adenylate kinase and either mitoplast ATPase or ouabain-sensitive (Na+ + K+ + Mg2+)-ATPase. Chloroform-released ATPase, however, was not capable of producing an ADPase activity when combined with adenylate kinase. Gel permeation chromatography of Triton-solubilised outer mitochondrial membranes was unable to resolve ADPase activity from contaminating ATPase. These results suggest that the majority of ADPase activity in rat liver mitochondria consists of the coupled activity of adenylate kinase and ATPase.     

Interaction of mammalian kidney membrane and mitochondria in vitro

1. In many parts of the kidney tubule, there exists a close physical association of membrane and mitochondria apparently related to salt transport. The present study examines the influence of adding rat kidney membrane fragments on mitochondrial respiration in vitro. The results are compared with previous findings on the effect of rat kidney membrane fragments on cytoplasmic glycolysis.

2. The membrane fraction stimulates mitochondrial respiration. The stimulus decreases with higher levels of ADP and is abolished by dinitrophenol. Stimulus of glycolysis by kidney membranes is related to a specific coupling of membrane ATPase activity and phosphoglycerate kinase. The stimulus of respiration appears to be due solely to the enhanced availability of ADP in presence of an ATPase system.     

Subcellular localization of vitamin D3 25-hydroxylase in human liver

Vitamin D3 25-hydroxylase activity was measured in subcellular and submitochondrial fractions of human liver. Quantitation of 25-hydroxyvitamin D3 was based on high performance liquid chromatography. Vitamin D3 25-hydroxylase activity was detected in the mitochondrial fraction only. The mitochondrial 25-hydroxylase activity was linear with time up to 60 min and with mitochondrial protein up to 1 mg/ml. An apparent Km value of about 10(-5) M was found. Substrate satuation level was not reached. In the presence of 2.4 X 10(-4) M vitamin D3, the rate of 25-hydroxyvitamin D3 formation was 0.19 nmol X mg of protein-1 X h-1 After fractionation of the mitochondria, 86% of the 25-hydroxylase activity was recovered in the mitoplast fraction. The outer membrane fraction was devoid of activity. It is concluded that human liver contains only one detectable vitamin D3 25-hydroxylase enzyme localized to the mitochondrial inner membrane.     

Functional Coupling between Nucleoside Diphosphate Kinase of the Outer Mitochondrial Compartment and Oxidative Phosphorylation

In rat liver mitochondria all nucleoside diphosphate kinase of the outer compartment is associated with the outer surface of the outer membrane (Lipskaya, T. Yu., and Plakida, K. N. (2003) Biochemistry (Moscow), 68, 1136-1144). In the present study, three systems operating as ADP donors for oxidative phosphorylation have been investigated. The outer membrane bound nucleoside diphosphate kinase was the first system tested. Two others employed yeast hexokinase and yeast nucleoside diphosphate kinase. The two enzymes exhibited the same activity but could not bind to mitochondrial membranes. In all three systems, muscle creatine phosphokinase was the external agent competing with the oxidative phosphorylation system for ADP. Determination of mitochondrial respiration rate in the presence of increasing quantities of creatine phosphokinase revealed that at large excess of creatine phosphokinase activity over other kinase activities (of the three systems tested) and oxidative phosphorylation the creatine phosphokinase reaction reached a quasi-equilibrium state. Under these conditions equilibrium concentrations of all creatine phosphokinase substrates were determined and K(eq)app of this reaction was calculated for the system with yeast hexokinase. In samples containing active mitochondrial nucleoside diphosphate kinase the concentrations of ATP, creatine, and phosphocreatine were determined and the quasi-equilibrium concentration of ADP was calculated using the K(eq)app value. At balance of quasi-equilibrium concentrations of ADP and ATP/ADP ratio the mitochondrial respiration rate in the system containing nucleoside diphosphate kinase was 21% of the respiration rate assayed in the absence of creatine phosphokinase; in the system containing yeast hexokinase this parameter was only 7% of the respiration rate assayed in the absence of creatine phosphokinase. Substitution of mitochondrial nucleoside diphosphate kinase with yeast nucleoside diphosphate kinase abolished this difference. It is concluded that oxidative phosphorylation is accompanied by appearance of functional coupling between mitochondrial nucleoside diphosphate kinase and the oxidative phosphorylation system. Possible mechanisms of this coupling are discussed.     

Effect of calcium ion on triiodothyronine binding to kidney outer mitochondrial membrane in vitro

The effect of calcium ion on 3,5,3'-triiodothyronine (T3) binding to rat kidney outer mitochondrial membranes was examined in vitro. The outer mitochondrial membranes were prepared by using a discontinuous sucrose density gradient centrifugation. The membrane fraction, which is enriched with monoamine oxidase activity, contained specific binding sites for T3. Scatchard analysis of T3 binding to outer mitochondrial membranes gave an association constant (Ka) of 0.53 X 10(10)M-1. The binding of [125I]-T3 to the membranes was inhibited by the addition of CaCl2(0.25 X 10(-4)--2.5 X 10(-3)M). 50% inhibition was obtained by 0.75 X 10(-4)M CaCl2 in the presence of 0.1 mM EGTA. When outer mitochondrial membranes were solubilized with Triton X-100, four main T3 binding activities were isolated by a gel filtration study. On the other hand, the binding of [125I]-T3 to the solubilized T3 receptors derived from outer mitochondrial membranes was not strongly inhibited by calcium. When outer mitochondrial membranes were preincubated in the presence of 1 mM calcium, the number of T3 binding sites in the membranes was decreased, and this was associated with an increase in the number of T3 binding sites in the supernatants of the incubation mixture. Scatchard analysis showed that the number of T3 binding sites in the membranes is decreased by calcium ion without any change in the association constant. In studies with gel filtration of receptors which are released by Ca2+ from outer mitochondrial membranes, three main T3 binding activities were isolated. Mg2+, Mn2+, Zn2+ and Cu2+ did not affect T3 binding to outer mitochondrial membranes. The results indicate that calcium ion regulates T3 binding to the outer mitochondrial membrane through the release of T3 receptors from the membranes.     

用荧光素磷脂酰乙醇胺直接测定线粒体内膜外表面pH

由磷脂极性头部基团和结合水分子组成的氢键网络有利于质子沿膜表面侧向快速扩散。因而在线粒体氧化磷酸化过程中,与呼吸链电子传递相偶联的跨膜转运质子是否滞留于线粒体内膜外表面即成为一个值得探讨的课题。本文采用荧光素磷脂酰乙醇胺标记于线粒体内膜外表面,首次建立了直接测定线粒体内膜外表面ｐＨ的方法。标记后,线粒体内膜体呼吸控制率,呼吸链电子传递驱动的质子跨膜转移活性及ＡＴＰ合成活性下降了近２８．０％,１１．     

Bax and heart mitochondria: uncoupling and inhibition of respiration without permeability transition

The effects of Bax (full-length, FL, and C-terminal truncated, DeltaC) on respiration rate, membrane potential, MgATPase activity and kinetics of regulation of respiration were studied in isolated rat heart mitochondria and permeabilized cardiomyocytes. The results showed that while both Bax-FL and Bax-DeltaC permeabilized the outer mitochondrial membrane, released cytochrome c and reduced the respiration rate, the latter could be fully restored by exogenous cytochrome c only in the case of Bax-DeltaC, but not in presence of Bax-FL. In addition, Bax-FL but not Bax-DeltaC increased the MgATPase activity, and their effects on the mitochondrial membrane potential were quantitatively different. None of these effects was sensitive to cyclosporin A (CsA).It is concluded that Bax-FL affects both the outer and the inner mitochondrial membranes by: (1) opening large pores in the outer membrane; (2) inhibiting some segments of the respiratory chain in the inner membrane; and (3) uncoupling the inner mitochondrial membrane by increasing proton leak without opening the permeability transition pore (PTP).     

Mitochondrial porin regulates the sensitivity of anion carriers to inhibitors

In mitoplasts, respiratory stimulation by ADP, palmitate, DNP and CCCP and sensitivity of respiration to carboxyatractylate are considerably less pronounced than in mitochondria. Addition of porin-containing preparations (purified outer membranes or solubilized mitochondrial porin) to mitoplasts results in partial restoration of the oxygen consumption and sensitivity to carboxyatractylate (CAT). The uncoupling effect of FCCP in mitoplasts is CAT-resistant and does not depend on added porin. It is suggested that mitochondrial porin may be a natural activator of ADP/ATP antiporter and succinate carrier in mitochondria.     

Photosensitivity of respiration in Neurospora mitochondria. A protective role for carotenoid.

1. The effect of visible light on respiratory activity was studied in two strains of Neurospora crassa, one a wild-type strain able to synthesize carotenoid and the other an albino mutant lacking carotenogenic activity. Light had no effect on growth under the conditions studied, but inhibited respiration of hyphal suspensions. the degree of inhibition being dependent on the carotenoid content of the hyphae. 2. In studies of respiration of isolated mitochondria, three types of photosensitive site were detected. These were the flavo-protein dehydrogenases themselves, a site separate from the latter also associated with the dehydrogenase but re-activatable by treatment with a thiol reagent, and the respiratory quinone, ubiquinone. Cytochrome oxidase, previously reported as photosensitive from many sources, was not appreciably affected by light in these preparations. 3. The degree of inactivation of the respiratory quinone was dependent on the amount of carotenoid in the preparation, high concentrations of the pigment in the mitochondrial membranes providing substantial protection against the effect of light. 4. Separation of the inner and outer membranes of mitochondria showed that under conditions where carotenoid appears to protect quinone, significant amounts are found in the inner mitochondrial membrane, oterhwise carotenoid is restricted to the outer membrane.     

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

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.     

Mitochondrial autonomy. Sialic acid residues on the surface of isolated rat cerebral cortex and liver mitochondria

N-acetylneuraminic acid at the surfaces of rat cerebral cortex and liver mitochondria and derived mitoplasts (inner membrane plus matrix particles) was studied biochemically and electrokinetically. Rat cerebral cortex mitochondria in 0.0145 M NaCl, 4.5% sorbitol, pH 7.2 ± 0.1, 0.6 mM NaHCO₃, had an electrophoretic mobility of - 2.88 ± 0.01 µ/sec per v per cm. In the same solution the electrophoretic mobility of rat liver mitochondria was - 2.01 ± 0.02, of rat liver mitoplasts was - 1.22 ± 0.07, and of rat cerebral cortex mitoplasts - 0.91 ± 0.04 µ/sec per v per cm. Treatment of these particles with 50 µg neuraminidase/mg particle protein resulted in the following electrophoretic mobilities in µ/sec per v per cm: rat cerebral cortex mitochondria, - 2.27; rat liver mitochondria, - 1.40; rat cerebral cortex mitoplasts, - 0.78; and rat liver mitoplasts, - 1.10. Rat liver mitochondria, mitoplasts, and outer mitochondrial membranes contained 2.0, 1.1, and 4.1 nmoles of sialic acid/mg protein, respectively. 10% of the liver mitochondrial protein and 27.5% of the sialic acid was solubilized in the mitoplast and outer membrane isolation procedure. Rat cerebral cortex mitochondria, mitoplasts, and outer mitochondrial membranes contained 3.1, 0.8, and 6.2 nmoles sialic acid/mg protein, respectively; 10% of the brain mitochondrial protein and 49 % of the sialic acid was solubilized in the mitoplast and outer membrane isolation solution procedure. Treatment of both the rat liver and cerebral cortex mitochondria with 50 µg neuraminidase (dry weight) /mg protein resulted in the release of about 50% of the available outer membrane sialic acid residues. Treatment of all of the particles with trypsin caused release of sialic acid but did not greatly affect the particle electrophoretic mobility. In each instance, curves of pH vs. electrophoretic mobility indicated that the particle surface contained an acid dissociable group, most likely a carboxyl group of sialic acid with pK_a ∼ 2.7. Treatment of either the rat liver or the cerebral cortex mitochondria with trypsinized concanavalin A did not affect the particle electrophoretic mobility but did cause a decrease in the electrophoretic mobility of L5178Y mouse leukemic cells.     

Long-chain acyl CoA synthetase,carnitine and β-oxidation in the pea-seed mitochondrion

Mitochondria from pea (Pisum sativum L.) seeds were separated into two fractions, mitoplasts (intact inner membrane) and the outer-membrane fraction. The mitoplasts only oxidised palmitate in the presence of carnitine and added outermembrane fraction. Mitoplasts were able to oxidise palmitoylCoA in the presence of carnitine and added outer-membrane fraction had no effect on this oxidation. It was concluded that a long-chain acylCoA synthetase (EC 6.2.1.3) was located on the outer membrane and that the activity of this enzyme in assays was more than sufficient to account for any observed rate of O₂ uptake during palmitate oxidation by pea mitochondria. The location of carnitine long-chain acyltransferase (carnitine palmitoyl transferase EC 2.3.1.21) would appear to be the mitoplast i.e. the inner mitochondrial membrane, and confirms the previous work at Newcastle.Abbreviation Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Mitochondrial outer membrane permeability change and hypersensitivity to digitonin early in staurosporine-induced apoptosis

We have shown here that the apoptosis inducer staurosporine causes an early decrease in the endogenous respiration rate in intact 143B.TK(-) cells. On the other hand, the activity of cytochrome c oxidase is unchanged for the first 8 h after staurosporine treatment, as determined by oxygen consumption measurements in intact cells. The decrease in the endogenous respiration rate precedes the release of cytochrome c from mitochondria. Moreover, we have ruled out caspases, permeability transition, and protein kinase C inhibition as being responsible for the decrease in respiration rate. Furthermore, overexpression of the gene for Bcl-2 does not prevent the decrease in respiration rate. The last finding suggests that Bcl-2 acts downstream of the perturbation in respiration. The evidence of normal enzymatic activities of complex I and complex III in staurosporine-treated 143B.TK(-) osteosarcoma cells indicates that the cause of the respiration decrease is probably an alteration in the permeability of the outer mitochondrial membrane. Presumably, the voltage-dependent anion channel closes, thereby preventing ADP and oxidizable substrates from being taken up into mitochondria. This interpretation was confirmed by another surprising finding, namely that, in staurosporine-treated 143B.TK(-) cells permeabilized with digitonin at a concentration not affecting the mitochondrial membranes in naive cells, the outer mitochondrial membrane loses its integrity; this leads to a reversal of its impermeability to exogenous substrates. The loss of outer membrane integrity leads also to a massive premature release of cytochrome c from mitochondria. Most significantly, Bcl-2 overexpression prevents the staurosporine-induced hypersensitivity of the outer membrane to digitonin. Our experiments have thus revealed early changes in the outer mitochondrial membrane, which take place long before cytochrome c is released from mitochondria in intact cells.     

呼吸链底物和抑制剂对线粒体内膜流动性的影响

用DPH和ANS标记大鼠肝线粒体内膜,以稳态荧光偏振法,研究了呼吸链底物和抑制剂对内膜流动性的影响。1.苹果酸+谷氨酸、琥珀酸分别为底物,均能引起内膜流动性增加。2.琥珀酸对含心磷脂的脂质体的膜流动性无影响。3.在鱼藤酮存在的条件下,苹果酸+谷氨酸对内膜流动性的增加作用消失,但琥珀酸的作用仍然存在。有氰化钾时则琥珀酸的作用消失。4.不论外加底物存在与否,鱼藤酮使内膜的流动性下降,而氰化钾则使之增加。抗霉素A亦可使内膜的流动性增加。上述结果表明:线粒体内膜流动性与其功能密切相关。电子沿呼吸链传递使线粒体内膜流动性增加,这种变化可能与呼吸链成分的氧化还原态有关。