Creatine kinase and mitochondrial respiration in hearts of trout,cod and freshwater turtle

The importance of the creatine kinase system in the cardiac muscle of ectothermic vertebrates is unclear. Mammalian cardiac muscle seems to be structurally organized in a manner that compartmentalizes the intracellular environment as evidenced by the substantially higher mitochondrial apparent K_m for ADP in skinned fibres compared to isolated mitochondria. A mitochondrial fraction of creatine kinase is functionally coupled to the mitochondrial respiration, and the transport of phosphocreatine and creatine as energy equivalents of ATP and ADP, respectively, increases the mitochondrial apparent ADP affinity, i.e. lowers the K_m. This function of creatine kinase seems to be absent in hearts of frog species. To find out whether this applies to hearts of ectothermic vertebrate species in general, we investigated the effect of creatine on the mitochondrial respiration of saponin-skinned fibres from the ventricle of rainbow trout, Atlantic cod and freshwater turtle. For all three species, the apparent K_m for ADP appeared to be substantially higher than for isolated mitochondria. Creatine lowered this K_m in trout and turtle, thus indicating a functional coupling between mitochondrial creatine kinase and respiration. However, creatine had no effect on K_m in cod ventricle. In conclusion, the creatine kinase-system in trout and turtle hearts seems to fulfil the same functions as in the mammalian heart, i.e. facilitating energy transport and communication between cellular compartments. In cod heart, however, this does not seem to be the case.Abbreviations ACR acceptor control ratio - CK creatine kinase - PCr creatine phosphate - V_ADP ADP-stimulated respiration rate - V_max maximal respiration rate - V₀ respiration rate in the absence of ADPCommunicated by: G. Heidmaier     

The action of bothrops neuwiedii phospholipase A2 on mitochondrial phospholipids and electron transfer

Summary Incubation of heart-muscle mitochondrial fragments (Keilin-Hartree preparation, henceforth KH particles) withBothrops neuwiedii phospholipase A₂ ((EC 3.1.1.4); isoenzyme P-1;Vidal, J. C. et al., Arch. Biochem. Biophys. 151, 168 (1972)) in the presence of Ca²⁺, affected both the phospholipid content and catalytic properties of particles. Phospholipid analysis revealed the hydrolysis of phosphatidylcholine, phosphatidalcholine, phosphatidylethanolamine and phosphatidalethanolamine (with concomitant increase of lysophosphatides); diphosphatidyl glycerol (cardiolipin) was not attacked. Investigation of digested particles with adequate electron acceptors (or donors) showed inhibitions at the NADH-ubiquinone segment and the cytochromeb-c segment of the respiratory chain; a third less sensitive site was at (or near) cytochrome oxidase. In contrast to these inhibitory effects, the activities of succinate dehydrogenase and NADH-ferricyanide reductase were increased. The inhibition of electron transfer was due to the action of the products of phospholipid hydrolysis (unsaturated fatty acids and lysophosphatides) since (a), lipid extracts from phospholipase digested particles inhibited intact particles in much the same manner as phospholipase digestion; (b), unsaturated fatty acids inhibited NADH-oxidase and activated succinate and NADH-dehydrogenases; (c), lysophosphatidyl choline inhibited NADH- and succinate oxidases; (d), washing of digested particles with BSA completely restored NADH-oxidase activity; (e), after extraction of the digested lipids with aqueous acetone, rebinding of ubiquinone and non-digested lipid completely restablished electron transfer from succinate to cytochromec; (f) the content of ubiquinone, cytochromesb andc +c ₁ was not affected by phospholipase digestion. The summarized data taken together are similar to the effects ofB. neuwiedii andCrotalus adamanteus phospholipases on electron transfer particles. On the other hand, the contrast with theNaja naja phospholipase (s) was remarkable.Abbreviations KH particles Keilin-Hartree particles (or preparation) - LPC lysophosphatidyl choline - BSA bovine serum albumin - tlc thin layer chromatography - UQ_o 2,3-dimethoxy-5-methyl-1,4-benzoquinone - K₃H₂ menadiol - PMS phenazine methosulfate     

A durohydroquinone oxidation site in the mitochondrial transport chain

Although duroquinone had little effect upon NADH oxidation in neutral lipid depleted mitochondria, durohydroquinone was oxidized by ETP at a rate sensitive to antimycin A. Fractionation of mitochondria into purified enzyme systems showed durohydroquinone: cytochromec reductase to be concentrated in NADH: cytochromec reductase, absent in succinate:cytochromec reductase, and decreased in reduced coenzyme Q:cytochromec reductase. Durohydroquinone oxidation could be restored by recombining reduced coenzyme Q:cytochromec reductase with NADH:coenzyme Q reductase. Pentane extraction had no effect upon either durohydroquinone or reduced coenzyme Q₁₀ oxidation, indicating lack of a quinone requirement between cytochromesb andc. Both chloroquine diphosphate and acetone (96%) treatment irreversibly inhibited NADH but not succinate oxidation. Neither reagents had any effect upon durohydroquinone oxidation but both inhibited reduced coenzyme Q₁₀ oxidation 50%, indicating a site of action between Q₁₀ and duroquinone sites. Loss of chloroquine sensitive reduced coenzyme Q₁₀ oxidation after acetone extraction suggests two sites for Q₁₀ before cytochromeb.     

Inhibition of electron transfer in the cytochromeb-c 1 segment of the mitochondrial respiratory chain by a synthetic analogue of ubiquinone

A synthetic analogue of ubiquinone, 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, inhibits oxidation of succinate and NADH-linked substrates by rat liver mitochondria. Inhibition occurs both in the presence (state 3) and absence (state 4) of ADP. With isolated succinate-cytochromec reductase complex from bovine heart mitochondria the quinone analogue inhibits succinate-cytochromec reductase and ubiquinol-cytochromec reductase activities but does not inhibit succinate-ubiquinone reductase activity. Inhibition of cytochromec reductase activities is markedly dependent on pH in the range pH 7–8. At pH 7.0 inhibition occurs with an apparentK _i1×10^–8 M, while at pH 8.0 the apparentK _i is more than an order of magnitude greater than this. Spectrophotometric titrations of 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole show a visibly detectable pK _a at pH 6.5 attributable to ionization of the 6-hydroxy group. These results indicate that this quinone derivative is a highly specific and potent inhibitor of electron transfer in theb-c ₁ segment of the respiratory chain. Because of the structural analogy, it is likely that the mechanism of inhibition involves disruption of normal ubiquinone function. In addition, this inhibition depends on protonation of the ionizable hydroxy group of the inhibitory analogue or on protonation of a functional group in theb-c ₁ segment.     

On the role of K+ on succinic dehydrogenase activity

The effect of potassium ions on succinic dehydrogenase activity of mitochondria was studied. The results showed that in these organelles K⁺ induces inhibition of the respiratory control; moreover, in submitochondrial particles potassium inhibits the rate of oxidation of succinate. The results showed also that K⁺ does not changes theK _m for succinate but diminishes theV _max. In addition, the data provide evidence that mitochondria oxidizing glutamatemalate in a sucrose medium show a higher activity of succinate dehydrogenase than mitrochondria incubated in KCl.     

Activation and inhibition of mitochondrial adenosine triphosphatase by various anions and other agents

The activating or inhibiting actions of a variety of anion species and of oligomycin, aurovertin and Dio-9 on the ATPase of a sonic particle preparation of rat liver mitochondria have been characterized by measurements of the relevantV _max,K _i andK _m values.The normalV _max was increased by a factor near 7 by the anions: dichromate, chromate, pyrophosphate, orthophosphate, orthoarsenate and sulphate. The fully activating concentration varied from about 2 mM for dichromate to 150 mM for sulphate. The increase inV _max was accompanied by a time-dependent decrease in (K _i)ADP, but there was no change in (K _m)ATP. The increase inV _max by the activating anions was abolished by aurovertin; but in presence of oligomycin, the lowV _max was increased by the activating anions by the same factor as theV _max in absence of oligomycin.Certain anions, notably azide, decreasedV _max, but did not affect (K _i)ADP or (K _m)ATP. The decrease inV _max by azide and oligomycin were approximately additive. Even at high concentration, Dio-9 was without detectable effect on the ATPase, but it had a gramicidinlike effect on the intact mitochondria.The specificity of the ATPase for ATP relative to GTP was found to be attributable to the high value of (V _max)ATP compared with (V _max)GTP. The values of (K _m)ATP and (K _m)GTP were virtually the same.Some rationalization of these and other supporting observations is attempted in terms of present knowledge of the constitution of the ATPase complex.     

Dissecting the molecular mechanism of ion-solute cotransport: Substrate specificity mutations in theputP gene affect the kinetics of proline transport

Summary Rare mutations that alter the substrate specificity of proline permease cluster in discrete regions of theputP gene, suggesting that they may replace amino acids at the active site of the enzyme. IfputP substrate specificity mutations directly alter the active site of proline permease, the mutants should show specific defects in the kinetics of proline transport. In order to test this prediction, we examined the kinetics of threeputP substrate specificity mutants. One class of mutation increases theK _m over 120-fold but only decreases theV _max fourfold. SuchK _m mutants may be specifically defective in substrate recognition, thus identifying an amino acid critical for substrate binding. Another class of mutation decreases theV _max 80-fold without changing theK _m .V _max mutants appear to alter the rate of substrate translocation without affecting the substrate binding site. The last class of mutation alters both theK _m andV _max of proline transport. These results indicate that substrate specificity mutations alter amino acids critical for Na⁺/proline symport.     

Cytochromec oxidase fromParacoccus denitrificans in Triton X-100: Aggregation state and kinetics

Cytochromec oxidase fromParacoccus denitrificans was homogenously dispersed in Triton X-100. Using gel exclusion chromatography and sucrose gradient centrifugation analysis a molecular weight of the detergent-protein complex of 155,000 was determined. After subtraction of the bound detergent (111 mol/mol hemeaa ₃) a molecular weight of 85,000 resulted, which agreed well with the model of a monomer containing two subunits. This monomer showed high cytochromec oxidase activity when measured spectrophotometrically in the presence of Triton X-100 (V _max=85 s^–1). The molecular activity, plotted according to Eadie-Hofstee, was monophasic as a function of the cytochromec concentration. AK _m of 3.6×10^–6 M was evaluated, similar to theK _m observed in the presence of dodecyl maltoside [Naeczet al. (1985).Biochim. Biophys. Acta 808, 259–272].     

Coenzyme Q-pool function in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria

We have investigated the role of the Coenzyme Q pool in glycerol-3-phosphate oxidation in hamster brown adipose tissue mitochondria. Antimycin A and myxothiazol inhibit glycerol-3-phosphate cytochromec oxidoreductase in a sigmoidal fashion, indicating that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III. The inhibition of ubiquinol cytochromec reductase is linear at low concentrations of both inhibitors, indicating that sigmoidicity of antimycin A and myxothiazol inhibition is not a direct property of antimycin A and myxothiazol binding. Glycerol-3-phosphate cytochromec oxidoreductase is strongly stimulated by added CoQ₃, indicating that endogenous CoQ is not saturating. Application of the pool equation for nonsaturating ubiquinone allows calculation of theK _m for endogenous CoQ of glycerol-3-phosphate dehydrogenase of 3.14mM. The results of this investigations reveal that CoQ behaves as a homogeneous pool between glycerol-3-phosphate dehydrogenase and complex III in brown adipose tissue mitochondria; moreover, its concentration is far below saturation for maximal electron transfer activity in comparison with other branches of the respiratory chain connected with the CoQ pool. HPLC analysis revealed a lower amount of CoQ in brown adipose mitochondria (0.752 nmol/mg protein) in comparison with mitochondria from other tissues and the presence of both CoQ₉ and CoQ₁₀.     

Transport of 3-methoxy-4-hydroxymandelic acid by isolated rat choroid plexus

3-Methoxy-4-hydroxymandelic acid (VMA) is transported into the isolated choroid plexus against a concentration gradient by a saturable, energy-dependent system. The apparentK _m for transport is 35 nM and theV _max is 1 pmol/mg tissue/hr. Concentrations of probenecid (0.1 mM) that block the transport of other acidic biogenic amine metabolites did not block the transport of VMA. The transport system of the choroid plexus probably plays a role in clearing VMA from the CSF.     

A comparative study of the transport of pyruvate in liver mitochondria from normal and diabetic rats

A comparative study of the transport of pyruvate in liver mitochondria from normal and diabetic rats has been carried out. TheK _m for net pyruvate uptake in diabetic, ketotic mitochondria is practically equal to that measured in normal mitochondria, while theV _max is significantly lower. The lower activity of the pyruvate translocator in diabetic mitochondria compared to normal mitochondria is also shown by swelling experiments as well as by following the rate of pyruvate-supported respiration. Pre-exposure of mitochondria from normal rats to the ketone body acetoacetate and to 2-oxobutyrate results in a decrease of theK _m for pyruvate uptake. This effect is impaired in mitochondria from diabetic animals. The results indicate that the activity and the properties of the mitochondrial pyruvate translocator are modified in the diabetic, ketotic condition.Supported by a joint grant from Consiglio Nazionale delle Ricerche, Rome, Italy, and the Polish Academy of Sciences, Warsaw, Poland.     

Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A-insensitive NADH-cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)^?1 min^?1; K_m= 3.1 ± 0.8 μM) or antimycin A-insensitive NADH-ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)^?1 min^?1; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH-cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally-facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol-(β-aminoethylether)-N,N,-tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone-10 analogues, duroquinone and ubiquinone-1, but had no effect on the antimycin A-insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.     

Thyroid Hormone-Induced Alterations in Membrane Structure-Function Relationships: Studies on Kinetic Properties of Rat Kidney Microsomal Na+,K+-ATPase and Lipid/Phospholipid Profiles

The effects of thyroidectomy (Tx) and subsequent treatment with 3,5,3′-triiodothyronine (T₃) or combined replacement therapy (T_R) with T₃ and thyroxine (T₄) on the substrate and temperature kinetics properties of Na⁺,K⁺-ATPase and lipid/phospholipid makeup of rat kidney microsomes were examined. Enzyme activity was somewhat high in the hypothyroid (Tx) animals and increased significantly following T₃ treatment, while T_R treatment caused a decrease. In the Tx and T₃ groups enzyme activity resolved in two kinetic components, while in the T_R group the enzyme showed allosteric behavior up to 0.5 mm ATP concentration. The K _m and V _max values of both the components decreased in Tx animals without affecting the catalytic efficiency. T₃ treatment caused a significant increase in the V _max of both the components, with a significant increase in the catalytic efficiency, while the K _m values were not upregulated. The T_R regimen lowered the K _m and V _max of component II but improved the catalytic efficiency. Thyroid status-dependent changes were also noted in the temperature kinetics of the enzyme. Regression analysis revealed that changes in the substrate and temperature kinetics parameters correlated with specific phospholipid components.     

A correlation between newly induced gene activity and an enhancement of mitochondrial enzyme activity in the salivary glands of Drosophila

A comparison was made between some respiratory characteristics of mitochondria isolated from larval salivary glands of Drosophila hydei displaying chromosome puffs induced by anaerobiosis and mitochondria from non-treated glands. Mitochondria from anaerobically treated glands displayed a K_m of the respiration in the presence of isocitrate (2.4 mM) which is half that of the K_m found in control glands (5.6 mM). The V_max of respiratory activity in the presence of isocitrate is similar for mitochondria of treated and non-treated glands. The apparent V_max of the NADH dehydrogenase (E.C. 1.6.99.3) activity in mitochondria isolated from treated glands was 70% higher than in the control glands. Neither the change in K_m of the respiratory activity in the presence of isocitrate, nor the change in app. V_max of the NADH-dehydrogenase in the anaerobically treated glands was apparent when puff induction occurred in the presence of actinomycin D or cycloheximide in the incubation medium. The present results indicate that the changes in the pattern of active genes (the occurrence of new puffs) may be related with a change in the respiration of isocitrate and a change in NADH-dehydrogenase activity.     

Nystatin affects zinc uptake in human fibroblasts

The mechanism(s) by which zinc is transported into cells has not been identified. Since zinc uptake is inhibited by reducing the temperature, zinc uptake may depend on the movement of plasma membrane micoenvironments, such as endocytosis or potocytosis. We investigated the potential role of potocytosis in cellular zinc uptake by incubating normal and acrodermatitis enteropathica fibroblasts with nystatin, a sterol-binding drug previously shown to inhibit potocytosis. Zinc uptake was determined during initial rates of uptake (10 min) following incubation of the fibroblasts in 50 μg nystatin/mL or 0.1% dimethyl-sulfoxide for 10 min at 37°C. The cells were then incubated with 1 to 30 μM ⁶⁵zinc. Michaelis-Menten kinetics were observed for zinc uptake. Nystatin inhibited zinc uptake in both the normal and AE fibroblasts. Reduced cellular uptake of zinc was associated with its internalization, not its external binding. In normal fibroblasts, nystatin significantly reduced theK _m 56% and theV _max 69%. In the AE fibroblasts, nystatin treatment significantly reduced theV _max 59%, but did not significantly affect theK _m. The AE mutation alone affected theV _max for cellular zinc uptake. The control AE fibroblasts exhibited a 40% reduction inV _max compared to control normal fibroblasts. We conclude that nystatin exerts its effect on zinc uptake by reducing the velocity at which zinc traverses the cell membrane, possibly through potocytosis. Furthermore, the AE mutation also effects zinc transport by reducing zinc transport.     

Influence of 8-azido-ATP and other anions on the activity of cytochromec oxidase

The effect of ATP and other anions on the kinetics of cytochromec oxidation by reconstituted bovine heart cytochromec oxidase was investigated. The following results were obtained: (1) ATP and other polyvalent anions increase theK _m for cytochromec and theV _max (if assayed by the photometric method). The magnitude of the effect is proportional to the charge of the anion as follows from the series of increasing effectiveness: P_iii. (2) The kinetic effects are obtained in the millimolar physiological concentration range. (3) The kinetic changes are not saturated at high concentrations. (4) A specific interaction site for ATP at the cytosolic domain of the enzyme is concluded from the increase ofK _m for cytochromec after photolabelling of proteoliposomes with 8-azido-[-³²P]-ATP, which is protected by ATP but not by ADP. (5) No specific binding site for ATP could be identified by photolabelling with 8-azido-[-³²P]-ATP. The labelling is only partly protected by ATP or ADP.Abbreviations CCP carbonylcyanide-m-chlorophenylhydrazone - TMPD N,N,N,N-tetramethyl-1,4-phenylenediamine dihydrochloride - 8-N₃-ATP 8-azido-adenosine-5-triphosphate Dedicated to Professor Dr. Friedhelm Schneider on the occasion of his 60th birthday.     

The uptake of serotonin and dopamine by homogenates of frozen rat and human brain tissue

A recently described procedure of freezing and thawing, which allows retention of metabolic and functional integrity, has been applied in the study of serotonin and dopamine uptake into frozen rat and post mortem human frozen tissue. TheK _m andV _max for the serotonin uptake into human hypothalamus were estimated to be 0.12 M and 0.03 nmol/g/min respectively. TheK _m andV _max for the dopamine uptake into human putamen were estimated to be 0.28 M and 0.13 nmol/g/min respectively. The results indicate that the freezing procedure does not affect the uptake sites for these transmitters. The storage time before freezing is however of importance for theV _max value. TheK _m value for the uptake, on the other hand, seems to be rather resistant to storage time before freezing.     

Potassium modulation of methionine uptake in astrocytes in vitro

Methionine participates in a large variety of metabolic pathways in brain, and its transport may play an important regulatory role. The properties of methionine uptake were examined in a preparation of neonatal rat brain astrocytes. Uptake is linear for 15 minutes, up to 2.5 M. At steady state conditions, methionine is concentrated 30–50-fold. Measured methionine homoexchange accounts for a significant fraction of uptake at concentrations greater than 10 M. We recently reported that methionine uptake is decreased by elevations in extracellular K⁺. Potassium induced efflux cannot account for this apparent effect; and thus for concentrations less than 2.5M, and for short times of incubation, measured rates of methionine uptake represent unidirectional flux. At extracellular concentrations of K⁺ equal to 6.9 mM, the apparentV _max of methionine transport is 182 pmol/min/mg protein, and theK _m is 1.3 M. Where K⁺ is shifted to 11.9 mM, theK _m remains unchanged, and theV _max is reduced by half.     

Differential exposure of components of cytochromeb−c 1 region in beef heart mitochondria and electron transport particles

The reduction of cyctochromesc +c ₁ by durohydroquinone and ferrocyanide in electron transport particles (ETP) and intact cytochromec-depleted beef heart mitochondria has been studied. At least 94% of the ETP are in an inverted orientation. Durohydroquinone reduces 80% ofc +c ₁ in ETP but less than 20% in mitochondria; sonication of mitochondria allows reduction of cytochromesc +c ₁ (80%). Addition of ferrocyanide (effective redox potential +245 mV) to electron transport particles results in 30% reduction of cytochromesc +c ₁. Addition of ferrocyanide to intact cytochromec-depleted mitochondria does not reduce cytochromec ₁; treatment withN,N,N,N-tetramethylphenylenediamine, Triton X-100, or sonic oscillation results in 30% reduction of cytochromesc +c ₁. TheK _m value of ferrocyanide oxidase for K-ferrocyanide is pH-dependent in ETP only, increasing with increasing pH. The extent of reduction of cytochromec ₁ is also pH-dependent in ETP only, the extent of reduction increasing with decreasing pH. On the basis of these data cytochromec ₁ is exposed to the matrix face and cytochromec is exposed to the cytoplasmic face. No redox center other than cytochromec in the segment between the antimycin site and cytochromec is exposed on the C-side.Abbreviations Used: MES, 2(N-morpholino)-ethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; TMPD,N,N,N,N-tetramethylphenylenediamine; ETP, electron transport particles; NAD-NADH, nicotinamide adenine dinucleotide; PMS, phenazine methosulfate.     

Succinate cytochrome <Emphasis Type="Italic">c</Emphasis> reductase in schistosomiasis: in vitro inhibition by some schistosomicidal drugs

Enzymes in mitochondria play an important role in biological oxidation and energy production. To understand the effect of schistosomiasis on these important processes, succinate cytochrome c reductase (SCR) from control and Schistosoma-infected mice was subjected for investigation. In this article, we report that SCR from Schistosoma-infected mouse showed a significant decrease in its V _max and K _m compared to control using both cytochrome c and 2,6-dichlorophenolindophenol as substrates. Furthermore, the kinetic studies of the purified SCR in the absence and presence of the schistosomicidal drugs praziquantel and Commiphora extract reveal that both drugs have an inhibitory action on the enzyme from the control and Schistosoma-infected mice and praziquantel changes the type of inhibition of SCR towards cytochrome c from mixed type in control to a competitive one in the case of the infection.