Mitochondrial ryanodine‐sensitive Ca2+ channels of rat liver

To examine ryanodine‐sensitive Ca²⁺ channels in mitochondria of rat hepatocytes and their role in energy state of the cells via investigation of the ryanodine effect on mitochondrial membrane potential. Oxygen consumption was measured by polarography using the Clark electrode. The substrates of oxidation such as pyruvate (5mM), α‐ketoglutarate (5mM), or succinate (5mM) were used. Oxidative phosphorylation was stimulated by the addition of adenosine diphosphate (200nM). Mitochondrial membrane potential was measured using a voltage‐sensitive fluorescent probe tetramethylrhodamine‐methyl‐ester (0.1μM) and was analyzed by a flow cytometer. To evaluate the intact mitochondria, we used carbonil cyanide m‐chlorophenyl hydrazone (CCCP, 10μM). Changes in the ionized calcium concentration in rat liver mitochondria were measured using a fluorescent probe Fluo‐4 AM. Effect of ryanodine on oxygen consumption of rat liver mitochondria depends on the oxidation substrate and the incubation time. Oxidation of pyruvate in the presence of ryanodine (0.05μM) decreased the membrane potential of rat liver mitochondria by 38.4%. At higher concentrations, ryanodine (0.1μM or 1μM) led to decrease of membrane potential by 51.7% and 42.8%, respectively. In contrast, oxidation of α‐ketoglutarate in the presence of ryanodine (0.05μM) increased mitochondrial membrane potential by 16.8%. However, at higher concentrations, ryanodine (0.1μM or 1μM) triggered a decreasing of membrane potential by 42.5% and 31.0%, respectively. Therefore, ryanodine at various concentrations (0.05μM, 0.1μM, or 1μM) causes differential effects on Ca²⁺ concentration in the mitochondria matrix under oxidation of pyruvate or α‐ketoglutarate. The data suggest the presence of ryanodine receptors in mitochondrial membrane of rat hepatocytes. Their inhibition with higher concentrations of ryanodine leads to decreasing of intra‐mitochondrial Ca²⁺ concentration and affecting the energy state of mictochondria in hepatocytes.     

The effect of some micronutrients and heavy metals on phosphate absorption by maize root cortex segments

The effect of salts (nitrates, chlorides, and sulfates) of microelements, Cd²⁺, Ni²⁺, and Co²⁺ and the effect of boric acid and ammonium molybdate on phosphate uptake by maize root cortex segments were tested. Higher concentration (0.1 mM) of Cu²⁺ salts caused enhancement of phosphate efflux to the extent that efflux was higher than influx. Inhibitory action on phosphate uptake by maize root cortex segments was exerted by following salts: 0.01 mM Cu²⁺ salts (20–30% inhibition), 0.5 mM ZnSO₄ (9.7%), 0.5 and 0.05 mM ZnCl₂ (34.3% and 20.8%), 0.1 mM salts of Cd²⁺, Ni²⁺, Co²⁺ (35–78%). 1 mM FeS_O4 had significant stimulatory effect (92%) on phosphate uptake. Much weaker stimulatory effect was exerted by 1 mM FeCl₃ (14%), 0.05 mM ZnS_O4 (9.6%), 0.005 mM ZnCla and ZnS_O4 (8.4 and 18.5%) and 0.001 mM CdCl2 and CdS_O4 (20.8 and 12.4%). All other tested salts-salts of Mn²⁺ (0.1 and 0.01 mM), 0.01 and 0.001 mM salts of Co²⁺ and Ni²⁺, 0.001 mM salts of Cu²⁺, 0.001–10 mM boric acid, and 0.001–0.1 mM ammonium molybdate left phosphate uptake unaffected.     

Effects of palmityl coenzyme A and palmitylcarnitine on phosphorylating respiration in heart mitochondria

Glutamate-supported respiration in mitochondria is inhibited by palmityl-CoA in the presence of carnitine. Palmityl-CoA-induced lag phase and depressed state 3 rates increase with increasing ADP. Palmityl-CoA inhibition of state 3 respiration with glutamate shows an increased I₅₀ for palmityl-CoA (three to fourfold) when ADP increases and carnitine is present. ADP alone has a small effect. Glutamate-supported respiration is more profoundly inhibited by palmityl-CoA (+carnitine) than palmityl-CoA oxidation. With palmityl-CoA (+ carnitine) alone, the I₅₀ for palmityl-CoA is two-to threefold greater than when glutamate is also present. Active respiration with palmityl-CoA as substrate demonstrates a 2.5-fold greater apparent affinity for ADP than when glutamate is also present. The kinetics are competitive in both cases. Palmitylcarnitine, above 30 μm, produces inhibition of glutamate-supported respiration, concomitant with mitochondrial swelling and eventual lysis. At 15 μm palmitylcarnitine (minimal swelling), succinate (+ rotenone)-supported respiration decreases with a decrease in K_app for ADP; no effect of 15–20 μm palmitylcarnitine on glutamate-supported respiration is observed. However, palmityl-CoA (+ carnitine)-inhibited respiration with glutamate is further decreased with 15 and 20 μm palmitylcarnitine, i.e., by 13 and 29%, respectively. Inhibition is competitive with ADP. With 3 μm palmitylCoA and 20 μm palmitylcarnitine, a decrease in carnitine (1.5 to 0.25 mm) decreases the apparent K_i for palmityl-CoA from 2.6 to 1.8 μm. The results suggest that glutamate increases the palmityl-CoA available to inhibit adenine nucleotide transport. Inhibition may take place external to the inner membrane. Competition of carnitine and palmitylcarnitine for substrate sites may explain the decreased apparent K_i for palmityl-CoA as carnitine decreases.     

Inhibition of bovine heart Na+, K+-ATPase by palmitylcarnitine and palmityl-CoA.

The activity of a partially purified bovine heart Na⁺,K⁺-ATPase is inhibited by DL- and L- palmitylcarnitine (I₅₀=44–48μM). Palmitylcarnitine with a I₅₀ of 25μM also markedly inhibits K⁺-phosphatase activity. Palmityl-CoA decreases Na⁺,K⁺-ATPase activity, but to a lesser extent (I₅₀=80μM). Both palmitic acid and hexanoic acid produce 10 to 15% inhibition of activity at concentrations of 70μM and 3–5mM, respectively. These free fatty acids protect the enzyme against inhibition by 40μM palmitylcarnitine. However, at 50μM palmitylcarnitine, the protective effect by hexanoic acid is no longer apparent. Addition of 40μM palmitylcarnitine to the Na⁺,K⁺-ATPase in the presence of varying concentrations of palmityl-CoA produces an additive inhibition of enzyme activity, suggesting two different sites on the enzyme susceptible to inhibition by the two ester forms of the fatty acid.     

The regulation of pyruvate dehydrogenase by fatty acids in isolated rabbit heart mitochondria.

The rate of pyruvate oxidation by isolated rabbit heart mitochondria was inhibited by fatty acylcarnitine derivatives. The extent of inhibition by pyruvate oxidation in State 3 was greatest with palmitylcarnitine and only a minimal inhibition was observed with acetylcarnitine, while octanoylcarnitine or octanoate caused an intermediate extent of inhibition. Analyses of the intramitochondrial $\text{ATP}\text{ADP}$ and $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratios under the different conditions of incubation indicated that it is unlikely that changes in either or both of these parameters were the primary negative effectors of the rate of pyruvate oxidation. A positive correlation between the decrease in the rate of pyruvate oxidation and the decrease in the level of free CoASH in the mitochondria was observed. Extraction and assay of the pyruvate dehydrogenase from rabbit heart mitochondria during the time course of the fatty acid-mediated inhibition of pyruvate oxidation indicated that pyruvate dehydrogenase was strongly inactivated when palmitylcarnitine was the fatty acid, while incubation with octanoate and acetylcarnitine resulted in less extensive inactivation of pyruvate dehydrogenase. Measurement of the effects of NADH, NAD⁺, acetyl-CoA, and CoASH on the inactivation of pyruvate dehydrogenase extracted from rabbit heart mitochondria indicated that NADH and acetyl-CoA activated the pyruvate dehydrogenasee kinase while CoASH strongly inhibited the kinase and NAD⁺ was without effect. In addition, palmityl-CoA and octanoyl-CoA had little, if any, effect on the pyruvate dehydrogenase kinase activity. It was observed that palmityl-CoA but not octanoyl-CoA strongly inhibited the activity of the extracted pyruvate dehydrogenase. Hence, it is concluded that (a) decreased mitochondrial CoASH levels, which essentially remove a potent inhibitor of the pyruvate dehydrogenase kinase, (b) possibly a diminished free CoASH supply, which may be utilized as a substrate for the active complex, and (c) direct inhibitory effects of palmityl-CoA on the active form of the pyruvate dehydrogenase complex combine to make palmitylcarnitine a much more potent inhibitor of mitochondrial pyruvate oxidation than shorter chain length acylcarnitine derivatives.     

Palmityl-CoA synthetase activity in the muscle of dystrophic mice.

Palmityl-CoA synthetase activity (acid CoA ligase (AMP), E C 6.2.1.3.) was determined using the radioassay method. The rate of formation of palmityl-CoA under the optimal conditions established was 20 nmoles per mg protein per min for mitochondria and 5.8 nmoles for the 9000 × g supernatant. The activity of palmityl-CoA synthetase in mitochondria from skeletal muscle of dystrophic mice was not significantly different from that obtained in normal littermate controls, whereas the activity of this enzyme in the 9000 × g supernatant fraction from dystrophic muscle preparation was found to be significantly higher than for the corresponding controls. It is concluded that the previously observed decrease in palmitate-1-¹⁴C oxidation in dystrophic muscle mitochondria was not due to a defect in the activation of palmitic acid.     

Effect of monovalent cations on the inhibition by NAD+ of NADH oxidation in submitochondrial particles.

Oxidation of NADH in submitochondrial particles, with O₂ or ferricyanide as electron acceptor, was inhibited by micromolar concentrations of NAD⁺ when measured in 240 mM sucrose or, in a lesser extent, in 120 mM NaCl or LiCl. In 120 mM solutions of either KCl, RbCl, CsCl or NH₄Cl the inhibition by up to 100 μM concentrations of NAD⁺ did not occur. The inhibition observed in the sucrose medium disappeared after solubilization of the particles with detergents and re-appeared when the membranes were reconstituted. The inhibitory effect was potentiated by palmitoyl-CoA. The possibility is discussed that the inhibition of NADH oxidation by low concentrations of NAD⁺ and its release by K⁺, Rb⁺, Cs⁺ and NH₄⁺ depend on the interaction between NAD⁺ and the negatively charged mitochondrial membrane.     

Inhibition of palmitoyl carnitine oxidation by 3-mercaptopropionic acid in mitochondria isolated from tobacco hornworm (Manduca sexta) midgut

Mitochondria were isolated from the posterior region of the midgut of the tobacco hornworm, Manduca sexta. Measurements of mitochondrial oxygen consumption revealed that the oxidation of palmitoyl carnitine plus malate was inhibited by 3-mercaptopropionic acid (MPA) in a dose-dependent manner. The maximal percent inhibition was 65% and the I₅₀ was 0.15mM. When exposed to a dose which maximally inhibits the oxidation of palmitoyl carnitine (0.5 mM), mitochondrial oxidation of octanoate and pyruvate were inhibited by 30 and 8%, respectively. Oxidation of succinate was unaffected under these conditions. These results indicate that MPA is an effective inhibitor of fatty acid oxidation in midgut mitochondria.     

Ca2+ transport in mitochondria of the ciliate protozoan Tetrahymena pyriformis

Mitochondria isolated from the late-exponential non-shaken culture of the ciliate protozoan Tetrahymena pyriformis GL was investigated. The presence of energy-dependent Ca²⁺ transport system was shown. In the main the properties of this system have been essentially the same as in mitochondria of vertebrate organisms. The isolated mitochondria contained 23±5 ng-ion Ca²⁺ per mg of protein. The intramitochondrial free concentration of Ca²⁺ was measured in the presence of uncoupler FCCP with the use of fluorescent Ca²⁺ chelator chlortetracycline and null point titration method. In the absence of phosphate, free [Ca²⁺] varied from 1 to 2.5 mM depending on the internal Ca²⁺ content. In the presence of 2 mM phosphate, free [Ca²⁺]_in has not exceeded 0.1–0.3 mM. It was shown that ruthenium red and Mg²⁺ in different manner have an inhibitory effect on Ca²⁺ transport. Besides this, Mg²⁺ also has a stabilizing effect on mitochondria, possibly, by preventing passive ions leaks across the membrane.     

Effect of oxidative processes in mitochondria on contractility of heart muscle of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis>. Actions of Cd<Superscript>2+</Superscript>

The inotropic Cd²⁺ action on frog heart is studied with taking into account its toxic effects upon mitochondria. Cd²⁺ at concentrations of 1, 10, and 20 mM is established to decrease dose dependently (21.3, 50.3, and 72.0%, respectively) the muscle contraction amplitude; this is explained by its competitive action on the potential-controlled Na²⁺-channels of the L-type (Ca_v 1.2). In parallel experiments on isolated rat heart mitochondria (RHM) it was shown that Cd²⁺ at concentrations of 15 and 25 mM produces swelling of non-energized and energized mitochondria in isotonic (with KNO₂ and NH₂NO₃) and hypoosmotic (with 25 mM CH₃COOK) media. Study of oxidative processes in RHM by polarographic method has shown 20 mM Cd²⁺ to disturb activity of respiratory mitochondrial chain. The rate of endogenous respiration of isolated mitochondria in the medium with Cd²⁺ in the presence of malate and succinate was approximately 5 times lower than in control. In experimental preparations, addition into the medium of DNP—uncoupler of oxidation and phosphorylation did not cause an increase of the oxygen consumption rate. Thus, the obtained data indicate that a decrease in the cardiac muscle contractility caused by Cd²⁺ is due not only to its direct blocking action on Ca²⁺-channels, but also is mediated by toxic effect on rat heart mitochondria, which was manifested as an increase in ion permeability of the inner mitochondrial membrane (IMM), acceleration of the energy-dependent K⁺ transport into the matrix of mitochondria, and inhibition of their respiratory chain.     

Cytoplasmic streaming and ionic regulation inNitella flexilis having artificial cell saps of low ionic strength

The cell sap of the internode ofNitella flexilis was replaced with the isotonic artificial pond water of high Ca²⁺-concentration (0.1 mM KCl, 0.1 mM NaCl, 10 mM CaCl₂ and 275 mM mannitol) and changes in osmotic value and concentrations of K⁺, Na⁺ and Cl⁻ of the cells were followed. When the operated cells were incubated in the artificial pond water containing 0.1 mM each of KCl, NaCl, CaCl₂, they survived for only a short period of time (<10 hr). The cells did not absorb ions from the artificial pond water and showed a conspicuous decrease in the rate of cytoplasmic streaming. In such cell the concentration of K⁺ in the protoplasm decreased significantly. In order to reverse normal concentration gradients of K⁺ and Na⁺ across the protoplasmic layer, the cells of low vacuolar ionic concentrations were incubated in the artificial cell sap (90 mM KCl, 40 mM NaCl, 15 mM CaCl₂, 10 mM MgCl₂). It was found that the cells rapidly absorbed much K⁺, Na⁺ and Cl⁻ and survived for a longer period (1–2 days). During this period the rate of cytoplasmic streaming was nearly normal. Furthermore, the cell lost much mannitol, indicating an enormous increase in permeability to it. Since both absorption of ions and leakage of mannitol at 1 C occurred at nearly the same rates as at 22 C, the processes are assumed to be passive.     

Action of La3+ on the systems providing contractility of vertebrate myocardium

The inotropic action of La³⁺ on frog myocardium was studied with taking into account its effect on mitochondria of cardiomyocytes (CM). It has been established that in the range of studied concentrations (0.2–6.0 mM), La³⁺ decreases dose-dependently the force of cardiac contractions (by 3.3–92.2%). In parallel experiments on isolated rat heart mitochondria (RHM), La³⁺ at a concentration of 25 μM has been shown to cause swelling of non-energized and energized mitochondria incubated in isotonic medium with 125 mM NH₄NO₃ and in hypotonic medium with 25 mM CH₃COOK. The study of oxidative processes in mitochondria with aid of polarographic method of measurement of oxygen concentration has shown that La³⁺ at concentrations of 50 and 100 μM increases the oxygen consumption rate by mitochondria in the state 2. However, La³⁺ does not decrease the respiration rate of isolated mitochondria in the state 3, as this takes place in the case of use of Cd²⁺ or at the Ca²⁺-overloading of mitochondria. The rate of endogenous respiration of isolated mitochondria in the medium with La³⁺ was higher than in control, which suggests its effect on ion permeability of the inner membrane. The data obtained in this work indicate that the La³⁺-produced decrease of contractility of cardiac muscle is not only due to the direct blocking effect on the potential-controlled Ca²⁺-channels, but is also mediated by its unspecific action on the CM mitochondria. This action is manifested as an acceleration of the energy-dependent K⁺ transport in matrix and as an increase of ion permeability of the inner mitochondrial membrane (IMM).     

Isolation and partial characterization of rat brain synaptic plasma membranes

Abstract— Synaptic plasma membranes from the cortices of adult rat brain were isolated from synaptosomes prepared by flotation of a washed mitochondrial pellet (P2) in a discontinuous Ficoll-sucrose gradient. Contamination of the synaptosome fraction by microsomes was estimated by enzymic and chemical analysis to be less than 15 per cent. (2) The purified synaptosome fraction was subjected to osmotic shock, subfractionated on a discontinuous sucrose gradient and the distribution of enzymic and chemical markers for synaptic plasma membranes, microsomal membranes and mitochondria was determined. (3) Comparison of synaptosome subfractions prepared in the presence and absence of 1 mM NaH₂ PO₄/0.1 mM EDTA buffer pH 7.5, indicated that the ionic composition of the isolation medium markedly affected the distribution and enzymic composition of the subfractions. (4) Synaptic plasma membranes prepared in the presence of PO₄/EDTA exhibited a 10-fold enrichment in [Na⁺+ K⁺] ATPase and were characterized by less than 15 and 10 per cent contamination by microsomes and mitochondria respectively. (5) The polypeptide composition of the purified synaptic plasma membranes was compared with the microsomes and mitochondria by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. No differences between the protein and glycoprotein composition of the synaptic plasma membranes and microsomes were detected. The mitochondria, in contrast, possessed a unique protein composition.     

Cyclic AMP induced inhibition of pyruvate kinase flux in the intact liver cell.

The rate of pyruvate kinase flux in the intact cell is estimated by a new procedure, involving trapping of ¹⁴C from NaH¹⁴CO₃ in a large pyruvate + lactate pool, and calculation of the specific activity of phosphoenol pyruvate. With high concentrations of pyruvate as substrate for isolated rat liver cells, cyclic AMP (0.1 mM) depresses pyruvate kinase flux by about 45%, in addition to inhibiting both glucose and lactate formation. The inhibition of pyruvate kinase may cause an inhibition of hydrogen translocation from the mitochondria to the cytosol.     

Involvement of cation channels and NH4+-sensitive K+ transporters in Na+ uptake by cowpea roots under salinity

Na⁺ accumulation was investigated in the roots of 11-d-old cowpea [Vigna unguiculata (L.) Walp.] plants. The relative contribution of different membrane transporters on Na⁺ uptake was estimated by applying Ca²⁺, K⁺, NH₄ ⁺, and pharmacological inhibitors. Na⁺ accumulation into the root symplast was decreased by half in the presence of 1 mM Ca²⁺ and it was almost abolished by 100 mM K⁺. The inhibitory effect of external NH⁴⁺ on Na⁺ accumulation was more pronounced in the roots of NH₄ ⁺-free growing plants. Na⁺ accumulation was reduced about 73 % by 0.1 mM flufenamate and it was almost blocked by 2 mM quinine. In addition, 20 mM tetraethylammonium and 1.0 mM Cs⁺ decreased Na⁺ accumulation by 28 and 30 %, respectively. These results evidenced that low-affinity Na⁺ uptake by cowpea roots depends on Ca²⁺-sensitive and Ca²⁺-insensitive pathways. The Ca²⁺-sensitive pathway is probably mediated by nonselective cation channels and the Ca²⁺-insensitive one may involve K⁺ channels and to a lesser extent NH₄ ⁺-sensitive K⁺ transporters.     

Phosphorylation of deoxyguanosine in intact and fractured mitochondria

The phosphorylation of deoxyguanosine was measured in fractured and intact mitochondria and an apparent K_m of 16 M for deoxyguanosine was calculated using fractured mitochondria. The effects of various deoxynucleotides on the phosphorylating activity in fractured organelles was tested at both a high and low ratio of NXP/ATP and at two pH values, 7.0 and 5.5. Exogenous dGTP, dGDP or dITP were inhibitory under all conditions tested. With a NXP/ATP ratio of 0.08 at pH 7.0, TTP, TDP, dADP, ADP, UTP and UDP were stimulatory, but at pH 5.5 only TTP elicited that response. When the NXP/ATP ratio was 10 at pH 5.5, TTP and UTP increased the activity more than 10-fold, whereas, at pH 7.0 TTP, TDP, dADP, ADP, UTP, UDP caused stimulation, but to a much lesser extent. When exogenous Mg²⁺, Mn²⁺ or Ca²⁺ were added to intact mitochondria, the rates of phosphorylation were lowered. In fractured mitochondria in the absence of exogenous ATP, little phosphorylation occurs, hence these metal ions caused little change. ATP-Mg, ATP-Mn and ATP-Ca, each at 0.05 mM caused a small inhibition with intact mitochondria, whereas, these compounds supported phosphorylation with fractured organelles. ATP-Mn (10 mM) or ATP-Ca (10 mM) stimulated phosphorylation in both intact and fractured mitochondria. Intact mitochondria synthesized dGMP, dGDP and dGTP when metal ion or ATP-Me concentrations were low (0.05 mM) or when Mg²⁺ concentration was high (10 mM). Additions of ATP-Ca, ATP-Mn, ATP-Mg, Mn²⁺ or Ca²⁺ at 10 mM cause the loss of dGDP and dGTP formation and, in most cases, an increase in the synthesis of dGMP. Fractured mitochondria make only dGMP and the levels of its synthesis are greater than that observed for intact mitochondria. These data suggest that intact mitochondria are required for the synthesis of dGTP and that its synthesis is regulated by mitochondria nucleotides.     

Glutamate dehydrogenase from Pisum sativum L.

A 2–8-fold increase in the activity of glutamate dehydrogenase (GDH), accompanied by an alteration of the GDH isoenzyme pattern, was observed in detached pea shoots floated on tap water (preincubated shoots). Sugars supressed the process, whereas NH ₊ ⁴ and various metabolites as well as inhibitors of energy metabolism and protein synthesis were ineffective. The subcellular distribution pattern revealed evidence that the GDH isoenzymes are exclusively located in the mitochondrial matrix. The alterations in GDH activity occurring in preincubated shoots are restricted to the mitochondria.An experimental device suitable for studying the GDH function in isolated intact mitochondria has been established. Using [¹⁴C] citrate as the carbon source and hydrogen donor, the mitochondria synthesized considerable amounts of glutamate upon addition of NH ₊ ⁴ . The rates of glutamate formation in dependency of increasing NH ₊ ⁴ levels follow simple Michaelis-Menten kinetics. Half-saturation concentrations of NH ₊ ⁴ of 3.6±1.2 mM; 1.9±0.06 mM and 1.6±0.1 mM were calculated for the mitochondria isolated from pea shoots, roots, and preincubated shoots, respectively. The results are discussed in relation to the possible role of GDH in NH_+/4 assimilation at elevated intracellular NH_+/4 levels.Abbreviations GDH Glutamate dehydrogenase - MDH malate dehydrogenase - GOT aspartate aminotransferase - SDH succinate dehydrogenase - HEPES 4-(2-hydroxyethyl)-1-piperazineethan-sulfonic acid - BSA bovine serum albumin - TPP thiamine pyrophosphate - DNP 2,4-dinitrophenol - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCPIP 2,6-dichlorophenolindophenol Dedicated to Professor Dr. Maximilian Steiner on the occasion of his 75th birthday     

Kinetic advantage of the interaction between the fatty acid β-oxidation enzymes and the complexes of the respiratory chain

Respiration-linked oxidation of 3-hydroxybutyryl-CoA, crotonyl-CoA and saturated fatty acyl (C₄, C₈ and C₁₄)-CoA esters was studied in different mitochondrial preparations. Oxidation of acyl-CoA esters was poor in intact mitochondria; however, it was significant, as well as, NAD⁺ and CoA-dependent in gently and in vigorously sonicated mitochondria. The respiration-linked oxidation of crotonyl-CoA and 3-hydroxybutyryl-CoA proceeded at much higher rates (over 700%) in gently disrupted mitochondria than in completely disrupted mitochondria. The redox dye-linked oxidation of crotonyl-CoA (with inhibited respiratory chain) was also higher in gently disrupted mitochondria (149%) than in disrupted ones. During the respiration-linked oxidation of 3-hydroxybutyryl-CoA the steady-state NADH concentrations in the reaction chamber were determined, and found to be 8 μM in gently sonicated and 15 μM in completely sonicated mitochondria in spite of the observation that the gently sonicated mitochondria oxidized the 3-hydroxybutyryl-CoA much faster than the completely sonicated mitochondria. The NAD⁺-dependence of 3-hydroxybutyryl-CoA oxidation showed that a much smaller NAD⁺ concentration was enough to half-saturate the reaction in gently disrupted mitochondria than in completely disrupted ones. Thus, these observations indicate the positive kinetic consequence of organization of β-oxidation enzyme in situ. Respiration-linked oxidation of bytyryl-, oxtanoyl- and palmitoyl-CoA was also studied and these CoA intermediates were oxidized at approx. 50% of the rate of crotonyl- and 3-hydroxybutyryl-CoA in the gently disrupted mitochondria. In vigorously disrupted mitochondria the oxidation rate of these saturated acyl-CoA intermediates was hardly detectable indicating that the connection between the acyl-CoA dehydrogenase and the respiratory chain had been disrupted.     

Effect of ethephon and daminozide on the respiration of isolated leaf cells

A combined foliar application of ethephon (2-chloroethylphosphonic acid) at 0.8 kg/ha and daminozide (butanedioic acid mono (2,2 dimethylhydrazide) at 3.2 kg/ha inhibited the vegetative growth of Black Valentine bean (Phaseolus vulgaris L.) without the leaf chlorosis and necrosis caused by ethephon alone. This antagonistic interaction was further evaluated by examining the effect of ethephon and daminozide on respiration and lipid synthesis of isolated leaf cells. Ethephon (1.0 mM) promoted¹⁴CO₂ evolution from cells incubated with¹⁴C-glucose for 14 h by approximately 75%. Characterization of this response with Black Valentine bean mitochondria indicated that the observed stimulation could not be attributed to the existence of a major cyanide insensitive pathway or the possibility of ethephon acting as an uncoupler, which supports the view that ethephon (or ethylene) acts in the cytosol rather than in mitochondria. Daminozide at 30.0 and 60.0 mM inhibited¹⁴CO₂ evolution of isolated cells by 30 and 70%, respectively. Ethephon in combination with daminozide (1.0+60 mM) resulted in a 32% inhibition of respiration. Daminozide (60.0 mM) inhibited the incorporation of¹⁴C-glucose into chloroform-methanol soluble products by 47%, but did not affect the incorporation of¹⁴C-acetate. The results suggest that daminozide may reduce or overcome any stimulatory effect of ethephon on respiration and support an active inhibitory site for daminozide in mitochondria.     

Formation of palmityl-[13'-32P]coenzyme A from [gamma-32P]ATP in mitochondrial extracts of guinea pig liver.

Investigations of the incorporation of ³²P into acyl-coenzyme A (CoA) in incubation mixtures containing a soluble protein preparation derived from mitochondria, [γ-³²P]ATP, and palmityl-CoA have led to the discovery of an enzymatic activity which catalyzes the exchange of palmityl groups between molecules of CoA: CoA¹ + palmityl-CoA ? palmityl-CoA¹ + CoA. The preparation also contains dephospho-CoA kinase and palmityl-CoA thiolester hydrolase activities. The initial detection of the exchange reaction resulted from the formation of [3′-³²P]CoA via the dephospho-CoA kinase reaction with exogenous [γ-³²P]ATP. The described preparation of palmityl-[3′-³²P]CoA and palmityl-[³⁵S]CoA facilitated demonstration of the reversibility of the reaction and ruled out the possibility that the exchange of fragments of the CoA molecule mediated the observed incorporation. The reversible palmityl group exchange does not appear to be catalyzed by a previously described enzyme. None of the possible acyl group acceptors considered in these studies participated in the reaction as efficiently as CoA itself. The possibility is discussed that the exchange reaction may explain reports of an unknown lipid formed by an oligomycin-sensitive mitochondrial ATPase preparation.