Effect of Adenine Nucleotides on the Respiration of Carrot Root Slices

Sodium pyruvate and dinitrophenol stimulated O(2) uptake of freshly cut phloem parenchyma from carrot roots by 63 and 120% at optimal concentrations, indicating that production of pyruvate by glycolysis regulates over-all respiratory rate. Adding 0.5 to 6.7 mm Na(3)ADP and Na(3)ATP to slices rapidly stimulates respiration rate by 20 to 85%. The effect is greater at the lower end of this concentration range and is not due to change in pH or active cation uptake. It is suggested that treating tissue with both nucleotides stimulates pyruvate kinase, the rate-limiting step in respiration of freshly cut slices, by increasing the concentration of endogenous ADP. Adenosine diphosphate continued to stimulate O(2) uptake until the peak of induced respiration, but ATP inhibited respiration during development and decline of this peak. Absence of respiratory stimulation by NaH(2)PO(4) and of respiratory inhibition by added nucleosides confirms that inorganic phosphate is not a limiting factor of respiration in freshly cut slices. The stimulation of respiration rate of these slices by dinitrophenol is consistent with results from experiments in which ADP and ATP were applied to the tissue.     

Analysis of adenine nucleotides at the picomole level with 32P-phosphoenolpyruvate and pyruvate kinase.

A new sensitive method for adenine nucleotide analysis is described. The key reaction is the phosphorylation of ADP by [³²P]PEP in a reaction catalyzed by pyruvate kinase, with the extent of transfer of ³²P to ADP being determined by adsorbing the nucleotides onto charcoal. The nonadenine nucleoside diphosphates which also react in the pyruvate kinase reaction are corrected for by determining the ³²P retained in the nucleotide fraction after a second incubation with hexokinase and excess glucose. ATP is determined as ADP, after it is quantitatively converted by hexokinase in the presence of excess glucose. Similarly, AMP is analyzed by its conversion to ADP in an incubation with excess ATP and adenylate kinase. The sensitivity of the method for ADP and ATP is 0.05–0.5 pmoles while for AMP it is 5 pmoles.     

Regulation of oxidative phosphorylation in mitochondria of epididymal bull spermatozoa

The regulation of oxidative phosphorylation was studied with digitonin-treated epididymal bull spermatozoa in which mitochondria are directly accessible to low molecular compounds in the extracellular medium. Due to the high extramitochondrial ATPase activity in this cell preparation, it was possible to stimulate respiration to a small extent only by added hexokinase in the presence of glucose and adenine nucleotides. Added pyruvate kinase plus phosphoenol pyruvate, however, strongly suppressed the respiration. Under these conditions, the respiration was found to depend on the extramitochondrial [ATP]/[ADP] ratio in the range of 1-100. The contribution of the adenine nucleotide translocator to this dependence was determined by titration with the irreversible inhibitor carboxyatractyloside in the presence of ADP. Using lactate plus malate as substrate, the active state respiration was controlled to about 30% by the translocator, whereas 12 and 4% were determined in the presence of L-glycerol-3-phosphate and malate alone, respectively. In order to compare the results with those for intact cells, the adenine nucleotide patterns were determined in intact and digitonin-treated spermatozoa under conditions of controlled respiration in the presence of vanadate and carboxyatractyloside, respectively. About 21% of total cellular adenine nucleotides were found in digitonin-treated cells representing the mitochondrial compartment. While allowing for the intramitochondrial amount of adenine nucleotides, the cytosolic [ATP]/[ADP] ratio was estimated to be 6-times higher than the mitochondrial ratio in intact cells. It is concluded from the data presented that the principal mechanism by which oxidative phosphorylation in sperm mitochondria is regulated via the extramitochondrial [ATP]/[ADP] ratio is the same as that demonstrated for other isolated mitochondria.     

Dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space of rat-heart mitochondria

To investigate whether or not the mitochondrial intermembrane space together with the extramitochondrial space form a homogeneous pool for adenine nucleotides, rat-heart mitochondria were studied in reconstituted systems with pyruvate kinase and ADP-producing enzymes with varied localization. In the hexokinase system, ADP is produced extramitochondrially by added yeast hexokinase, whereas in the creatine kinase system mitochondrial creatine kinase is responsible for ADP regeneration in the intermembrane space. The dependence of mitochondrial respiration on the extramitochondrial [ATP]/[ADP] ratio in both systems was investigated experimentally and by means of computer simulation. Near the resting state, higher [ATP]/[ADP] ratios were found in the creatine kinase system than in the hexokinase system at the same rate of respiration. This and the maintaining of a substantial creatine kinase-stimulated respiration in the presence of pyruvate kinase in excess is explained by a two-compartment model considering diffusion limitations of adenine nucleotides. A diffusion rate constant of (8.7 +/- 4.7) 10(4) microliters X mg-1 X min-1 for ADP and ATP was estimated, resulting in rate-dependent concentration differences up to 13.7 microM AdN between the extramitochondrial space and the AdN-translocator at the maximum rate of oxidative phosphorylation of rat-heart mitochondria. The results support the assumption that ADP diffusion towards the AdN-translocator is limited if its extramitochondrial concentration is low, resulting in a dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space.     

The relation of sodium and potassium ion transport to the respiration and adenine nucleotide content of liver slices treated with inhibitors of respiration

1. The dependence of the net transport of Na(+) and K(+) by rat liver on the respiration has been determined by incubating slices in the presence of varying concentrations of respiratory inhibitors. 2. Neither the rate of net transport nor the total amount of each ion transported was inhibited unless the rate of endogenous respiration was decreased below a critical value of about 330mmol of O(2)/h per kg of protein (i.e. 50% of the total endogenous respiration). 3. The uninhibited rate of respiration could be varied over a twofold range (380-770mmol of O(2)/h per kg of protein) by the use of different substrates, but the critical value for the onset of transport inhibition was quite constant (290-360mmol/h per kg of protein) under these different conditions. 4. Slices incubated at 38 degrees C without inhibitors showed an increase of their ATP content and the concentration ratio ATP/ADP. The final ATP content and concentration ratio, ATP/ADP, of slices treated with different concentrations of inhibitors were closely related to the rate of respiration. 5. The increased ATP content of the control slices during incubation was equal to the increase of total adenine nucleotides. At increasing degrees of respiratory inhibition the relative contributions of ADP and AMP to the total adenine nucleotide content increased. 6. The critical rate of respiration for the onset of inhibition of ion transport and the corresponding contents of adenine nucleotides provide estimates of the maximal values of certain parameters of energy metabolism required for the support of alkali-cation transport in the liver slices.     

Radioisotopic assay of femtomole quantities of total adenine nucleotides,ATP plus ADP,and AMP

AMP is converted to ATP by incubating overnight with pyruvate kinase, phosphoenolpyruvate and adenylate kinase in th prensence of endogenous ATP (ADP) as primer. In a subsequent incubation in the presence of pyruvate kinase, phosphoenolpyruvate, radioactive glucose and hexokinase. ATP and ADP are estimated together by coupling their recycling to the formation of glucose 6-phosphate. The latter is separated by precipitation using 76% (v/v) acetone for radioactivity measurement in the same Eppendorf tube. The sensitivity of these simple procedures matches or exceeds those of luciferase methods of nucleotide determination.     

Control of pyruvate dehydrogenase activity in intact cardiac mitochondria. Regulation of the inactivation and activation of the dehydrogenase.

The control of pyruvate dehydrogenase activity by inactivation and activation was studied in intact mitochondria isolated from rabbit heart. Pyruvate dehydrogenase could be completely inactivated by incubating mitochondria with ATP, oligomycin, and NaF. This loss in dehydrogenase activity was correlated with the incorporation of 32P from [gamma-32P]ATP into mitochondrial protein(s) and with a decrease in the mitochondrial oxidation of pyruvate. ATP may be supplied exogenously, generated from endogenous ADP during oxidative phosphorylation, or formed from exogenous ADP in carbonyl cyanid p-trifluoromethoxyphenylhydrazone-uncoupled mitochondria. With coupled mitochondria the concentration of added ATP required to half-inactivate the dehydrogenase was 0.24 mM. With uncoupled mitochondria the apparent Km was decreased to 60 muM ATP. Inactivation of pyruvate dehydrogenase by exogenous ATP was sensitive to atractyloside, suggesting that pyruvate dehydrogenase kinase acts internally to the atractyloside-sensitive barrier. The divalent cation ionophore, A23187, enhanced the loss of dehydrogenase activity. Pyruvate dehydrogenase activity is regulated additionally by pyruvate, inorganic phosphate, and ADP. Pyruvate, in the presence of rotenone, strongly inhibited inactivation. This suggests that pyruvate facilitates its own oxidation and that increases in pyruvate dehydrogenase activity by substrate may provide a modulating influence on the utilization of pyruvate via the tricarboxylate cycle. Inorganic phosphate protected the dehydrogenase from inactivation by ATP. ADP added to the incubation mixture together with ATP inhibited the inactivation of pyruvate dehydrogenase. This protection may result from a direct action on pyruvate dehydrogenase kinase, as ADP competes with ATP, and an indirect action, in that ADP competes with ATP for the translocase. It is suggested that the intramitochondrial [ATP]:[ADP] ratio effects the kinase activity directly, whereas the cytosolic [ATP]:[ADP] ratio acts indirectly. Mg2+ enhances the rate of reactivation of the inactivated pyruvate dehydrogenase presumably by accelerating the rate of dephosphorylation of the enzyme. Maximal activation is obtained with the addition of 0.5 mM Mg2+..     

Rapid Metabolic Changes in the Wounding Response of Leaf Discs following Excision

The dark respiration rate of discs from fully expanded tobacco leaves (Nicotiana tabacum) increased linearly with decreasing diameter, the relative increase being independent of leaf age. The wound respiration responsible for this situation reached a plateau within 15 minutes of excision. Metabolite analysis gave evidence for two independent effects, also unrelated to age. The first was a forward crossover between phosphoenolpyruvate and pyruvate which was found as early as 1 minute after excision and persisted for up to 40 minutes. It was attributed to activation of pyruvate kinase by a changed ionic balance resulting from membrane damage, was accompanied by a reverse crossover between triose phosphates and 3-phosphoglycerate, and was localized in the outer region of the discs. The second effect was a rapid rise in hexose monophosphate and ATP levels throughout the discs. After 1 to 10 minutes the ATP/ADP ratio rose strongly for at least 3 hours; after 20 to 40 minutes there was net synthesis of adenine nucleotide as ATP. These results indicate that extrapolation from leaf discs to intact leaves is highly inadvisable.     

Changes in intramitochondrial adenine nucleotides in blowfly flight-muscle mitochondria

1. With freshly isolated blowfly mitochondria 38% of the intramitochondrial adenine nucleotide was present as AMP. 2. On incubation with oxidizable substrates the AMP and ADP concentrations fell and that of ATP rose; with pyruvate together with proline the ATP concentration reached its maximum value at 6min; with glycerol phosphate the phosphorylation of endogenous nucleotide was more rapid. 3. Addition of the uncoupling agent carbonyl cyanide phenylhydrazone caused a rapid fall of ATP and a parallel rise in ADP, then ADP was converted into AMP. 4. This was in contrast with rat liver mitochondria endogenous AMP concentrations, which were always lower than those of blowfly mitochondria and changed little under different metabolic conditions. 5. Evidence is presented that adenylate kinase (EC 2.7.4.3) has a dual distribution in blowfly mitochondria, a part being located in the matrix space and a part in the space between the outer and inner mitochondrial membranes, as in liver and other mitochondria. 6. The possible regulatory role of changing AMP concentrations in the mitochondrial matrix was investigated. Partially purified pyruvate carboxylase (EC 6.4.1.1) and citrate synthase (EC 4.1.3.7) were inhibited 30% by 2mm-AMP, whereas pyruvate dehydrogenase (EC 1.2.4.1) was unaffected. 7. AMP activated the NAD(+)-linked isocitrate dehydrogenase (EC 1.1.1.41) activity of blowfly mitochondria in the absence of ADP, but in the presence of ADP, AMP caused inhibition. 8. It is suggested that AMP may exert a controlling effect on the oxidative activity of blowfly mitochondria.     

Histochemical technique for the demonstration of pyruvate kinase activity

We describe an enzyme histochemical multistep technique for the demonstration of pyruvate kinase activity. In this technique, a semipermeable membrane is interposed between the incubation medium and the tissue sections, thus preventing diffusion of the enzyme into the medium during the incubation period. In this histochemical system, phosphoenolpyruvate (PEP) donates its phosphate group to ADP in a reaction catalysed by pyruvate kinase. Next, exogenous and endogenous hexokinase catalyses the reaction between ATP and D-glucose to yield D-glucose-6-phosphate and ADP. The D-glucose-6-phosphate is oxidized by exogenous and endogenous D-glucose-6-phosphate dehydrogenase, and concomitantly, the generated electrons are transported via NADP+, phenazine methosulphate and menadione to nitro-BT, which is finally precipitated as formazan. Sodium azide and amytal are included to block electron transfer to cytochromes. The method proved to be of value for the qualitative demonstration of pyruvate kinase activity in tissue sections of kidneys, heart muscle and skeletal muscle. For quantitative studies and for investigating the activity of this enzyme in liver sections, the method cannot be recommended.     

Mechanism of peroxide-induced cellular injury in cultured adult cardiac myocytes

Reactive oxygen species contribute to the tissue injury seen after reperfusion of ischemic myocardium. We propose that toxicity originates from the effect that mitochondrial peroxide metabolism has on substrate entry into oxidative pathways. To support our contention, cultured adult rat cardiomyocytes were incubated with physiological concentrations of peroxide. The cellular extract and incubation medium were analyzed for adenine nucleotides and purines by reverse-phase high-pressure liquid chromatography. Cellular glutathione efflux was determined by enzymatic analysis of the incubation medium. Pyruvate dehydrogenase (PDH) activity was determined in the cultured myocytes as well as in freshly isolated cardiac mitochondria using [1-C14]pyruvate. Extracellular glutathione rose 3.3-fold in response to small doses of peroxide (approximately 108 nmol/mg protein). Likewise, small quantities of peroxide reduced total cellular adenine nucleotides to 50-60% of control values with only a modest (0.95-0.91) reduction in energy charge [ATP + 1/2 ADP)/(ATP + ADP + AMP]. Peroxide-treated myocytes selectively release inosine and adenosine, as only these two purine degradation products were detected in the incubation medium. The most dramatic response was a peroxide dose-dependent inhibition of PDH activity in cultured myocytes as well as freshly isolated mitochondria; just 65 and 30 nmol peroxide/mg protein induced a 50% reduction in cellular and mitochondrial PDH activity, respectively. In conclusion, physiological quantities of peroxide potently inhibit PDH in cultured cardiomyocytes and isolated cardiac mitochondria. PDH inhibition blocks the aerobic oxidation of glucose and inhibits the oxidative phosphorylation of ADP, which in turn leads to cellular adenine nucleotide degradation.     

Control of adenine nucleotide metabolism in hepatic mitochondria from rats with ethanol-induced fatty liver

Male rats developed fatty liver after being fed on an ethanol-containing diet for 31 days. Liver mitochondria from these animals catalysed ATP synthesis at a slower rate when compared with mitochondria from pair-fed control rats (control mitochondria), and demonstrated lowered respiratory control with succinate as substrate, owing to a decrease in the State-3 respiratory rate. Respiration in the presence of uncoupler was comparable in mitochondria from both groups of rats. Translocation of both ATP and ADP was decreased in mitochondria from ethanol-fed rats, with ADP uptake being lowered more dramatically by ethanol feeding. Parameters influencing adenine nucleotide translocation were investigated in mitochondria from ethanol-fed rats. Experiments performed suggested that lowered adenine nucleotide translocation in these mitochondria is not the result of inhibition of the translocase by either long-chain acyl-CoA derivatives or unesterified fatty acids. Analysis of endogenous adenine nucleotides in these mitochondria revealed lowered ATP concentrations, but no decrease in total adenine nucleotides. In experiments where the endogenous ATP in these mitochondria was shifted to higher concentrations by incubation with oxidizable substrates or defatted bovine serum albumin, the rate of ADP translocation was increased, with a linear correlation being observed between endogenous ATP concentrations and the rate of ADP translocation. The depressed ATP concentration in mitochondria from ethanol-fed rats suggests that the ATP synthetase complex is replenishing endogenous ATP at a slower rate. The lowered ATPase activity of the ATP synthetase observed in submitochondrial particles from ethanol-fed animals suggests a decrease in the function of the synthetase complex. A decrease in the rate of ATP synthesis in mitochondria from ethanol-fed rats is sufficient to explain the decreased ADP translocation and State-3 respiration.     

Synaptosomal bioenergetics. The role of glycolysis, pyruvate oxidation and responses to hypoglycaemia

The bioenergetic interaction between glycolysis and oxidative phosphorylation in isolated nerve terminals (synaptosomes) from guinea-pig cerebral cortex is characterized. Essentially all synaptosomes contain functioning mitochondria. There is a tight coupling between glycolytic rate and respiration: uncoupler causes a tenfold increase in glycolysis and a sixfold increase in respiration. Synaptosomes contain little endogenous glycolytic substrate and glycolysis is dependent on external glucose. In glucose-free media, or following addition of iodoacetate, synaptosomes continue to respire and to maintain high ATP/ADP ratios. In contrast to glucose, the endogenous substrate can neither maintain high respiration in the presence of uncoupler nor generate ATP in the presence of cyanide. Pyruvate, but not succinate, is an excellent substrate for intact synaptosomes. The in-situ mitochondrial membrane potential (delta psi m) is highly dependent upon the availability of glycolytic or exogenous pyruvate; glucose deprivation causes a 20-mV depolarization, while added pyruvate causes a 6-mV hyperpolarization even in the presence of glucose. Inhibition of pyruvate dehydrogenase by arsenite or pyruvate transport by alpha-cyano-4-hydroxycinnamate has little effect on ATP/ADP ratios; however respiratory capacity is severely restricted. It is concluded that synaptosomes are valuable models for studying the control of mitochondrial substrate supply in situ.     

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.     

The tricarboxylic acid cycle and glycolysis in relation to ion transport by the ciliary body

1. The respiration and aerobic glycolysis of pig ciliary processes in oxygenated phosphate and bicarbonate buffers have been investigated. 2. Significant amounts of lactic acid are produced only in the presence of added glucose, but this does not change the endogenous respiration rate. 3. Succinate and citrate increase the oxygen uptake considerably, but pyruvate has almost no effect; oxaloacetate and fumarate stimulate slightly in the presence of glucose. Aspartate and fumarate together stimulate pyruvate utilization and are oxidized as fast as citrate. 4. Ouabain inhibits the oxidation of glucose and other substrates by limiting the ADP supply from the sodium transport system. Cyanide and azide inhibit respiration and stimulate glycolysis. 5. The transport mechanism depends largely on ATP from oxidative phosphorylation and regulates the rate of respiration and glycolysis by controlling ADP production from the Na(+)-K(+)-activated adenosine triphosphatase.     

Criteria of viability of isolated liver cells.

36.4 +/Various cellular parameters were measured with regard to their usefulness as criteria of viability of isolated cells. Stainability by trypan blue and release of lactate dehydrogenase indicate only severe irreversible damage of cells. Neither endogenous respiration nor even the ATP/ADP ratio is a sensitive criterion of viability. On aging of cells, the ATP/ADP ratio remains high, even though the membrane potential, the intracellular K concentration and the content of adenine nucleotides decrease considerably. A sensitive, easily performed test is the stimulation of cellular respiration by 1mM succinate. Only a damaged plasma membrane allows succinate permeation of a rate sufficient to stimulate respiration. The membrane potential and the intracellular Na and K concentrations are the most sensitive criteria of viability, since they indicate the earliest changes on aging. (For freshly isolated cells, we found a membrane potential of 36.4 "/- 3.4 mv [n = 5], an intracellular K concentration of 109.0 +/- 9.1 mM, and an intracellular Na concentration of 47.0 +/- 13.4mM.) The incorporation of [14C]uridine also sensitively reflects cellular damage.     

Interrelation between salvage of purine nucleotides and protein synthesis in rat heart cells

The effect of transition from a respiring to a respiration-inhibited state on the rate of protein synthesis was investigated in glycolyzing, cultured rat heart cells. The rate was found to be significantly lower after blocking respiration, and it was further decreased by L-lactate. In contrast, pyruvate or phenazine methosulfate prevented the drop in the rate caused by lack of respiration. The changes in the respiratory state also affected the steady-state concentration of ATP, which varied in the same sense as the rate of protein synthesis. Pyruvate or phenazine methosulfate induced an increment in the concentration of ATP of respiration-inhibited cells. This increment could not be accounted for by more extensive phosphorylation of the available purine nucleotides, but required repletion of the pool by synthesis of purine nucleotides through the salvage pathway. Pyruvate and phenazine methosulfate were found to stimulate incorporation of labeled hypoxanthine into the purine nucleotide fraction in general, and into the nucleotide triphosphates in particular. Under similar incubation conditions an increase in the ATP/ADP ratio was also noted. The stimulatory effect of pyruvate on protein synthesis and on the cellular level of ATP was also observed in respiration-inhibited 3T6 cells and in human fibroblasts, but not in human fibroblasts deficient in the salvage enzyme, hypoxanthine-guanine-phosphoribosyltransferase. Based on the demonstrated influence of L-lactate, pyruvate, and phenazine methosulfate on the salvage synthesis of purine nucleotides [K. Ravid, P. Diamant, and Y. Avi-Dor, (1984) Arch. Biochem. Biophys. 229, 632-639] and on the present findings, the connection between protein synthesis and the salvage activity is discussed.     

Persistence of the effect of insulin on pyruvate dehydrogenase activity in rat white and brown adipose tissue during the preparation and subsequent incubation of mitochondria.

Increases in the amount of the active non-phosphorylated form of pyruvate dehydrogenase in rat epididymal adipose tissue, as a result of incubation with insulin, persist not only during the preparation of mitochondria but also during subsequent incubation of coupled mitochondria in the presence of respiratory substrates. No effect on insulin was found if the hormone was added directly to mitochondria in the presence or absence of added plasma membranes. Concentrations of several possible regulators of pyruvate dehydrogenase kinase (ATP, ADP, NADH, NAD+, acetyl-CoA, CoA and potassium) were measured in rat epididymal-adipose-tissue mitochondria incubated under conditions where differences in pyruvate dehydrogenase activity persist as a result of insulin action. No alterations were found, and it is suggested that inhibition of the kinase is not the principal means by which insulin activates pyruvate dehydrogenase. The intramitochondrial concentration of magnesium was also unaffected. Differences in pyruvate dehydrogenase activity in interscapular brown adipose tissue associated with manipulation of plasma insulin concentrations of cold-adapted rats were also shown to persist during the preparation and subsequent incubation of mitochondria in the presence or absence of GDP. It is pointed out that the persistence of the effect of insulin on pyruvate dehydrogenase in incubated mitochondria will facilitate the recognition of the mechanism of this action of the hormone. Evidence that the short-term action of insulin involves an increase in pyruvate dehydrogenase phosphate phosphatase activity rather than inhibition of that of pyruvate dehydrogenase kinase is discussed.     

Control of heart mitochondrial oxygen consumption by creatine kinase: The importance of enzyme localization

The route of movement of ADP produced in the mitochondrial creatine kinase reaction was investigated by recording the rate of ADP-dependent oxygen consumption in the presence of phosphoenolpyruvate and pyruvate kinase. This pyruvate kinase system completely abolished activation of respiration by ADP added or by ADP produced in the hexokinase reaction in the medium, but was not able to inhibit the creatine kinase activated respiration when creatine kinase was bound to the inner mitochondrial membrane. These different responses of oxidative phosphorylation were observed at equal $\text{ATP}\text{ADP}$ ratios in the medium. The data obtained evidence direct channeling of ADP from heart mitochondrial creatine kinase to the adenine nucleotide translocase without its prompt release into the medium.     

Light-Dependent Incorporation of Adenine Nucleotide into Noncatalytic Sites of Chloroplast ATP Synthase

The binding of ADP and ATP to noncatalytic sites of dithiothreitol-modified chloroplast ATP synthase was studied. Selective binding of nucleotides to noncatalytic sites was provided by preliminary light incubation of thylakoid membranes with [¹⁴C]ADP followed by its dissociation from catalytic sites during dark ATP hydrolysis stimulated by bisulfite ions (“cold chase”). Incorporation of labeled nucleotides increased with increasing light intensity. Concentration-dependent equilibrium between free and bound nucleotides was achieved within 2–10 min with the following characteristic parameters: the maximal value of nucleotide incorporation was 1.5 nmol/mg of chlorophyll, and the dissociation constant was 1.5 μM. The dependence of nucleotide incorporation on Mg²⁺ concentration was slight and changed insignificantly upon substituting Ca²⁺ for Mg²⁺. Dissociation of nucleotide from noncatalytic sites was illumination dependent. The dissociation kinetics suggested the existence of at least two nucleotide-binding sites with different dissociation rate constants. __________ Translated from Biokhimiya, Vol. 70, No. 11, 2005, pp. 1514–1520. Original Russian Text Copyright ? 2005 by Malyan.