Cytoplasmic ATP Research Articles

Properties of red cell ghost preparations susceptible to invasion by malaria parasites

Experimental procedures are described that lead to the formation of sealed red cell ghosts capable of efficient invasion by malaria parasites. Both human and monkey cells have been studied with respect to invasion by Plasmodium falciparum and P. knowlesi respectively. Resealed ghosts containing about half the normal concentration of haemoglobin are prepared by osmotic lysis and resealing at a haematocrit of 70%. When examined in the scanning electron microscope, populations of these ghosts contain few echinocytes, but have an abundance of stomatocytic forms. When undiluted haemolysate is substituted for the aqueous saline diluent, in which the cells are suspended for lysis and resealing, discocytes result, with a morphology very similar to that of the original cells. Invasion is somewhat, but not dramatically higher for this material. An investigation of the effect of intracellular potassium concentration revealed that this had no major effect on invasion. A small increase in invasion resulted from addition of 50 or 100 mg/ml of albumin to the medium in which the cells were resealed, but intra-erythrocytic protein content per se is evidently not a major factor in determining the efficiency of invasion. Augmentation of the cytoplasmic ATP concentration during lysis and resealing increased the level of parasitaemia significantly, and the parasites in these ghosts developed normally and gave rise to viable merozoites. It was found that albumin at certain concentrations passed freely into the lysed cells and reached equilibrium with that in the external medium. By contrast, the high molecular weight solute Blue Dextran 2000 was completely excluded from the lysed cells.

A novel technique for rapid determination of energy consumption in platelets. Demonstration of different energy consumption associated with three secretory responses.

A novel method has been developed for rapid and quantitative determination of the rate of energy consumption in platelets. In platelets suspended in a cyanide-containing medium. ATP resynthesis is abruptly blocked by addition of 2-deoxyglucose and D-glucono-1,5-lactone. We demonstrate that the subsequent changes in the levels of cytoplasmic ATP and ADP reflect the velocity of energy consumption in the platelets immediately before addition of the inhibitors. Despite the arrest in ATP resynthesis the platelets remain responsive to stimulation by thrombin (5 units x ml-1) which triggers the secretion of the contents of dense, alpha- and acid hydrolase granules. Unstimulated platelets were found to consume about 3.5 and 0.5 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at 37 degrees C and 15 degrees C, respectively; the thrombin-treated platelets consumed respectively 16 and 2 mumol of ATP equivalents x min-1 x (10(11) cells)-1 at these temperatures. When the velocity of energy consumption was varied by (a) changing the temperature and (b) preincubation with glyco(geno)lytic inhibitors, it was found to be linearly related to the initial rate of secretion from the three types of granules. The precise nature of this relationship differed between the three types of secretion responses and indicated an increasing requirement for metabolic energy for secretion from the three types of granules in the order: dense granule less than alpha-granule less than acid hydrolase granule. The results obtained with changes in temperature were superimposable on those obtained with the glyco(geno)lytic inhibitors for dense granule secretion and alpha-granule secretion, suggesting an apparent coupling between energy consumption and the rate of these secretion responses. The rate of secretion of acid hydrolase was always higher when energy consumption was varied by temperature changes than when glyco(geno)lytic inhibitors were used, probably as a result of metabolic changes prior to induction of secretion. On the basis of these experiments, we calculated an incremental energy consumption during complete secretion of dense, alpha- and acid hydrolase granule contents of 2.5, 4.2 and 6.7 mumol of ATP equivalents x (10(11) platelets)-1, respectively.

Effects of ATP or phosphate on passive rubidium fluxes mediated by Na‐K‐ATPase reconstituted into phospholipid vesicles

1. The passive Rb fluxes mediated by the Na-K pump in reconstituted vesicles, described by Karlish & Stein (1982), are affected by ATP or by phosphate acting separately.2. Rb-Rb exchange through inside-out pumps is stimulated by ATP at low concentrations and is inhibited at high concentrations. There are mutual effects of Rb at cytoplasmic sites and ATP. The higher is the Rb concentration, the greater is the degree of stimulation and the less is the inhibition of exchange by ATP, and the higher are the concentrations of ATP required to produce effects. ATP stimulates Rb-Rb exchange maximally by about 5-fold.3. There are similar effects of ATP on zero-trans net Rb uptake through inside-out pumps. However, much lower degrees of stimulation and greater inhibition of the net flux by ATP are observed, and much lower concentrations of ATP are required for these effects, by comparison with those on Rb-Rb exchange.4. Rb uptake on inside-out pumps in Na-loaded vesicles shows only inhibition by ATP.5. Phosphate effects require the presence of Mg(0) ions. At low Mg(0) concentrations (up to 100 muM) phosphate moderately stimulates Rb uptake into Rb-free or Rb-loaded vesicles (about 50%), but has no effect on Rb uptake into Na-loaded vesicles. At millimolar concentrations of Mg(0) ions, phosphate strongly inhibits the Rb uptake into Rb-free or Na-loaded vesicles but has no effect on Rb uptake into Rb-loaded vesicles.6. The separate effects of ATP and of phosphate are explained in terms of the model proposed by Karlish & Stein (1982), modified to take into account stimulation of the conformational transition E(2)(Rb)(occ) --> E(1) Rb by ATP, and stimulation of the conformational transition E(2)(Rb)(occ) --> E(2) Rb by phosphate due to phosphorylation of the protein.

31P NMR saturation transfer measurements of the steady state rates of creatine kinase and ATP synthetase in the rat brain

There are two theories concerning the function of phosphocreatine (PCr) in cellular energy metabolism. In one PCr is seen as a reservoir, functioning to maintain constant cytoplasmic ATP concentrations. The alternate theory postulates that PCr acts as an energy shuttle between the site of ATP production in the mitochondria and the site of utilization in the cytoplasm (reviews [ 1,2]). Both theories require that the exchange flux in the creatine kinase (CPK) reaction be considerably greater than the rate of ATP hydrolysis. This is necessary in order to stabilize ATP concentrations during transitions in workload, or during periods when ATP synthesis is impaired, such as ischaemia. Calculations of cytoplasmic phosphorylation potential [3] and measurements of intracellular pH [4] have been based on the assumption that the CPK reaction is near equilibrium. It is now possible to test this assumption in vivo using the technique of 31P NMR saturation transfer; the flux through the CPK reaction was 5 times the steady state rate of ATP turnover in the Langendorff perfused rat heart 151. The total extractable activities of CPK in the rat brain and heart are similar; 500 pmol . g-’ . min-’ in heart [6] and 200 pmol . g-’ min-’ in brain [7], measured at 25’C. The mitochondrial form of the enzyme accounts for 30-40% of the total activity in the heart [l] and 5% in brain [7]. However, mitochondrial CPK activity in both organs is very similar on a per gram mitochondrial protein basis [8]. Despite these similarities, the metabolic response to a decrease in the efficiency of cellular energy transduction,

5-hydroxytryptamine and adenine uptake by human blood platelets following the release reaction

Human blood platelets were incubated with drugs which either liberated only 5-HT (incomplete release reaction) or 5-HT as well as ATP/ADP (complete release reaction) from subcellular storage organelles. The incomplete release reaction induced by tyramine, tryptamine, and 1-aminoadamantane was followed by an increased 5-HT uptake which was enhanced up to 350% as compared with the untreated platelets. After treatment with drugs like 1-amino-3,5-dimethyladamantane, imipramine, and reserpine, which induce the complete release reaction, platelets took up significantly less 5-HT as compared with the controls. - Apparently the metabolically inert ATP within the storage organelles is necessary for 5-HT uptake and storage. The uptake of radioactively labeled adenine and the synthesis of metabolically active adenine nucleotides were increased after preincubation with both types of release-inducing substances. The newly synthesized ATP may serve to restore the metabolically active, cytoplasmic ATP which is consumed either for the extrusion process during the complete release reaction or for the energy-requiring 5-HT reuptake observed after treatment with 5-HT displacing agents.

A proposal for the role of ecto-enzymes and adenylates in traumatic shock

It is proposed that the biochemical mechanisms which lead to the pathology of irreversible traumatic shock are based on the vasodilatory action of adenylates and failure of ecto-enzymes to clear these substances from the vascular bed. Following massive injury, or another assault which produces significant cytolysis, cytoplasmic ATP is released into the circulation. Ecto-phosphoesterhydrolases on erythrocytes normally control plasma adenylate concentrations to be maintained below 10 −7 M. If a critical ATP (or ADP) concentration between 10 −5 and 10 −4 M is reached, erythrocytes become semi-permeable and themselves release additional ATP into the plasma compartment. When ecto-ATPase activity becomes insufficient in metabolizing plasma ATP at rates which are needed to compensate for the release of cytoplasmic ATP, the vasodilatory effects of adenylates will manifest themselves in a sustained manner. It is suggested that ecto-phosphoesterhydrolases on blood formed elements and endothelial cells serve a protective function. Circulatory shock ensues under conditions when these ecto-enzymes are rendered incompetent or when plasma adenylate concentrations exceed their catabolic capacity.

Studies on frost hardiness in Chlorella ellipsoidea. V. The role of glucose and related compounds

Chlorella ellipsoidea cells at an intermediate stage in the ripening phase of the life cycle were hardened at 3°C. d-Glucose added during hardening in the dark induced a tolerance to freezing. Sucrose, d-galactose and d-fructose were also capable of increasing hardiness, but pyruvate, succinate and citrate were not. These results suggest the involvement of the pentose-phosphate cycle in the hardening process. The amounts of monosaccharide and dextrin in Chlorella cells did not change during hardening. Sucrose concentration increased during hardening in both the light and the dark in the presence of glucose. Starch concentration remained unchanged in the dark in the presence of glucose, but increased remarkably in the light. These results suggest that the hardening process involves an accumulation of sucrose, and indicate that there is no direct correlation between starch content and frost hardiness. Glucose 6-phosphate dehydrogenase activity increased greatly during the early phase of hardening; 6-phosphogluconate dehydrogenase activity also increased with hardiness. These results suggest that pentose-phosphate cycle activity occurs at a high level during hardening. Cycloheximide and oligomycin completely inhibited the hardiness increase in the dark in the presence of glucose; chloramphenicol and DCMU had no effect. These results suggest that protein synthesis on cytoplasmic ribosomes and ATP synthesis in mitochondria are involved in the process of hardening in the dark in the presence of glucose, but protein and ATP synthesis in chloroplasts are not. The inhibition of the hardiness increase by sodium dodecyl sulfate was greater in the light than in the dark in the presence of glucose. Triton X-100 inhibited completely the hardiness increase in both the light and the dark in the presence of glucose. It seems likely, therefore, that membrane alterations during hardening in the dark in the presence of glucose may occur; these alterations are different from those which occur in the light.

Role of cytoplasmic ATP in the restoration and maintenance of a membrane permeability barrier in transformed mammalian cells.

Addition of ATP to medium surrounding intact, transformed 3T3 cells activates the formation of aqueous channels in the plasma membrane. This results in efflux of nucleotide pools and ions and entry into the cytosol of charged, phosphorylated species. In such permeabilized cells, glycolysis is totally dependent on the external addition of glucose, inorganic phosphate, ADP, K+, Mg2+ and NAD+ which restore lactic acid formation to levels found in untreated cells. As expected, such reconstitution of glycolytic activity is found to restore intracellular ATP levels. This is accompanied by sealing of the membrane channels so that efflux of nucleotide pools ceases. Pyruvate, a substrate for mitochondrial ATP synthesis, when provided along with ADP and inorganic phosphate also produces sealing of the membrane channels. On the other hand, reactivation of pentose phosphate shunt activity, which does not lead to ATP synthesis, does not induce restoration of the membrane permeability barrier. Furthermore, compounds which lower the internal ATP pool prevent sealing, and also render the plasma membrane more sensitive to external ATP (Rozengurt and Heppel, '79). Sealing of aqueous channels following restoration of the internal ATP pool is associated with phosphorylation of the inner membrane surface, and is unaffected by inhibitors of protein synthesis, microfilament or microtubular assembly. These results indicate the probable role of intracellular ATP in the restoration and/or maintenance of an active membrane barrier against efflux of small molecules and ions in transformed 3T3 cells.

Transient 45Ca uptake and release in isolated rat-liver cells during recovery from deenergized states.

1. Aerobic incubation of isolated rat liver cells--after dilution from the anaerobic stock suspension--transiently brings about a state, during which a reversible calcium uptake can be observed on addition of a respiratory substrate. Uptake varies greatly and can reach more than 50 nmol/mg protein, but declines to zero on prolonged preincubation, especially at higher temperature. Repeated additions of succinate or 3-hydroxybutyrate evoke new calcium transients. If ATP is simultaneously added, if greatly potentiates succinate-initiated reversible uptake. 2. If rotenone is present during the preincubation phase, calcium transients are strongly enhanced. Uptake is blocked by uncouplers and respiratory inhibitors, indicating the involvement of mitochondria. 3. Calcium uptake is not accompanied by increased oxygen consumption. The actual respiration cannot account sufficiently for the energy need of calcium uptake. Participation of cytoplasmic ATP is likely, as inhibitors of adenine nucleotide translocase affect uptake. 4. Lanthanum enhances calcium uptake in contrast to its action on mitochondria. 5. Pulse-labeling experiments indicate that the calcium taken up is removed from a rapidly exchangeable calcium pool by withdrawal into the mitochondria as a deep compartment. 6. Calcium uptake is accelerated either by increasing the phosphate level or by high temperature. It is prolonged by low temperature, high pH or high ATP concentration. Calcium release accelerates with increasing temperature, decreasing pH and a further rise in phosphate concentration. 7. The dependency on phosphate and temperature reveals a delicately poised equilibrium of uptake and release. At ambient temperature, phosphate increases uptake up to a concentration of 0.5 mM. Higher concentrations accelerate both uptake and release. At lower temperature, the accelerating effect on uptake predominates. A temperature shift during incubation results in adaptation of the calcium equilibrium to the new temperature, i.e. release of calcium at high temperature, uptake at low temperature. 8. Oxidizing metabolites inhibit succinate-stimulated calcium uptake and promote release of previously accumulated calcium. An increased sensitivity to phosphate is established. 9. With respect to isolated mitochondria, isolated liver cells appear to be a more realistic model for studying the physiological mechanism of mitochondrial calcium release, since compartmental constraints and regulations are maintained.

Nigericin-induced death of an acidophilic bacterium.

At an external pH of 3.5, nigericin (which catalyses an electroneutral H+/K+ exchange) abolished the transmembrane proton gradient (delta pH) of Bacillus acidocaldarius, causing a rapid acidification of the cytoplasm from approximately pH 6.0 to pH 3.5. A pronounced loss of viability and fine-structural changes rapidly followed treatment with nigericin. A marked decline in respiration and an even more rapid decrease in cytoplasmic ATP were observed. Activity of at least one cytoplasmic enzyme decreased more slowly. There was no generalized loss in the integrity of the cytoplasmic membrane, as assayed by permeability to inulin or Na+ or by release of ultraviolet light-absorbing compounds. The loss of viability upon treatment with carbonyl cyanide m-chlorophenylhydrazone was similar to what observed with nigericin, so proton influx alone, rather than together with K+ efflux, was probably involved in the death of the organism. Moreover, acidification of the cytoplasm rather than abolition of the delta pH was the lethal event, since no loss of viability was observed when the delta pH was abolished by elevation of the external pH.

GTP hydrolysis in intact rod outer segments and the transmitter cycle in visual excitation

THE light-sensitive dark current of vertebrate retinal rods is the main metabolic load on the receptor cells, turning over the cytoplasmic Na+ every 1–6 min (refs 1–4) and the cytoplasmic ATP as often as every 20 s (refs 5,6). However, a second process in the rod outer segment (ROS), the transmitter cycle, probably demands metabolic free energy during visual excitation. Photons absorbed in the intracellular rod disks reduce the dark current traversing the nearby plasma membrane by altering the cytoplasmic level of a diffusible intracellular transmitter substance7–10 that may be free Ca2+ (refs 11–14). As tens to thousands of transmitter particles must be released and removed in a single photon response15–17, the transmitter cycle should cause fast metabolic events in ROSs even when they are isolated from those parts of the receptor cells that sustain the dark current. We report here that light causes ROSs to destroy GTP and form GDP at a rate sufficient to meet the predicted requirement of the transmitter cycle. Briefly, ROSs with intact plasma membranes and normal cytoplasmic nucleotide content were isolated within a few seconds from live frog retinas; aliquots of the ROS suspension were exposed to flashes of light, and the cytoplasmic levels of ROS nucleotides monitored as a function of time. The experimental procedure permits changes in cytoplasmic nucleotides in ROS to be distinguished from those involving nucleotides in the suspending medium or bound to rod disk membranes that are not enclosed within an intact plasma membrane.

Comparative study on the effects of exogenous palmitate and erucate on intracellular electric properties of cultured beating heart cells

The effects of the palmitate- and erucate-enriched culture medium (0.2 m m) on the electrical and on the rhythmic activities of dissociated newborn rat cardiac cells maintained in vitro were analyzed. Both free fatty acids have no significant effect on the resting potential, but decrease the beating frequency. Palmitate shortens the action potential, while erucate elicits the opposite response. Palmitate and erucate induce specific alteration of the frequency-dependence of the action potential duration. Only K-erucate depresses overshoot potential level. It is suggested that the electrophysiological responses to high palmitate concentration are probably related to disturbed mitochondrial supply of cytoplasmic ATP, while the erucate would more specifically affect numerous membrane properties because of its chain length and unsaturated structure. The observed membrane potential changes may account for an arrhythmogenic effect of these fatty acids on the whole heart.

Adenine nucleotide storage and secretion in platelets as studied by 31P nuclear magnetic resonance.

Suspensions of human and pig blood platelets have been studied by 31P NMR at 145.7 MHz and by chemical and radiochemical determination of nucleotide levels. In both types of platelets the cytoplasmic nucleotide pool, which was prelabeled by incubation with [14C]adenine, was selectively reduced by addition of H2O2/NaN3 or 2-deoxyglucose/antimycin A. After the reduction of cytoplasmic ATP in human platelets, the 31P NMR spectra showed an almost complete loss of the nucleoside di- and triphosphate resonances at temperatures examined (4--50 degrees C), indicating that only the cytoplasmic nucleotides had been observed, with no detectable contributions from the granular ATP, ADP, and pyrophosphate. Slow tumbling of the granular nucleotides, possibly due to aggregation, is the probable explanation of their undetectability at 145.7 MHz. Similar experiments showed that in pig platelets, granular ATP and ADP were not detected by 31P NMR at 4 degrees C but were observed at higher temperatures, indicating that aggregation may be occurring at the lower temperatures. Upon thrombin stimulation of human platelets, the NMR spectra and the chemical and radioactivity analyses showed that the granular adenylates and pyrophosphate were secreted, and that cytoplasmic ATP levels were appreciably reduced.

Modulation of the organization of erythrocyte membrane phospholipids by cytoplasmic ATP. The susceptibility of isoionic human erythrocyte ghosts to attack by detergents and phospholipase C

Human erythrocyte ghosts were prepared in media of physiological ionic composition, and these “isoionic” ghosts were then lysed and resealed in media of varying Ca 2+, Mg 2+ and ATP concentrations. The susceptibilities of these ghosts to limited attack by various detergents and by phospholipases C were then compared with the susceptibilities of intact cells to similar attack: attack was assessed by measurements of lysis and of phospholipid hydrolysis. Ghosts were more readily attacked than cells by anionic detergents (cholate, glycocholate, dodecyl sulphate) and by phospholipases C, but Triton X-100 and cetyltrimethylammonium attacked cells and ghosts to the same extent. Mg · ATP 2− partially protected ghosts from attack by the anionic detergents and by the phospholipases C of Bacillus cereus and of Clostridium perfringens. Protection by Mg · ATP 2− occurred only if Mg · ATP 2− had access to the cytoplasmic surface of the membrane. Adenylyl(β-γ-methylene)diphosphonate, a non-hydrolysable ATP analogue, protected as effectively as did Mg · ATP 2−. Internal Mg · ATP 2− caused a marked reduction in the hydrolysis by phospholipases of phosphatidylethanolamine and sphingomyelin, but had no appreciable effect upon the simultaneous hydrolysis of phosphatidylcholine. It therefore seems that interaction of ATP with sites on the cytoplasmic surface of the erythrocyte membrane can, without ATP hydrolysis, cause changes in the organization of the outer surface of the membrane that specifically render phosphatidylethanolamine and sphingomyelin less accessible to attack by extracellular phospholipases.

Ecto-ATPase deficiency in glia of seizure-prone mice.

A DEFICIENCY of calcium-stimulated adenosine triphosphatase (Ca2+-ATPase) is seen in brains of mice which are susceptible to audiogenic seizures1. After studying five other animal models of idiopathic epilepsy we concluded that brain nucleotide metabolism is altered in the epileptic animals and that the lesion might not be confined to Ca2+-ATPase2. We speculated that the deficiency of Ca2+-ATPase, which we observed in membrane-enriched brain homogenates, was the expression of incompetent brain cell ecto-ATPases. We have suggested that the genesis of seizures was related to a protracted action of translocated cytoplasmic ATP on cell membranes3 when the latter were deficient in this enzyme. We report here a significant deletion of ecto-ATPase in cultured glia cells raised from neonatal, seizure-prone mice. Between the ages of 20 to 35 d, mice of the DBA/2N strain convulse after repeated exposure to loud noise. Before and after this period, susceptibility to induced convulsions is minimal or absent. Mice of the C57 B1/6N strain are not afflicted in this manner.

Human deoxycytidine kinase purification and characterization of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia patients

A procedure for purifying human cytoplasmic and mitochondrial deoxycytidine kinase (NTP:deoxycytidine 5′-phosphotransferase, EC 2.7.1.74) was developed. Both purified isozymes have a similar molecular weight, activation energy and catalyze the reaction by a sequential mechanism. These two isozymes differ with respect to their substrate specificities. With cytoplasmic deoxycytidine kinase, ATP, GTP and TTP have the highest reaction velocity. Pyrimidine nucleoside triphosphates have higher affinity but lower V than purine nucleoside triphosphates. Cytidine and arabinosylcytidine can serve as substrates. With mitochondrial isozyme only ATP gives the highest reaction velocity. ATP and dATP have the same K m but different V values. Besides deoxycytidine, also deoxythymidine but not cytidine or arabinosylcytidine can serve as substrates. There are also differences between these two isozymes with respect to their sensitivity to inhibition. For cytoplasmic enzyme, Br 5dCyd and Iodo 5dCyd are not inhibitory. Both dCTP and UTP are competitive inhibitors ( K i 0.25 and 0.5 μM, respectively) with respect to ATP. For mitochondrial isozyme both Br 5dCyd and Iodo 5dCyd are inhibitory and dCTP and TTP are competitive inhibitors ( K i 2 and 10 μM, respectively) with respect to ATP.

Evidence for ATP action on the cell surface.

IT seems that Mg2+–ATPase (EC 3.6.1.3) and 5′-nucleotidase (EC 3.1.3.5) are prevalent plasma membrane marker enzymes in eukaryotic cells1. Our studies on the ‘sidedness’ of plasma membrane macromolecules suggested that the two enzymes function as ecto-enzymes2, although the substrates presumably were not present on the cell surface. We assumed that the substrates were present, but obscured within the intercellular spaces and by rapid metabolism. The specificity of the ecto-ATPase is low but we suspected that translocated cytoplasmic ATP might be a primary substrate.

The control of membrane-bound Ca 2+ by ATP

1. 1. It is known that the viscosity of venous blood is greater than that of arterial blood, and that an ATP-depleted erythrocyte loses its deformability. In order to test the hypothesis that these effects may be explained by an intracellular chelation of membrane-bound Ca 2+ by ATP, the effect of ATP on the membrane-bound Ca 2+ of erythrocyte ghost membranes was examined by adding 45Ca 2+ and ATP to the isolated membranes. 2. 2. The membrane-bound Ca 2+ was reduced to zero at 1.5 mM ATP, pH 7. 3. 3. A complete adsorption isotherm for ATP at 21° indicated that there was a very small binding component of about 540 molecules of ATP per single erythrocyte membrane which became saturated at about 10 −7 M ATP. The majority of the ATP binding sites, however, did not indicate any saturation up to 10 −3 M ATP. 4. 4. 10 −5 M Ca 2+ or Mg 2+ depressed the membrane-bound ATP, but increased it at 1 mM. 5. 5. ATP binding was increased at low pH. 6. 6. Calculations indicated that the oxygenated erythrocyte would have a cytoplasmic free ATP level of 1.45 mM, and that the ATP concentration in the erythrocyte of venous blood (60% oxygenated) would be 0.94 mmole per l water. The level of membrane-bound Ca 2+, therefore, would tend to be higher in the deoxygenated cell, explaining its greater rigidity and the higher viscosity of venous blood.

Direct and Indirect Transfer of ATP and ADP across the Chloroplast Envelope

1. In intact leaves of Elodea densa illumination resulted in a large increase in the levels of chloroplastic and cytoplasmic ATP and a decrease in chloroplastic and cytoplasmic ADP. Reverse changes were observed on darkening. The kinetics of the fluctuations were similar in chloroplasts and cytoplasm suggesting effective transfer of ATP and ADP between chloroplasts and cytoplasm. Ratios of ATP to ADP were significantly lower in the chloroplasts than in the cytoplasm in the dark and in the light. This may indicate different phosphate potentials in chloroplasts and cytoplasm. Transfer of ATP across the chloroplast envelope as calculated from the light-dependent cytoplasmic ATP increase was 7 to 9 μmoles/mg chlorophyll per hour. Actual transfer rates are probably higher. 2. The determination of the rate of adenylate transfer across the envelope of intact isolated chloroplasts requires information on the composition of chloroplast preparations. Intact chloroplasts were quantitatively separated from envelope-free chloroplasts by density gradient centrifugation on Ludox gradients. The percentage of envelope-free chloroplasts in preparations of intact chloroplasts was also determined from measurements of light-dependent ferricyanide reduction. 3. During isolation in sorbitol buffer ca. 50% of the adenylates were lost from intact chloroplasts which were still capable of high rates of phosphoglycerate reduction and photosynthesis. 4. Adenylate transfer across the envelope of isolated chloroplasts as measured by the light-dependent phosphorylation of added ADP in the absence of cofactors was slow and occured at a rate of 0 to 4 µmoles/mg chloroplyll per hour. In the dark chloroplastic adenylate kinase reacted only very slowly with added AMP and ATP to form ADP. Breakage of the chloroplast envelope stimulated reaction rates. 5. Indirect transfer of ATP and ADP across the chloroplast envelope occurred via a shuttle transfer of phosphoglycerate and dihydroxyacetone phosphate. 3-phosphoglyceraldehyde is not involved as a transport metabolite. Maximum transfer rates of phosphoglycerate and dihydroxyacetone phosphate across the chloroplast envelope were higher than maximum rates of photosynthesis and reached 300 μmoles/mg chlorophyll per hour. Indirect transfer of ATP was somewhat slower than rates of phosphoglycerate reduction by isolated chloroplasts. In vivo transfer of phosphate energy by this transport metabolite system is under control of the redox state of pyridine nucleotides and of the phosphate potential.