Plasmodium falciparum: ATP/ADP transport across the parasitophorous vacuolar and plasma membranes

Previous studies have shown that ATP is required for the growth of the intracellular parasite, Plasmodium, outside its host cell, the erythrocyte, and that bongkrekic acid, an inhibitor of mitochondrial ATP/ADP transporter, inhibits intraerythrocytic Plasmodium maturation. We have characterized ATP/ADP transport of Plasmodium falciparum, isolated by either immune lysis or N2-cavitation. [3H]ATP uptake was due to ATP/ADP exchange since ADP efflux was dependent on exogenous ATP in an approximate 1:1 stoichiometry and both ATP influx and ADP efflux were equally inhibited by atractyloside (Ki = 100 nM). ATP uptake was not inhibited by the nucleoside transport inhibitor, nitrobenzylthioinosine. Conversely, adenosine and hypoxanthine transport were insensitive to atractyloside. ATP influx was characterized by a Km = 0.14 mM and Vmax = 1.2 nmol ATP/min/10(6) cells. Substrate specificity studies for nucleotide-induced ADP efflux indicated a preference for an adenosine ring and triphosphate, but transport did not require a hydrolyzable phosphate bond. Protein synthesis was measured with free parasites starved of glucose. Addition of 1.0 mM ATP resulted in a 40% recovery of total protein synthetic capacity in a process inhibited by 500 nM atractyloside, suggesting that uptake of erythrocyte-derived ATP by P. falciparum may be essential for maintaining maximal rates of protein synthesis during specific stages of intra-erythrocytic parasite maturation.     

The pho regulon-dependent Ugp uptake system for glycerol-3-phosphate in Escherichia coli is trans inhibited by Pi.

sn-Glycerol-3-phosphate (G3P) or glyceryl phosphoryl phosphodiesters, the substrates of the phoB-dependent Ugp transport system, when transported exclusively through this system, can serve as a sole source of phosphate but not as a sole source of carbon (H. Schweizer, M. Argast, and W. Boos, J. Bacteriol. 150:1154-1163, 1982). In order to explain this phenomenon, we tested two possibilities: repression of the pho regulon by Ugp-mediated transport and feedback inhibition by internal G3P or its degradation product Pi. Using an ugp-lacZ fusion, we found that the expression of ugp does not decline upon exposure to G3P, in contrast to the repressing effect of transport of Pi via the Pst system. This indicated that the Ugp system becomes inhibited after the uptake and metabolism of G3P. Using 32P-labeled G3P, we observed that little Pi is released by cells taking up G3P via the Ugp system but large amounts of Pi are released when the cells are taking up G3P via the GlpT system. Using a glpD mutant that could not oxidize G3P but which could still phosphorylate exogenous glycerol to G3P after GlpF-mediated transport of glycerol, we could not find trans inhibition of Ugp-mediated uptake of exogenous 14C-G3P. However, when allowing uptake of Pi via Pst, we observed a time-dependent inhibition of 14C-G3P taken up by the Ugp transport system. Inhibition was half maximal after 2 min and could be elicited by Pi concentrations below 0.5 mM. Cells had to be starved for Pi in order to observe this inhibition. We conclude that the activity of the Ugp transport system is controlled by the level of internal phosphate.     

Dependence of Streptococcus lactis phosphate transport on internal phosphate concentration and internal pH.

Uptake of phosphate by Streptococcus lactis ML3 proceeds in the absence of a proton motive force, but requires the synthesis of ATP by either arginine or lactose metabolism. The appearance of free Pi internally in arginine-metabolizing cells corresponded quantitatively with the disappearance of extracellular phosphate. Phosphate transport was essentially unidirectional, and phosphate concentration gradients of up to 10(5) could be established. Substrate specificity studies of the transport system indicated no preference for either mono- or divalent phosphate anion. The activity of the phosphate transport system was affected by the intracellular Pi concentration by a feedback inhibition mechanism. Uncouplers and ionophores which dissipate the pH gradient across the cytoplasmic membrane inhibited phosphate transport at acidic but not at alkaline pH values, indicating that transport activity is regulated by the internal proton concentration. Phosphate uptake driven by arginine metabolism increased with the intracellular pH with a pKa of 7.3. Differences in transport activity with arginine and lactose as energy sources are discussed.     

Effects of hormones and N6O2''-dibutyryl-adenosine 3'' :5''-cyclic monophosphate, administered in vivo, on phosphate transport and metabolism in isolated rat liver mitochondria.

1. The administration of glucagon or N6O2'-dibutyryl cyclic AMP to fed rats by intraperitoneal injection was associated with a 2-fold increase in the amounts of endogenous Pi and ATP, and an increase in the rate and extent of transport of exogenous Pi (measured in either the presence or the absence of Ca2+) in mitochondria subsequently isolated from the liver. No change was observed in either the maximum rate of transport of exogenous Pi or in the rate of 32Pi exchange. 2. The changes induced by glucagon and dibutyryl cyclic AMP were markedly decreased by the co-administration of cycloheximide. 3. The administration of insulin to rats resulted in an increase of about 1.3-fold in the concentration of endogenous mitochondrial Pi 4. The amounts of endogenous Pi in mitochondrial isolated from the livers of starved rats were 3 times those in mitochondria isolated from fed animals. 5. It is concluded that the liver mitochondrial phosphatetransport system may be an important site of hormone action. 6. In the course of these experiments, it was shown that Ca2+ markedly stimulates mitochondrial phosphate transports.     

Phosphate exchange in the pit transport system in Escherichia coli.

The Pit system of phosphate transport in Escherichia coli catalyzes a rapid exchange between the external inorganic phosphate and internal phosphate pools, including some ester phosphates which are in rapid equilibrium with the internal Pi pool. Unlike net energized uptake, the Pi exchange proceeds in energy-depleted cells in the presence of uncouplers and is not accompanied by the movement of potassium ions. In the absence of externally added phosphate, the exit of Pi from the cells is insignificant. The apparent Km for external Pi in the exchange reaction is about 7 mM (2 orders of magnitude higher than that of energized uptake), but the maximal velocity is about the same. The exchange is temperature sensitive and is affected by thiol reagents. The combined observations suggest the operation of a facilitator which is part of the Pit system. The exchange is repressed in cells grown on glucose and other phosphotransferase system substrates, but not in cells grown on other carbohydrate sources. The repression can be reversed by the addition of cyclic AMP to the medium.     

Characterization of the Rhizobium (Sinorhizobium) meliloti high- and low-affinity phosphate uptake systems.

Genetic studies have suggested that Rhizobium (Sinorhizobium) meliloti contains two distinct phosphate (Pi) transport systems, encoded by the phoCDET genes and the orfA-pit genes, respectively. Here we present data which show that the ABC-type PhoCDET system has a high affinity for Pi (Km, 0.2 microM) and that Pi uptake by this system is severely inhibited by phosphonates. This high-affinity uptake system was induced under Pi-limiting conditions and was repressed in the presence of excess Pi. Uptake via the OrfA-Pit system was examined in (i) a phoC mutant which showed increased expression of the orfA-pit genes as a result of a promoter-up mutation and (ii) a phoB mutant (PhoB is required for phoCDET expression). Pi uptake in both strains exhibited saturation kinetics (Km, 1 to 2 microM) and was not inhibited by phosphonates. This uptake system was active in wild-type cells grown with excess Pi and appeared to be repressed when the cells were starved for Pi. Thus, our biochemical data show that the OrfA-Pit and PhoCDET uptake systems are differentially expressed depending on the state of the cell with respect to phosphate availability.     

A novel ATP-driven glucose transport system in Escherichia coli.

In Escherichia coli wild-type cells and in ATPase-deficient cells (unc mutants), glucose was found to be transported mainly by an ATP-driven system. The evidence is based on experiments involving interference at different sites of energy metabolism with the use of uncouplers, arsenate, and starved cells. Furthermore, addition of succinate to starved cells increased glucose uptake only in the wild-type cells, where ATP could be regenerated. Glucose transport was also ATP-dependent in cells deficient in methyl-beta-galactoside transport (a system that carries glucose specificity). It was found to be shock-sensitive in all strains tested. The NOVEL ATP-driven glucose transport is a high-affinity (Km 3-10 microM) and high-capacity (V 240-330 Mmol . min-1 . mg cell protein-1) uptake system.     

Increased adenine nucleotides in liver mitochondria after mannoheptulose injection in vivo

In adult rats, mannoheptulose injection causes a transient decrease in the serum insulin-to-glucagon ratio and a concomitant increase in serum glucose concentration. These effects attain a maximum 1 h after the injection and then decline toward normal. Correlated with the hormone changes is a dramatic increase in the adenine nucleotide content (ATP + ADP + AMP) of liver mitochondria, which peaks to over 50% of control values at 1 h. The increase in mitochondrial adenine nucleotides must occur by uptake from the cytosol, because the adenine nucleotide content of the whole tissue remains constant. The accumulation of adenine nucleotides by the mitochondria probably occurs over the recently characterized carboxyatractyloside-insensitive transport pathway that allows exchange of ATP-Mg for Pi. The actual mechanism by which net uptake is regulated after mannoheptulose injection has not yet been elucidated; however, changes in the Km or Vmax of the carrier and an increase in the tissue ATP/ADP ratio were eliminated as possibilities. The increase in matrix adenine nucleotide content in response to hormone changes brought about by mannoheptulose was much greater and more reproducible than what is achieved with glucagon injection. Mannoheptulose treatment may therefore be preferable as a model for further study of hormone effects on mitochondrial function.     

Regulation of arginine-ornithine exchange and the arginine deiminase pathway in Streptococcus lactis.

Streptococcus lactis metabolizes arginine by the arginine deiminase (ADI) pathway. Resting cells of S. lactis grown in the presence of galactose and arginine maintain a high intracellular ornithine pool in the absence of arginine and other exogenous energy sources. Addition of arginine results in a rapid release of ornithine concomitant with the uptake of arginine. Subsequent arginine metabolism results intracellularly in high citrulline and low ornithine pools. Arginine-ornithine exchange was shown to occur in a 1-to-1 ratio and to be independent of a proton motive force. The driving force for arginine uptake in intact cells is supplied by the ornithine and arginine concentration gradients formed during arginine metabolism. These results confirm studies of arginine and ornithine transport in membrane vesicles of S. lactis (A. J. M. Driessen, B. Poolman, R. Kiewiet, and W. N. Konings, Proc. Natl. Acad. Sci. USA, 84:6093-6097). The activity of the ADI pathway appears to be affected by the internal concentration of (adenine) nucleotides. Conditions which lower ATP consumption (dicyclohexylcarbodiimide, high pH) decrease the ADI pathway activity, whereas uncouplers and ionophores which stimulate ATP consumption increase the activity. The arginine-ornithine exchange activity matches the ADI pathway most probably by adjusting the intracellular levels of ornithine and arginine. Regulation of the ADI pathway and the arginine-ornithine exchanger at the level of enzyme synthesis is exerted by glucose (repressor, antagonized by cyclic AMP) and arginine (inducer). An arginine/ornithine antiport was also found in Streptococcus faecalis DS5, Streptococcus sanguis 12, and Streptococcus milleri RH1 type 2.     

Specificity and properties of the nucleotide carrier in chromaffin granules from bovine adrenal medulla.

1. The influence of various substances on the uptake of [3H]ATP and [14C]-noradrenaline into isolated bovine chromaffin granules was investigated. The carrier-mediated [3H]ATP uptake is specifically inhibited by SO42-, PO43- and phosphoenolpyruvate. Compounds with carboxylic acid or sulphonic acid groups had no significant inhibitory effects on either uptake. 2. 35SO42-, 32PO43- and phosphoenol[14C]pyruvate are taken up into chromaffin granules by a temperature-dependent process that is inhibited by atractyloside, uncouplers of oxidative phosphorylation and lipid-permeant anions. The apparent Km of 35SO42- uptake is 0.4 mM. 3. These results indicate that the nucleotide carrier in chromaffin granules has a broad specificity, transporting compounds with two strong negative charges. 4. Amino acid probes influence the uptake of ATP and catecholamines differently. Pyridoxal phosphate inhibits both uptake processes, 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid preferentially blocks ATP uptake, whereas phenylglyoxal blocks only ATP transport. It is suggested that the nucleotide carrier possesses arginine residues in a functionally important position. 5. The significance of these results obtained on isolated granules for the function of chromaffin granules within the cell is discussed.     

Pyrophosphate inhibition of carbon dioxide fixation in isolated pea chloroplasts by uptake in exchange for endogenous adenine nucleotides

Carbon dioxide-dependent O(2) evolution by isolated pea (Pisum sativum) chloroplasts was inhibited by inorganic pyrophosphate (PPi). Oxygen evolution was also inhibited by high concentrations of orthophosphate (Pi) and the inhibition was relieved by 3-phosphoglycerate. In contrast, the inhibition by PPi was not relieved by 3-phosphoglycerate, indicating that hydrolysis of PPi and accumulation of inhibitory concentrations of Pi were not occurring. In agreement with this suggestion, the percentage of (14)C-labeled products diffusing out of the chloroplasts was increased by Pi but not by PPi. The inhibition of O(2) evolution by PPi was reversed by ATP. The concentration of PPi required for 50% inhibition was 1.2 to 1.4 mm and the subsequent stimulation by ATP was half-maximal at 16 to 25 mum. Carbon dioxide-dependent O(2) evolution by spinach chloroplasts, or chloroplasts isolated from older pea plants, was not significantly inhibited by PPi.Chloroplasts were preloaded with (14)C-ATP and release of the labeled nucleotides was measured to assess the activity of adenine nucleotide transport across the inner chloroplast envelope membrane. A rapid exchange was promoted by the addition of exogenous ATP. Addition of PPi also resulted in a release of endogenous nucleotides. We suggest that PPi inhibits CO(2) fixation by entering the chloroplast in exchange for endogenous adenine nucleotides via the transporter on the inner envelope membrane. The subsequent depletion of the internal adenine nucleotide pool would result in decreased CO(2) fixation due to insufficient ATP. Addition of ATP to PPi-inhibited chloroplasts apparently results in uptake of catalytic amounts of ATP and restoration of the internal adenine nucleotide pool thus relieving the inhibition of CO(2) fixation.     

Mechanisms of Ca2+ transport in plasma membrane vesicles prepared from cultured pituitary cells. II. (Ca2+ + Mg2+)-ATPase-dependent Ca2+ transport activity

Purified plasma membrane vesicles from GH3 rat anterior pituitary cells exhibit a Mg2+-ATP-dependent Ca2+ transport activity. Concentrative uptake of Ca2+ is abolished by exclusion of either Mg2+ or ATP or by inclusion of the Ca2+ ionophore A23187. Furthermore, addition of A23187 to vesicles which have reached a steady state of ATP-supported Ca2+ accumulation rapidly and completely discharges accumulated cation. Ca2+ uptake is unaffected by treatment of vesicles with oligomycin, the uncoupler CCCP, or valinomycin and is greatly reduced in non-plasma membrane fractions. Likewise, Ca2+ accumulation is not stimulated by oxalate, consistent with the plasma membrane origin of this transport system. (Na+, K+)-ATPase participation in the Ca2+ transport process (i.e. via coupled Na+/Ca2+ exchange) was eliminated by omitting Na+ and including ouabain in the reaction medium. Ca2+ transport activity in GH3 vesicles has a similar pH dependence as that seen in a number of other plasma membrane systems and is inhibited by orthovanadate in the micromolar range. Inhibition is enhanced if the membranes are preincubated with vanadate for a short time. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ and ATP are 0.7 and 125 microM, respectively. The average Vmax is 3.6 nmol of Ca2+/min/mg of protein at 37 degrees C. Addition of exogenous calmodulin or calmodulin antagonists had no significant effect on these kinetic properties. GH3 plasma membranes also contain a Na+/Ca2+ exchange system. The apparent Km for Ca2+ is almost 10-fold higher in this system than that for ATP-driven Ca2+ uptake. When both processes are compared under similar conditions, the Vmax of the exchanger is approximately 2-3 times that of ATP-dependent Ca2+ accumulation. Similar results are obtained when purified plasma membranes from bovine anterior pituitary glands were investigated. It is suggested that both Na+/Ca2+ exchange and the (Ca2+ + Mg2+)-ATPase are important in controlling intracellular levels of Ca2+ in anterior pituitary cells.     

Fructose transport in Neurospora crassa.

A specific fructose uptake system (Km = 0.4 mM) appeared in Neurospora crassa when glucose-grown mycelia were starved. Fructose uptake had kinetics different from those of intramycelial fructose phosphorylation, and uptake appeared to be carrier mediated. The only sugar which competitively inhibited fructose uptake was L-sorbose (Ki = 9 mM). Glucose, 2-deoxyglucose, mannose, and 3-O-methyl glucose were noncompetitive inhibitors of fructose uptake. Incubation of glucose-grown mycelia with glucose, 2-deoxyglucose, or mannose prevented derepression of the fructose transport system, whereas incubation with 3-O-methyl glucose caused the appearance of five times as much fructose uptake activity as did starvation conditions.     

Transport of sugars in chick-embryo fibroblasts. Evidence for a low-affinity system and a high-affinity system for glucose transport.

The rate of D-glucose uptake by cells that had been deprived of sugar for 18-24h was consistently observed to be 15-20 times higher than that in control cells maintained for the same length of time in medium containing glucose. This increased rate of glucose transport by sugar-starved cells was due to a 3-5-fold increase in the Vmax. value of a low-affinity system (Km 1 mM) combined with an increase in the Vmax of a separate high-affinity system (Km 0.05-0.2 mM). The high-affinity system, which was most characteristic of starved cells, was particularly sensitive to low concentrations of the thiol reagent N-ethylmaleimide; 50% inhibition of uptake occurred at approx. 0.01 mM-N-ethylmaleimide. In contrast with the high-affinity system, the low-affinity system of either the fed cells or the starved cells was unaffected by N-ethylmaleimide. In addition to the increases in the rate of D-glucose transport, cells deprived of sugar had increased rates of transport of 3-O-methyl-D-glucose and 2-deoxy-D-glucose. No measurable high-affinity transport system could be demonstrated for the transport of 3-O-methylgucose, and N-ethylmaleimide did not alter the initial rate. Thus the transport of 3-O-methyglucose by both fed and starved cells was exclusively by the N-ethylmaleimide-insensitive low-affinity system. The low-affinity system also appeared to be the primary means for the transport of 2-deoxyglucose by fed and starved cells. However, some of the transport of 2-deoxyglucose by starved cells was inhibited by N-ethylmaleimide, suggesting that 2-deoxyglucose may also be transported by a high-affinity system. The results of experiments that measured transport kinetics strongly suggest that glucose can be transported by a least two separate systems, and 3-O-methylglucose and 2-deoxyglucose by one. Support for these interpretations comes from the analysis of the effects of N-ethylmaleimide and cycloheximide as well as from the results of competition experiments. The uptake of glucose is quite different from that of 2-deoxyglucose and 3-O-methylglucose. The net result of sugar starvation serves to emphasize these differences. The apparent de-repression of the transport systems studied presents an interesting basis for further studies of the regulation of transport in a variety of cells.     

The properties of arginine transport in vacuolar membrane vesicles of Neurospora crassa

We have measured the uptake of arginine into vacuolar membrane vesicles from Neurospora crassa. Arginine transport was found to be dependent on ATP hydrolysis, Mg2+, time, and vesicle protein with transported arginine remaining unmodified after entry into the vesicles. The Mg2+ concentration required for optimal arginine transport varied with the ATP concentration so that maximal transport occurred when the MgATP2- concentration was at a maximum and the concentrations of free ATP and Mg2+ were at a minimum. Arginine transport exhibited Michaelis-Menten kinetics when the arginine concentration was varied (Km = 0.4 mM). In contrast, arginine transport did not follow Michaelis-Menten kinetics when the MgATP2-concentration was varied (S0.5 = 0.12 mM). There was no inhibition of arginine transport when glutamine, ornithine, or lysine were included in the assay mixture. In contrast, arginine transport was inhibited 43% when D-arginine was present at a concentration 16-fold higher than that of L-arginine. Measurements of the internal vesicle volume established that arginine is concentrated 14-fold relative to the external concentration. Arginine transport was inhibited by dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl-hydrazone, and potassium nitrate (an inhibitor of vacuolar ATPase activity). Inhibitors of the plasma membrane or mitochondrial ATPase such as sodium vanadate or sodium azide did not affect arginine transport activity. In addition, arginine transport had a nucleoside triphosphate specificity similar to that of the vacuolar ATPase. These results suggest that arginine transport is dependent on vacuolar ATPase activity and an intact proton channel and proton gradient.     

Regulation of the glucose:H+ symporter by metabolite-activated ATP-dependent phosphorylation of HPr in Lactobacillus brevis.

Lactobacillus brevis takes up glucose and the nonmetabolizable glucose analog 2-deoxyglucose (2DG), as well as lactose and the nonmetabolizable lactose analoge thiomethyl beta-galactoside (TMG), via proton symport. Our earlier studies showed that TMG, previously accumulated in L. brevis cells via the lactose:H+ symporter, rapidly effluxes from L. brevis cells or vesicles upon addition of glucose and that glucose inhibits further accumulation of TMG. This regulation was shown to be mediated by a metabolite-activated protein kinase that phosphorylase serine 46 in the HPr protein. We have now analyzed the regulation of 2DG uptake and efflux and compared it with that of TMG. Uptake of 2DG was dependent on an energy source, effectively provided by intravesicular ATP or by extravesicular arginine which provides ATP via an ATP-generating system involving the arginine deiminase pathway. 2DG uptake into these vesicles was not inhibited, and preaccumulated 2DG did not efflux from them upon electroporation of fructose 1,6-diphosphate or gluconate 6-phosphate into the vesicles. Intravesicular but not extravesicular wild-type or H15A mutant HPr of Bacillus subtilis promoted inhibition (53 and 46%, respectively) of the permease in the presence of these metabolites. Counterflow experiments indicated that inhibition of 2DG uptake is due to the partial uncoupling of proton symport from sugar transport. Intravesicular S46A mutant HPr could not promote regulation of glucose permease activity when electroporated into the vesicles with or without the phosphorylated metabolites, but the S46D mutant protein promoted regulation, even in the absence of a metabolite. The Vmax but not the Km values for both TMG and 2DG uptake were affected. Uptake of the natural, metabolizable substrates of the lactose, glucose, mannose, and ribose permeases was inhibited by wild-type HPr in the presence of fructose 1,6-diphosphate or by S46D mutant HPr. These results establish that HPr serine phosphorylation by the ATP-dependent, metabolite-activated HPr kinase regulates glucose and lactose permease activities in L. brevis and suggest that other permeases may also be subject to this mode of regulation.     

Demembranated muscle fibers catalyze a more rapid exchange between phosphate and adenosine triphosphate than actomyosin subfragment 1

The rate of ATP in equilibrium with Pi exchange, that is, the incorporation of medium Pi into ATP during the net hydrolysis of ATP, has been measured for rabbit psoas muscle fibers, myofibrils, and actomyosin subfragment 1 (acto-S1). The maximum exchange rate in fibers at saturating [Pi] is 0.04 s-1 per myosin head at 8 degrees C, pH 7, and an ionic strength of 0.2 M. The dependence of the rate on Pi concentration can be approximated by a hyperbola with an apparent dissociation constant (Km) of 3 mM. Myofibrils catalyze ATP in equilibrium with Pi exchange with a similar Km but at a slightly lower rate. In contrast, the soluble acto-S1 system, in which ATP hydrolysis is not coupled to tension generation, catalyzes exchange at a rate 500 times lower than that of fibers at low Pi concentration, and the Km for Pi is greater than 50 mM. The difference between the ATP in equilibrium with Pi exchange of fibers and of acto-S1 is discussed in terms of a model in which Pi binds to a force-generating state AM'-ADP and, due to mechanical constraint, the average free energy of this state is higher in the fiber than in acto-S1.     

On the sidedness of membrane phosphorylation by Pi and ATP synthesis during reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles.

The membrane sidedness of Pi interaction in reactions which characterize reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was investigated. Vesicles previously loaded with calcium [32P]phosphate were incubated with 0.1 mM ADP and different concentrations of nonradioactive Pi. Alternatively, vesicles loaded with nonradioactive calcium phosphate were incubated in a medium containing 32Pi. The rates of Ca2+ efflux and ATP synthesis were siginficantly activated only when Pi was included in the assay medium. Although the Pi contained by the vesicles crosses the membrane at a rate proportional to the Ca2+ efflux, [gamma-32P]ATP was synthesized only when 32Pi interacted with the outer surface of the membrane. Similarly, ATP in equilibrium 32Pi or ITP in equilibrium 32Pi exchange could be measured only when the external pool of Pi was labeled. Both for ATP synthesis and for the ITP in equilibrium Pi exchange reaction, membrane phosphorylation by 32Pi was negligible unless the external pool of Pi was labeled. The ionophore X-537 A increased the rate of Ca2+ efflux but inhibited the synthesis of ATP. During reversal of the Ca2+ pump, Pi apparently interacts with the membrane only at the outer surface, and at a site different from that where Ca2+ crosses the membrane.     

A transport system for phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate in Salmonella typhimurium.

Salmonella typhimurium strain LT-2 was found to utilize phosphoenolpyruvate, 2-phosphoglycerate, and 3-phosphoglycerate as sole sources of carbon and energy for growth, but Escherichia coli strains did not. The following evidence suggests that this growth difference was due to the presence in Salmonella cells of an inducible phosphoglycerate permease distinct from previously studied transport systems: (a) The ability of cells to take up 3-phospho[14-C]glycerate was induced by growth in the presence of phosphoenolpyruvate, 2-phosphoglycerate, or 3-phosphoglycerate, but not glycerate, alpha-glycerophosphate, or other carbon sources tested. (b) Uptake of 3-phospho[14-C]glycerate was strongly inhibited by the three nonradioactive inducers of 3-phosphoglycerate uptake, but not by glycerate or alpha-glycerophosphate. (c) Mutants which lost the ability to utilize and take up 3-phosphoglycerate simultaneously lost the ability to utilize 2-phosphoglycerate and phosphoenolpyruvate, but not other compounds tested. (d) Mutant strains which constitutively synthesized the phosphoglycerate transport system could use both phosphoglycerates and phosphoenolpyruvate as sole sources of phosphate at low substrate concentrations. (e) A strain lacking alkaline and acid phosphatases could still grow with 3-phosphoglycerate as sole carbon source. Maximal rates of 3-phospho[14-C]glycerate uptake occurred at pH 6 in the presence of an exogenous energy source. The apparent Km for 3-phosphoglycerate uptake under these conditions was about 10-minus 4 M. The maximal uptake rate (but not the Km) was dependent on potassium ions. Although synthesis of the phosphoglycerate transport system appeared to be under adenosine 3:5-monophosphate control, glucose repressed induction only slightly. The genes controlling synthesis of the phosphoglycerate transport system (pgt genes) appeared to map at about 74 min on the Salmonella chromosome.     

Regulation of the oxidative phosphorylation rate in the intact cell

The mechanisms that underlie the balance between the consumption and oxidative generation of ATP in the intact cell are not well-defined. Cytosolic inorganic phosphate (Pi) and ADP levels, the cytosolic ATP/ADP ratio, and the cytosolic phosphorylation potential (PP) have all been proposed as major regulatory variables, the latter as a component of a "near-equilibrium" thermodynamic regulatory scheme. Therefore, the potential regulatory roles of these variables in the intact cell were evaluated with 31P NMR and Langendorff perfused rat hearts; in this preparation, the tissue oxygen consumption rate (MVO2) can be varied over a wide range. When the exogenous carbon source was varied, none of the proposed regulatory parameters, i.e., the ATP/ADP ratio, PP, or cytosolic ADP level, were found to be uniquely related to MVO2. Rather, ADP levels at a given MVO2 decreased progressively for the exogenous carbon sources in the following order: glucose, glucose + insulin, palmitate + glucose, lactate, pyruvate + glucose, and octanoate + glucose. In the octanoate and pyruvate groups, MVO2(-1) was linearly dependent upon [ADP]-1 with apparent Km values being in the range previously observed in isolated mitochondria. A similar trend was observed in the MVO2-[Pi] relationship. The present findings suggest that exogenous carbon sources which effectuate deregulation of intramitochondrial NADH generation lower cytosolic ADP and Pi to levels which are limiting to the rate of oxidative phosphorylation. For other carbon sources, the processes controlling the rate of NADH generation also participate in determining the rate of oxidative ATP synthesis. However, this control must be exerted kinetically rather than through a near-equilibrium thermodynamic mechanism as indicated by the present data and prior kinetic studies of the ATP synthetic process in both isolated mitochondria and intact myocardium [La Noue, K. F., et al. (1986) Biochemistry 25, 7667-7675; Kingsley-Hickman, P., et al. (1987) Biochemistry 26, 7501-7510].