Reconstitution of sugar phosphate transport systems of Escherichia coli

Studies with Escherichia coli cells showed that the transport systems encoded by glpT (sn-glycerol 3-phosphate transport) and uhpT (hexose phosphate transport) catalyze a reversible 32Pi:Pi exchange. This reaction could be used to monitor the glpT or uhpT activities during reconstitution. Membranes from suitably constructed strains were extracted with octylglucoside in the presence of lipid and glycerol, and proteoliposomes were formed by dilution in 0.1 M KPi (pH 7). Both reconstituted systems mediated a 32Pi:Pi exchange which was blocked by the appropriate heterologous substrate, sn-glycerol 3-phosphate (G3P) or 2-deoxyglucose 6-phosphate (2DG6P), with an apparent Ki near 50 microM. In the absence of an imposed cation-motive gradient, Pi-loaded proteoliposomes also transported the expected physiological substrate; Michaelis constants for the transport of G3P or 2DG6P were near 20 microM. The heterologous exchange showed a maximal velocity of 130 nmol/min/mg protein via the glpT system and 11 nmol/min/mg protein for the uhpT system. This difference was expected because the G3P transport activity had been reconstituted from a strain carrying multiple copies of the glpT gene. Taken together, these results suggest that anion exchange may be the molecular basis for transport by the glpT and uhpT proteins.     

Identification and functional reconstitution of phosphate: sugar phosphate antiport of Staphylococcus aureus

Resting cells of Staphylococcus aureus displayed a phosphate (Pi) exchange that was induced by growth with glucose 6-phosphate (G6P) or sn-glycerol 3-phosphate (G3P). Pi-loaded membrane vesicles from these cells accumulated 32Pi, 2-deoxyglucose 6-phosphate (2DG6P) or G3P by an electroneutral exchange that required no external source of energy. On the other hand, when vesicles were loaded with morpholinopropane sulfonic acid (MOPS), only transport of 32Pi (and L-histidine) was observed, and in that case transport depended on addition of an oxidizable substrate (DL-lactate). In such MOPS-loaded vesicles, accumulation of the organic phosphates, 2DG6P and G3P, could not be observed until vesicles were preincubated with both Pi and DL-lactate to establish an internal pool of Pi. This trans effect demonstrates that movement of 2DG6P or G3P is based on an antiport (exchange) with internal Pi. Reconstitution of membrane protein allowed a quantitative analysis of Pi-linked exchange. Pi-loaded proteoliposomes and membrane vesicles had comparable activities for the homologous 32Pi: Pi exchange (Kt's of 2.2 and 1.4 mM; Vmax's of 180 and 83 nmol Pi/min per mg protein), indicating that the exchange reaction was recovered intact in the artificial system. Other work showed that heterologous exchange from either G6P- or G3P-grown cells had a preference for 2DG6P (Kt = 27 microM) over G3P (Kt = 1.3 mM) and Pi (Kt = 2.2 mM), suggesting that the same antiporter was induced in both cases. We conclude that 32Pi: Pi exchange exhibited by resting cells reflects operation of an antiporter with high specificity for sugar 6-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of phosphate:hexose 6-phosphate antiport in membrane vesicles of Streptococcus lactis

Membrane vesicles of Streptococcus lactis were used to characterize a novel anion exchange involving phosphate and sugar 6-phosphates. For vesicles loaded with 50 mM phosphate at pH 7, homologous phosphate:phosphate exchange had a maximal rate of 130 nmol/min/mg of protein and a Kt of 0.21 mM external phosphate; among phosphate analogues tested, only arsenate replaced phosphate. Heterologous exchange was studied by 2-deoxyglucose 6-phosphate entry into phosphate-loaded vesicles; this reaction had a maximal velocity of 31 nmol/min/mg of protein and a Kt of 26 microM external substrate. Sugar phosphate moved intact during this exchange, since its entry led to loss of internal 32Pi without transfer of 32P to sugar phosphate. Inhibitions of phosphate exchange suggested that the preferred sugar phosphate substrates were (Kiapp): glucose, 2-deoxyglucose, and mannose 6-phosphates (approximately 20 microM) greater than fructose 6-phosphate (150 microM) greater than glucosamine 6-phosphate (420 microM) greater than alpha-methylglucoside 6-phosphate (740 microM). Stoichiometry for phosphate:2-deoxyglucose 6-phosphate antiport was 2:1 at pH 7, and since initial rates of exchange were unaffected by charge carrying ionophores (gramicidin, valinomycin, a protonophore), this unequal stoichiometry indicated the electroneutral exchange of two monovalent phosphates for a single divalent sugar phosphate.     

Transport in C4 mesophyll chloroplasts: evidence for an exchange of inorganic phosphate and phosphoenolpyruvate.

1. Mesophyll chloroplasts of the C4 plant Digitaria sanguinalis contain endogenous phosphoenolpyruvate which appears to distribute across the envelope according to the existing pH gradient. The phosphoenolpyruvate remaining in the stroma can be rapidly released by external inorganic phosphate or 3-phosphoglycerate while external pyruvate did not affect the distribution. 2. Phosphoenolpyruvate (PEP) was a competitive inhibitor (Ki (PEP) = 450 micrometer) of 32Pi uptake (Km(Pi)=200 micrometer) by chloroplasts in the dark and also reduced the steady-state internal concentration of 32Pi, which is consistent with phosphate and phosphoenolpyruvate sharing a common carrier. 3. Phosphoenolpyruvate formation by chloroplasts in the light in the presence of pyruvate but in the absence of inorganic phosphate was slow and the concentration ratio of phosphoenolpyruvate (internal/external) was high. Addition of 0.1 mM phosphate induced a high rate of phosphoenolpyruvate formation and the concentration ratio (internal/external) decreased 15-fold. It is proposed that external phosphate is required both for phosphoenolpyruvate formation and efflux from the chloroplast.     

Altered substrate selectivity in a mutant of an intrahelical salt bridge in UhpT, the sugar phosphate carrier of Escherichia coli

Site-directed and second site suppressor mutagenesis identify an intrahelical salt bridge in the eleventh transmembrane segment of UhpT, the sugar phosphate carrier of Escherichia coli. Glucose 6-phosphate (G6P) transport by UhpT is inactivated if cysteine replaces either Asp388 or Lys391 but not if both are replaced. This suggests that Asp388 and Lys391 are involved in an intrahelical salt bridge and that neither is required for normal UhpT function. This interpretation is strengthened by the finding that mutations at Lys391 (K391N, K391Q, and K391T) are recovered as revertants of the inactive D388C variant. Further work shows that although the D388C variant is null for G6P transport, movement of 32Pi by homologous Pi/Pi exchange is unaffected. This raises the possibility that this derivative may have latent function, a possibility confirmed by showing that D388C is a gain-of-function mutation in which phosphoenolpyruvate (PEP) is the preferred substrate. Added study of the Pi/Pi exchange shows that in wild type UhpT this partial reaction is readily blocked by G6P but not PEP. By contrast, in the D388C variant, Pi/Pi exchange is unaffected by G6P but is inhibited by both PEP and 3-phosphoglycerate. These latter substrates are used by PgtP, a related Pi-linked antiporter, which lacks the Asp388-Lys391 salt bridge but has instead an uncompensated arginine at position 391. For this reason, we conclude that in both UhpT and PgtP position 391 can serve as a determinant of substrate selectivity by acting as a receptor for the anionic carboxyl brought into the translocation pathway by PEP.     

Protein phosphatase 1 catalyses the direct hydrolytic cleavage of phosphate monoester in a ternary complex mechanism

The catalytic subunit of the Ser/Thr protein phosphatase 1 (PP1cat) hydrolyses N-acetyl Arg-Arg-Ala-phosphoThr-Val-Ala (K(M) = 3.7 mM) in a reaction that is inhibited competitively by inorganic phosphate (Pi, Ki = 1.6 mM) but unaffected by the product peptide alcohol at concentrations up to 3 mM. The enzyme does not catalyse the incorporation of 18O-label from 18O-labelled water into Pi whether, or not, the product alcohol is present. The dephosphorylated product alcohol of phosphorylated histone. an alternative substrate for the enzyme, serves as a competitive inhibitor for phosphopeptide hydrolysis (Ki = 60 microM) and co-mediates 18O-label exchange into Pi in a concentration-dependent manner (K(M) = 64 microM). These results indicate that hydrolysis occurs through the direct attack of an activated water molecule on the phosphate ester moiety of the substrate in a ternary complex mechanism.     

Bacterial anion exchange. Use of osmolytes during solubilization and reconstitution of phosphate-linked antiport from Streptococcus lactis

Membranes of Streptococcus lactis were solubilized with 1.1% octyl-beta-D-glucopyranoside in the presence of 0.37% acetone/ether-washed phospholipid from several sources. After adding excess Escherichia coli phospholipid as bath-sonicated liposomes, phosphate:sugar phosphate antiport was reconstituted in proteoliposomes by a 25-fold dilution in 0.1 M KPi (pH 7). Assays of 32Pi:Pi exchange showed that antiport was subject to an inactivation which varied in severity according to the lipid present at solubilization. Recovery of Pi-linked exchange was improved by the presence of 10-20% glycerol or other osmolyte during extraction. The osmolytes tested in this regard have included polyols (glycerol, erythritol, xylitol, sorbitol), sugars (glucose, trehalose), and two amino acids (glycine, proline). Each gave 10--20-fold increased recoveries of 32Pi:Pi antiport compared to controls using only detergent and lipid; these precautions were not required for the efficient reconstitution of F0F1-ATPase. Antiport in the artificial system was studied most carefully when glycerol was the stabilizing additive. For that case, the Kt values for Pi or 2-deoxyglucose 6-phosphate transport (275 and 25 microM, respectively) were the same as in native membranes. Maximal rates of Pi and 2-deoxyglucose 6-phosphate transport (200 and 42 nmol/min/mg of protein, respectively) and the turnover number for Pi exchange (25--50/s) suggested that antiporters were recovered without loss of activity. We conclude that the quantitative aspects of bacterial anion exchange are amenable to study in an artificial system, and that the use of osmolytes as general stabilants can be a valuable adjunct to current techniques for reconstitution of integral membrane transport proteins.     

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.     

Reaction intermediate analogues for enolase

A number of compounds that appear to be analogues of the aci form of the normal carbanion intermediate are good inhibitors of yeast enolase. These include (3-hydroxy-2-nitropropyl)phosphonate (I), the ionized (pK = 8.1) nitronate form of which in the presence of 5 mM Mg2+ has a Ki of 6 nM, (nitroethyl)phosphonate (III) (pK = 8.5; Ki of the nitronate in the presence of 5 mM Mg2+ = 1 microM), phosphonoacetohydroxamate (IV) (pK = 10.2; Ki with saturating Mg2+ for the ionized form = 15 pM), and (phosphonoethyl)nitrolate (VII) (Ki at 1 mM Mg2+ = 14 nM). The oxime of phosphonopyruvate (VI) has a pH-independent Ki of 75 microM. I, IV, VI, and VII are slow binding inhibitors. All of these compounds are trigonal at the position analogous to C-2 of 2-phosphonoglycerate and contain a phosphono group, but a negatively charged metal ligand at the position isosteric with the hydroxyl attached to C-3 of 2-phosphoglycerate (as in IV) appears to contribute more to binding than a nitro group isosteric with the carboxyl of 2-phosphoglycerate (I and III). These data support the carbanion mechanism for enolase and suggest that the 3-hydroxyl of 2-phosphoglycerate is directly coordinated to Mg2+ prior to being eliminated to give phosphoenolpyruvate.     

The Cyanelles (Organelles of a Low Evolutionary Scale) Possess a Phosphate-Translocator and a Glucose-Carrier in Cyanophora paradoxa

Cyanelles from Cyanophora paradoxa can easily be isolated and assayed for their carrier composition by the silicone oil filtering technique. The present investigation demonstrates a P_i-translocator transferring phosphate, dihydroxyacetone phosphate and 3-phosphoglycerate in a counter exchange mode in cyanelles as in chloroplasts of higher plants. The uptake of P_i is inhibited by dihydroxyacetone phosphate, phosphoglycerate and glucose-6-P, only poorly by phosphoenolpyruvate and not by 2-phosphoglycerate. The inhibitors pyridoxalphosphate and 4,4′diisothiocyanostilbene-2,2K'disulfonic acid at low concentration also affect P_i-uptake. Cyanelles probably transport photosynthate (reductant and ATP) by triosephosphates. This is the first demonstration of a phosphate translocator in an organism of a low evolutionary scale. Cyanelles also transport glucose which proceeds in two phases. In the lower concentration range (≤ 2.5 mM), glucose penetrates by facilitated diffusion, whereas transport follows first-order kinetics at higher amounts (> 2.5 mM). In the low concentration range, glucose-transport is affected by high concentrations of 3-O-methylglucose and fructose. The physiological role of the glucose-transport carrier in Cyanophora is doubtful. It may function in transporting glucose into cyanelles if the carbon level inside them becomes limiting, e.g. in dark periods.     

Transport of inorganic phosphate and C3- and C6-sugar phosphates across the envelope membranes of potato tuber amyloplasts

Amyloplasts have been isolated from tubers of potato plants (Solarium tuberosum. cv. Desirée). As it is difficult to isolate amyloplasts that have a high starch content, we used transformed plants in which the content of starch was reduced. This was achieved by decreasing the activity of ADP-glucose pyrophosphorylase by antisense techniques (Müller-Röber et al., 1992, EMBO. 11, 1229–1238). In the isolated plastids the activity of glutamine-oxoglutarate-aminotransferase (glutamate synthase, EC 2.6.1.53) was dependent upon the intactness of the plastids. For the supply of redox equivalents the addition of glucose-6-phosphate (Glc6P) was required. Glucose-1-phosphate (Glc1P) did not support glutamate synthesis. Plastids were treated with Triton X-100 and the solubilized proteins reconstituted into liposomes. Transport measurements with these liposomes revealed that inorganic phosphate (Pi), dihydroxyacetone phosphate (DHAP), 3-phosphoglycerate and Glc6P are transported in a counter-exchange mode. Transport of phosphoenolpyruvate was low and Glc1P was virtually not transported in exchange for Pi. Kinetic constants were determined for the Pi/Pi and Glc6P/Pi counter exchanges. For comparison, proteins of mitochondria from potato tubers and pea leaves were reconstituted into liposomes. As expected, the Pi/Pi exchange across the mitochondrial membrane was not affected by DHAP and Glc6P. Kinetic constants of the Pi/Pi counter exchange were determined for potato tuber mitochondria.Abbreviations DHAP dihydroxyacetone phosphate - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - PEP Phosphoenolpyruvate - 3-PGA 3-phosphoglycerate - Pi inorganic phosphate - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl] glycine This work was supported by Deutsche Forschungsgemeinschaft.     

Kinetics of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 ATPase

The rate of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 was measured as a function of the ATP concentration in the presence of inhibitors [ADP, Pi and 3'-O-(1-naphthoyl)ATP]. ATP hydrolysis can be described by Michaelis-Menten kinetics with Km(TF1) = 390 microM and Km (TF0F1) = 180 microM. The inhibition constants are for ADP Ki(TF1) = 20 microM and Ki(TF0F1) = 100 microM, for 3'-O-(1-naphthoyl)ATP Ki(TF1) = 150 microM and Ki(TF0F1) = 3 microM, and for Pi Ki(TF1) = 60 mM. From these results it is concluded that upon binding of TF0 to TF1 the mechanism of ATP hydrolysis catalyzed by TF1 is not changed qualitatively; however, the kinetic constants differ quantitatively.     

Transport of 3-phosphoglyceric acid,phosphoenolpyruvate, and inorganic phosphate in maize mesophyll chloroplasts,and the effect of 3-phosphoglyceric acid on malate and phosphoenolpyruvate production

Maize mesophyll chloroplasts loaded with radioactively labeled 3-phosphoglycerate or phosphoenolpyruvate exchange these compounds for externally provided inorganic phosphate, 3-phosphoglycerate, phosphoenolpyruvate, and dihydroxyacetone phosphate. These exchanges are inhibited by pyridoxal phosphate. 3-Phosphoglycerate uptake, which leads to accumulation of this substance in the stroma, is competitively inhibited by inorganic phosphate and phosphoenolpyruvate. These results are consistent with the transport of 3-phosphoglycerate, phosphoenolpyruvate, inorganic phosphate, and dihydroxyacetone phosphate being mediated by a common carrier (the phosphate translocator). The activation energy of 3-phosphoglycerate uptake as determined from its temperature dependence is 19.5 kcal (4–15 °C). In isolated chloroplasts malate and phosphoenolpyruvate production from oxalacetate and pyruvate, respectively, is inhibited by 3-phosphoglycerate, the extent of inhibition being dependent on the relative concentrations of inorganic phosphate and 3-phosphoglycerate. We propose that 3-phosphoglycerate from bundle-sheath cells may serve as a feedback regulator of mesophyll cell photosynthesis.     

Asymmetric orientation of the reconstituted aspartate/glutamate carrier from mitochondria

A partially purified preparation of the aspartate/glutamate carrier from bovine heart mitochondria was reconstituted into liposomal membranes by chromatography on hydrophobic ion exchange resins. Based on the favorable conditions of this reconstituted system the transmembrane orientation of the inserted carrier protein could be determined by functional analysis. For reliable measurement of the reconstituted aspartate-glutamate exchange activity an optimized inhibitor-stop technique using pyridoxal phosphate was developed. By simultaneous application of both forward and backward exchange experiments the practical usefulness of the reconstituted system could be extended to investigations including variation of internal and external substrate concentrations over a wide range. Thereby a complete set of Km values for both aspartate and glutamate at both the internal and external side of the proteoliposomes could be established. These experiments led to the following results and conclusions: (i) The observed substrate affinities are clearly different for the two different membrane sides both for aspartate (external 50 microM, internal 3 mM) and glutamate (external about 200 microM, internal 3 mM). (ii) The exclusive presence of only one type of transport affinity for every single substrate at one side of the liposomal membrane clearly demonstrates the asymmetric orientation of the functionally active carrier protein molecules. (iii) When comparing the values of these constants with published data obtained in mitochondria, an inside-out orientation of the aspartate/glutamate carrier after isolation and reinsertion into liposomes is strongly suggested.     

Mitochondrial phosphate transport. N-ethylmaleimide insensitivity correlates with absence of beef heart-like Cys42 from the Saccharomyces cerevisiae phosphate transport protein

The mitochondrial phosphate transport protein (PTP) has been purified in a reconstitutively active form from Saccharomyces cerevisiae and Candida parapsilosis. ADP/ATP carriers that copurify have been identified. The PTP from S. cerevisiae migrates as a single band (35 kDa) in sodium dodecyl sulfate gels with the same mobility as the N-ethylmaleimide-alkylated beef heart PTP. It does not cross-react with anti-sera against beef heart PTP. The CNBr peptide maps of the yeast and beef proteins are very different. The rate of unidirectional phosphate uptake into reconstituted proteoliposomes is stimulated about 2.5-fold to a Vmax of 170 mumol of phosphate min-1 (mg PTP)-1 (22 degrees C) by increasing the pHi of the proteoliposomes from 6.8 (same as pHe) to 8.0. The Km for Pi of this reconstituted activity is 2.2 mM. The transport is sensitive to mersalyl (50% inhibition at 60 microM) and insensitive to N-ethylmaleimide. We have purified peptides matching the highly conserved motif Pro-X-(Asp/glu)-X-X-(Lys/Arg)-X-(Arg/lys) (X is an unspecified amino acid) of the triplicate gene structure sequence of the beef heart PTP. The N-ethylmaleimide-reactive Cys42 of the beef heart protein, located between the two basic amino acids of this motif (Lys41-Cys42-Arg43), is replaced with a Thr in the yeast protein. This substitution most likely is responsible for the lack of N-ethylmaleimide sensitivity of the yeast protein and mersalyl thus reacts with another cysteine to inhibit the transport. Finally it is concluded that Cys42 has no essential role in the catalysis of inorganic phosphate transport by the mitochondrial phosphate transport protein.     

Effect of some glycolytic intermediates and palmitoyl-CoA on alpha-glycerophosphate dehydrogenase in mitochondria isolated from liver of triiodothyronine-treated rats

alpha-Glycerophosphate dehydrogenase (EC 1.1.99.5) in mitochondria from liver of the triiodothyronine-treated rats is competitively inhibited by phosphoenolpyruvate, glyceraldehyde 3-phosphate and 3-phosphoglycerate, the apparent Ki values for phosphoenolpyruvate being 0.76 mM at pH 7.0, 1.7 mM at pH 7.4 and 3.5 mM at pH 7.7. The apparent Ki values for glyceraldehyde 3-phosphate and 3-phosphoglycerate are also pH-dependent. Other glycolytic intermediates, such as 2-phosphoglycerate, 2,3-diphosphoglycerate, pyruvate, glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-diphosphate did not alter significantly alpha-glycerophosphate dehydrogenase activity. Palmitoyl-CoA is a competitive inhibitor of this enzyme, with Ki value of about 30 micron.     

Pyruvate kinase isozymes from the green alga, Selenastrum minutum. II. Kinetic and regulatory properties

The kinetic and regulatory properties of two pyruvate kinase isozymes, PKp and PKc (apparent chloroplastic and cytosolic isozymes, respectively) from the green alga Selenastrum minutum were studied. The two isozymes differed greatly in several kinetic properties. Although both isozymes showed hyperbolic substrate saturation kinetics, the apparent Michaelis constants for PEP and ADP were about twofold and fourfold lower, respectively, for PKc as compared with PKp. ADP was the preferred nucleotide substrate for both isozymes. However, PKc utilized alternate nucleotides far more effectively than did PKp. PKc and PKp also differed strongly in the effect of activators and inhibitors on the enzymes. Although both isozymes were activated by dihydroxyacetone phosphate (DHAP) with a similar activation constant of about 30 microM, this activator (0.5 mM) caused an approximate 30% increase in the Vmax of PKc, but had no effect on the Vmax of PKp. PKp, but not PKc, was inhibited by ribose 5-phosphate, ribulose 1,5-bisphosphate, 2-phosphoglycerate, phosphoglycolate, and malate. Both isozymes were inhibited by MgATP, Mg2citrate, Mg2oxalate, and Pi. PKc was far more sensitive to inhibition by Pi, as compared with PKp. Pi was a competitive inhibitor of PKc with respect to phosphoenolpyruvate (PEP) (Ki = 1.3 mM). Glutamate was a potent inhibitor of PKc, but had no effect on PKp. In contrast with Pi, glutamate was a mixed-type inhibitor of PKc with respect to PEP (Ki = 0.7 mM). DHAP facilitated the binding of PEP by both isozymes and reversed or relieved the inhibition of PKc by Pi and/or glutamate. The regulatory properties of PKp indicate that it is likely less active in the light and more active in the dark. The in vivo activity of PKc is probably regulated by the relative cytosolic levels of DHAP, Pi, and glutamate; this provides a rationale for the activation of algal cytosolic pyruvate kinase which occurs during periods of enhanced ammonia assimilation.     

Effect of arsenate on inorganic phosphate transport in Escherichia coli.

The effect of arsenate on strains dependent on the two major inorganic phosphate (Pi) transport systems in Escherichia coli was examined in cells grown in 1 mM phosphate medium. The development of arsenate-resistant Pi uptake in a strain dependent upon the Pst (phosphate specific transport) system was examined. The growth rate of Pst-dependent cells in arsenate-containing medium was a function of the arsenate-to-Pi ratio. Growth in arsenate-containing medium was not due to detoxification of the arsenate. Kinetic studies revealed that cells grown with a 10-fold excess of arsenate to Pi have almost a twofold increase in capacity (Vmax) for Pi, but maintained the same affinity (Km). Pi accumulation in the Pst-dependent strain was still sensitive to changes in the arsenate-to-Pi ratio, and a Ki (arsenate) for Pi transport of 39 microM arsenate was determined. The Pst-dependent strain did not accumulate radioactive arsenate, and showed only a transient decrease in intracellular adenosine triphosphate levels after arsenate was added to the medium. The Pi transport-dependent strain ceased growth in arsenate-containing media. This strain accumulated 74As-arsenate, and intracellular adenosine triphosphate pools were almost completely depleted after the addition of arsenate to the medium. Arsenate accumulation required a metabolizable energy source and was inhibited by N-ethylmaleimide. Previously accumulated arsenate could exchange with arsenate or Pi in the medium.     

Pseudomonas aeruginosa possesses two novel regulatory isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase

In Pseudomonas aeruginosa the initial enzyme of aromatic amino acid biosynthesis, 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase, has been known to be subject to feedback inhibition by a metabolite in each of the three major pathway branchlets. Thus, an apparent balanced multieffector control is mediated by L-tyrosine, by L-tryptophan, and phenylpyruvate. We have now resolved DAHP synthase into two distinctive regulatory isozymes, herein denoted DAHP synthase-tyr (Mr = 137,000) and DAHP synthase-trp (Mr = 175,000). DAHP synthase-tyr comprises greater than 90% of the total activity. L-Tyrosine was found to be a potent effector, inhibiting competitively with respect to both phosphoenolpyruvate (Ki = 23 microM) and erythrose 4-phosphate (Ki = 23 microM). Phenylpyruvate was a less effective competitive inhibitor: phosphoenolpyruvate (Ki = 2.55 mM) and erythrose 4-phosphate (Ki = 1.35 mM). DAHP synthase-trp was found to be inhibited noncompetitively by L-tryptophan with respect to phosphoenolpyruvate (Ki = 40 microM) and competitively with respect to erythrose 4-phosphate (Ki = 5 microM). Chorismate was a relatively weak competitive inhibitor: phosphoenolpyruvate (Ki = 1.35 mM) and erythrose 4-phosphate (Ki = 2.25 mM). Thus, each isozyme is strongly inhibited by an amino acid end product and weakly inhibited by an intermediary metabolite.     

Oxalate:formate exchange. The basis for energy coupling in Oxalobacter

In the Gram-negative anaerobe, Oxalobacter formigenes, the generation of metabolic energy depends on the transport and decarboxylation of oxalate. We have now used assays of reconstitution to study the movements of oxalate and to characterize the exchange of oxalate with formate, its immediate metabolic derivative. Membranes of O. formigenes were solubilized with octyl-beta-D-glucopyranoside in the presence of 20% glycerol and Escherichia coli phospholipid, and detergent extracts were reconstituted by detergent dilution. [14C]Oxalate was taken up by proteoliposomes loaded with unlabeled oxalate, but not by similarly loaded liposomes or by proteoliposomes containing sulfate in place of oxalate. Oxalate transport did not depend on the presence of sodium or potassium, nor was it affected by valinomycin (1 microM), nigericin (1 microM), or a proton conductor, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (5 microM) when potassium was at equal concentration on either side of the membrane. Such data suggest the presence of an overall neutral oxalate self-exchange, independent of common cations or anions. Kinetic analysis of the reaction in proteoliposomes gave a Michaelis constant (Kt) for oxalate transport of 0.24 mM and a maximal velocity (Vmax) of 99 mumol/min/mg of protein. A direct exchange of oxalate and formate was indicated by the observations that formate inhibited oxalate transport and that delayed addition of formate released [14C]oxalate accumulated during oxalate exchange. Moreover, [14C]formate was taken up by oxalate-loaded proteoliposomes (but not liposomes), and this heterologous reaction could be blocked by external oxalate. Further studies, using formate-loaded proteoliposomes, suggested that the heterologous exchange was electrogenic. Thus, for assays in which N-methylglucamine served as both internal and external cation, formate-loaded particles took up oxalate at a rate of 2.4 mumol/min/mg of protein. When external or internal N-methylglucamine was replaced by potassium in the presence of valinomycin, there was, respectively, a 7-fold stimulation or an 8-fold inhibition of oxalate accumulation, demonstrating that net negative charge moved in parallel with oxalate during the heterologous exchange. The work summarized here suggests the presence of an unusually rapid and electrogenic oxalate2-:formate1- antiport in membranes of O. formigenes. Since a proton is consumed during the intracellular decarboxylation that converts oxalate into formate plus CO2, antiport of oxalate and formate would play a central role in a biochemical cycle consisting of (a) oxalate influx, (b) oxalate decarboxylation, and (c) formate efflux.(ABSTRACT TRUNCATED AT 400 WORDS)