Correction

Phosphoribosylpyrophosphate synthetase (PRS; EC 2.7.6.1) from Hevea brasiliensis Mull. Arg. latex was located in the cytosol. After purification, its apparent molecular weight under nondenaturing conditions was estimated at 200,000 [plus or minus] 10,000; a single band at 57,000 [plus or minus] 3,000 was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme seemed to be a homotetramer. Its affinity constants were estimated at 200 [plus or minus] 30 [mu]M for adenosine triphosphate and 40 [plus or minus] 2 [mu]M for ribose-5-phosphate. The purified enzyme proved to be functional in a paraphysiological medium (cytosol deproteinized by ultrafiltration). Optimum pH was 7.5 in buffer and 6.5 in a paraphysiological medium. No PRS activity was detected in the absence of the Mg2+ ion. Of the numerous compounds tested, only Mn2+, phosphoribosylpyrophosphate and inorganic phosphate affected the enzymatic reaction. Mn2+ (inhibitor constant = 20 [mu]M) and phosphoribosylpyrophosphate (inhibitor constant = 30 [mu]M) were inhibitors. PRS responded allosterically (Hill's coefficient = 2.3) to ribose-5-phosphate in the presence of a physiological concentration of inorganic phosphate (10 mM). These results are set in the physiological context of laticifers.     

Acetylene Reduction by Symbiosomes and Free Bacteroids from Broad Bean (Vicia faba L.) Nodules (Role of Oxalate)

We report the presence of oxalate in the organic acid fraction of broad bean (Vicia faba L.) nodule cytosol. Using both high-performance liquid chromatography and enzymic assays, high levels of oxalate were detected (70.4 [plus or minus] 2.4 mM). To study the potential role of oxalate as an energy-yielding substrate for nitrogenase activity, free bacteroids were isolated from nodules and found to oxidize oxalate in support of C2H2 reduction under O2 tensions that were lower than those required to oxidize succinate, another dicarboxylate commonly detected in legume nodules. Symbiosomes of broad bean, isolated for the first time from amide-producing nodules, were provided with [14C]oxalate and found to have uptake kinetics with a lower affinity [Km(oxalate) = 330 [mu]M] than that for free bacteroids [Km(oxalate) = 130 [mu]M]. In anaerobic preparations of symbiosomes supplied with purified oxyleghemoglobin, O2 consumption was stimulated by oxalate from 20.2 [plus or minus] 0.8 nmol O2 min-1mg-1 protein to 24.5 [plus or minus] 1.1 nmol O2 min-1 mg-1 protein but always remained lower than the rate of O2 consumption in free bacteroids (32.2 [plus or minus] 1.4 nmol O2 min-1 mg-1 protein). Under these conditions, C2H2 reduction activity was 9.7 [plus or minus] 0.8 and 15.1 [plus or minus] 0.9 nmol C2H4 min-1 mg-1 protein for symbiosomes and bacteroids, respectively. These data support the suggestion that oxalate may play a role as a carbon substrate in support of N2 fixation in broad bean nodules.     

2,4-Dichlorophenoxyacetic Acid and Related Chlorinated Compounds Inhibit Two Auxin-Regulated Type-III Tobacco Glutathione S-Transferases

Two auxin-inducible glutathione S-transferase (GST, EC 2.5.1.18) isozymes from tobacco (Nicotiana tabacum, White Burley) were partially characterized. GST1-1 and GST2-1 are members of a recently identified new type of plant GST isozymes that we will here refer to as type III. Both enzymes were active, with 1-chloro-2,4-dinitrobenzene as a substrate, when expressed in bacteria as fusion proteins. The apparent Km for 1-chloro-2,4-dinitrobenzene was found to be 0.85 [plus or minus] 0.25 mM for GST1-1 and 0.20 [plus or minus] 0.15 mM for GST2-1. The apparent Km for glutathione was similar for both enzymes, 0.40 [plus or minus] 0.15 mM. The in vitro activity of both enzymes could be inhibited by the synthetic auxin 2,4-dichlorophenoxyacetic acid, with an apparent Ki of 80 [plus or minus] 40 [mu]M for GST1-1 and 200 [plus or minus] 100 [mu]M for GST2-1. The GST1-1 was also inhibited by structurally related substances, such as 2,4-dichlorobenzoic acid, with a roughly similar Ki. The nonchlorinated structures benzoic acid and phenoxyacetic acid did not inhibit. p-Chloroisobutyric acid, or clofibric acid, an auxin-transport inhibitor, was found to be an active inhibitor as well. The strongest inhibitor identified, however, was a phenylacetic acid derivative, ethacrynic acid, which showed an apparent Ki of 5 [plus or minus] 5 [mu]M for both enzymes. This substance is a known inducer as well as a substrate of specific mammalian GSTs. The results presented here indicate that the type III plant GSTs might be involved in the metabolism or transport of chlorinated substances that are structurally related to auxins. The possibility that auxins are endogenous ligands or substrates for GSTs is discussed.     

Characterization of Water Channels in Wheat Root Membrane Vesicles

The functional significance of water channels in wheat (Triticum aestivum L.) root membranes was assessed using light scattering to measure vesicle shrinking in response to osmotic gradients rapidly imposed in a stopped flow apparatus. Vesicles were obtained from both a plasma membrane fraction and a plasma membrane-depleted endomembrane fraction including tonoplast vesicles. Osmotic water permeability (Pos) in the endomembrane fraction was high (Pos= 86.0 [mu]m s-1) with a low activation energy (EA= 23.32 kJ mol-1 [plus or minus] 3.88 SE), and was inhibited by mercurials (K1= 40 [mu]M HgCl2, where K1 is the inhibition constant for half-maximal inhibition), suggesting participation of water channels. A high ratio of osmotic to diffusional permeability (Pd) (using D2O as a tracer, Pos/Pd = 7 [plus or minus] 0.5 SE) also supported this view. For the endomembrane fraction there was a marked decrease in Pos with increasing osmotic gradient that was not observed in the plasma membrane fraction. Osmotic water permeability in the plasma membrane fraction was lower (Pos= 12.5 [mu]m s-1) with a high activation energy (EA= 48.07 kJ mol-1 [plus or minus] 3.63 SE) and no mercury inhibition. Nevertheless, Pos/Pd was found to be substantially higher than one (Pos= 3 [plus or minus] 0.2 SE), indicating that water channels mediated water flow in this fraction, too. Possible distortion of the Pos/Pd value by unstirred layer effects was shown to be unlikely.     

Identification and Preliminary Characterization of a Ca2+- Dependent High-Affinity Binding Site for Inositol-1,4,5-Trisphosphate from Chenopodium rubrum

Using a radioligand-binding assay we have identified a Ca2+- dependent high-affinity D-myo-inositol-1,4,5-trisphosphate (InsP3) binding site in a membrane vesicle preparation from Chenopodium rubrum. Millimolar concentrations of Ca2+ were required to observe specific binding of [3H]InsP3. A stable equilibrium between bound and free ligand was established within 5 min and bound [3H]InsP3 could be completely displaced by InsP3 in a time- and concentration-dependent manner. Displacement assays indicated a single class of binding sites with an estimated dissociation constant of 142 [plus or minus] 17 nM. Other inositol phosphates bound to the receptor with much lower affinity. The glycosaminoglycan heparin was an effective competitor for the binding site (inhibitor concentration for 50% displacement = 534 nM). ATP at higher, although physiologically relevant, concentrations (inhibitor concentration for 50% displacement = 241 [mu]M) also displaced [3H]InsP3 from the receptor. Recent studies in animals have highlighted the importance of Ca2+ regulation of InsP3-induced Ca2+ release. The potential for the operation of similar regulatory mechanisms in plants is discussed.     

Oxidative Stress Affects [alpha]- Tocopherol Content in Soybean Embryonic Axes upon Imbibition and following Germination

The content of [alpha]-tocopherol ([alpha]T) in isolated soybean (Glycine max, var Hood) embryonic axes was measured upon germination. Dry, high-vigor axes contained 1.2 [plus or minus] 0.1, nmol/axis and after an increase during the initial 6 h of imbibition, there was a decline to 1.0 [plus or minus] 0.1 nmol/axis at 24 h of incubation. Incubation in the presence of the redox-cycling agent paraquat (4 mM) for 24 h increased the [alpha]T content to 1.9 [plus or minus] 0.2 nmol/axis. When the incubation medium was supplemented with 500 [mu]M Fe-EDTA over 24 h, the content of [alpha]T increased to 1.8 [plus or minus] 0.1 nmol/axis. Isolated axes from soybean seeds stored for 56 months contained 6.5 [plus or minus] 0.3 nmol of [alpha]T/axis after 24 h of imbibition as compared to 1.0 [plus or minus] 0.1 nmol of [alpha]T/axis in axes from soybean seeds stored for 8 months. In all of these experimental situations, oxidant production as assessed in vivo by a fluorometric assay was increased by 4 mM paraquat (8-fold), 500 [mu]M iron (2-fold), and 56 months of storage (4-fold) after 24 h of imbibition. The data presented here suggest that the cellular content of [alpha]T is physiologically adjusted as a response to conditions of oxidative stress.     

Characterization of vanadate-based transition-state-analogue complexes of phosphoglucomutase by spectral and NMR techniques

Near ultraviolet spectral studies were conducted on two inhibitor complexes obtained by treating the dephospho form of the phosphoglucomutase.Mg2+ complex with inorganic vanadate in the presence of either glucose 1-phosphate [cf. Percival, M. D., Doherty, K., & Gresser, M. J. (1990) Biochemistry (first of four papers in this issue)] or glucose 6-phosphate. Part of the spectral differences between the two inhibitor complexes arises because the glucose phosphate moiety in the complex derived from glucose 1-phosphate binds to the enzyme in a different way from the glucose phosphate moiety in the complex derived from glucose 6-phosphate and because these alternative binding modes produce different environmental effects on the aromatic chromophores of the dephospho enzyme. These spectral differences are strikingly similar to those induced by the binding of glucose 1-phosphate and glucose 6-phosphate to the phospho enzyme--which shows that the glucose 1-phosphate and glucose 6-phosphate moieties occupy positions in the inhibitor complexes closely related to those that they occupy in their respective catalytically competent complexes. This binding congruity indicates that in the inhibitor complexes the oxyvanadium grouping is bound at the site where (PO3-) transfer normally occurs. 31P NMR studies of the phosphate group in these complexes also provide support for this binding pattern. A number of other systems based on compounds with altered structures, such as the deoxysugar phosphates, or systems with different compositions, as in the case of the metal-free enzyme or of the glucose phosphates plus nitrate, also were examined for evidence that complexes analogous to the inhibitor complexes were formed, but none was found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbonic Anhydrase Activity in Isolated Chloroplasts of Wild-Type and High-CO2-Dependent Mutants of Chlamydomonas reinhardtii as Studied by a New Assay

In an assay of carbonic anhydrase (CA), NAH14CO3 soltution at the bottom of a sealed vessel releases 14CO2, which diffuses to the top of the vessel to be assimilated by photosynthesizing Chlamydomonas reinhardtii cells that have been adapted to a low-CO2 environment. The assay is initiated by illuminating the cells and is stopped by turning the light off and killing the cells with acid. Enzyme activity was estimated from acid-stable radioactivity. With bovine CA, 1.5 Wilbur-Anderson units (WAU) was consistently measured at 5- to 6-fold above background. Sonicated whole cells of air-adapted wild-type C. reinhardtii had 740 [plus or minus] 12.4 WAU/mg chlorophyll (Chl). Sonicated chloroplasts from a mixotrophically grown wall-less strain, cw-15, had 35.5 [plus or minus] 2.6 WAU/mg Chl, whereas chloroplasts from wall-less external CA mutant strain cia5/cw-15 had 33.8 [plus or minus] 1.9 WAU/mg Chl. Sonicated chloroplasts from the wall-less mutant strain cia-3/cw-15, believed to lack an internal CA, had 2.8 [plus or minus] 3.2 WAU/mg Chl. Sonicated whole cells from cia3/cw-15 had 2.8 [plus or minus] 7.8 WAU/mg Chl. Acetazolamide, ethoxyzolamide, and p-aminomethylbenzene sulfonamide (Mafenide) at 100 [mu]M inhibited CA in sonicated chloroplasts from cia-5/cw-15. Treatment at 80[deg]C for 10 min inhibited this CA activity by 90.8 [plus or minus] 3.6%. Thus, a sensitive 14C assay has confirmed the presence of a CA in cw-15 and cia-5/cw-15 chloroplasts and the lack of a CA in cia-3/cw-15 chloroplasts. Our results indicate that HCO3- is the inorganic carbon species that is accumulated by chloroplasts of Chlamydomonas and that chloroplastic CA is responsible for the majority of internal CA activity.     

Phosphoribosylpyrophosphate synthesis from glucose decreases during amino acid starvation of human lymphoblasts

When cultured human lymphoblasts are starved 3 h for an essential amino acid, rates of purine nucleotide synthesis decrease markedly because of a decrease in the intracellular phosphoribosylpyrophosphate concentration (Boss, G.R., and Erbe, R.W. (1982) J. Biol. Chem. 257, 4242-4247; Boss, G. R. (1984) J. Biol. Chem. 259, 2936-2941). In amino acid-starved cells, glucose transport was not changed, whereas total glucose consumption and lactate production decreased by approximately 25 and 10%, respectively. Carbon flow through the oxidative pentose phosphate pathway, measured by 14CO2 release from [1-14C]glucose, decreased by 18% during amino acid starvation. However, kinetic studies of ribulose-5-phosphate 3-epimerase and phosphoriboisomerase suggested that the ribulose 5-phosphate produced by this pathway is converted mostly to xylulose 5-phosphate instead of to ribose 5-phosphate so that this pathway produces little phosphoribosylpyrophosphate. The activity of the nonoxidative pentose phosphate pathway, measured by high performance liquid chromatography following the incorporation of [1-14C]glucose into phosphoribosylpyrophosphate, ATP, and GTP, decreased by approximately 55% during amino acid starvation. None of the enzymes of either pathway changed in specific activity during amino acid starvation. We conclude that the nonoxidative pentose phosphate pathway is the major source of phosphoribosylpyrophosphate for purine nucleotide synthesis and that this pathway is regulated by a metabolite which changes in concentration during amino acid starvation.     

Increased phosphoribosylpyrophosphate synthetase activity in fibroblasts of hypoxanthine-guanine phosphoribosyl transferase deficient patients.

An increased activity of phosphoribosylpyrophosphate synthetase at physiological levels of inorganic phosphate is demonstrated in extracts of skin fibroblast cultures derived from a patient with Lesch-Nyhan syndrome. This eccessive response of the phosphoribosylpyrophosphate synthetase at physiological levels of inorganic phosphate results in increased levels of phosphoribosylpyrophosphate and thus contributes to purine overproduction characteristic of this disorder. The level of enzyme response in skin fibroblast extracts from the carrier mother was between activity of the patient and normals, further suggesting the x-linkage of human phosphoribosylpyrophosphate synthetase.     

Substrate Kinetics of the Plant Mitochondrial Alternative Oxidase and the Effects of Pyruvate

The kinetics of alternative oxidase (AOX) of Arum italicum spadices and soybean (Glycine max L.) cotyledons were studied both with intact mitochondria and with a solubilized, partially purified enzyme. Ubiquinone analogs were screened for their suitability as substrates and ubiquinol-1 was found to be most suitable. The kinetics of ubiquinol-1 oxidation via AOX in both systems followed Michaelis-Menten kinetics, suggesting that the reaction is limited by a single-step substrate reaction. The kinetics are quite different from those previously described, in which the redox state of ubiquinone-10 was monitored and an increase in substrate was accompanied by a decrease in product. The difference between the systems is discussed. Pyruvate is a potent activator of the enzyme and its presence is essential for maximum activity. The addition of pyruvate to the solubilized enzyme increased the maximum initial velocity from 6.2 [plus or minus] 1.3 to 16.9 [plus or minus] 2.8 [mu]mol O2 mg-1 protein min-1 but had little effect on the Michaelis constant for ubiquinol-1, an analog of ubiquinol, which changed from 116 [plus or minus] 73 to 157 [plus or minus] 68 [mu]M. It is concluded that pyruvate (and presumably other keto acids) increases the activity of AOX but does not increase its affinity for its substrate. In agreement with this is the finding that removal of pyruvate (using lactate dehydrogenase and NADH) leads to an 80 to 90% decrease in the reaction rate, suggesting that pyruvate is important in the mechanism of reaction of AOX. The removal of pyruvate from the enzyme required turnover, suggesting that pyruvate is bound to the enzyme and is released during turnover.     

Effect of Mn2+ and Mg2+ ions on DNA conformation

The DNA conformation was studied at different relation between Na+ and Me2+ (Mn2+ or Mg2+) ions in solution at the fixed total ionic strength mu. At low mu the intrinsic viscosity of DNA [eta] decreased to the limited fixed value with the increasing of Mn2+ or Mg2+ concentration (CMe2+). At higher mu greater than or equal to 0.1 M [eta] doesn't depend on CMe2+. The presence of Mn2+ in solution caused a decrease of the optical anisotropy of DNA and the value of epsilon 260 (p) independent on ionic strengths. In contrary, these parameters of DNA didn't change in solution with Mg2+-concentration. The observed differences in the effects of Mn2+ and Mg2+ on the optical properties of the macromolecule suggest that there are different modes of binding of these ions to DNA. It has been concluded, that Mn2+ interacts with bases and phosphate groups of DNA, but Mg2+--only with phosphates. The persistence length of DNA doesn't depend on Me2+ concentration under the conditions of the experiment (mu greater than or equal to 0.005 M).     

Purification and Characterization of Geranyl Diphosphate Synthase from Vitis vinifera L. cv Muscat de Frontignan Cell Cultures

A geranyl diphosphate synthase (EC 2.5.1.1), which catalyzes the formation of geranyl diphosphate from dimethylallyl diphosphate and isopentenyl diphosphate, was isolated from Vitis vinifera L. cv Muscat de Frontignan cell cultures. Purification of the enzyme was achieved successively by ammonium sulfate precipitation and chromatography on DEAE-Sephacel, hydroxylapatite, Mono Q, Phenyl Superose, Superose 12, and preparative nondenaturing polyacrylamide gels. The enzyme formed only geranyl diphosphate as a product. In all cases, neither neryl diphosphate, the cis isomer, nor farnesyl diphosphate was detected. The enzyme showed a native molecular mass of 68 [plus or minus] 5 kD as determined by gel permeation. On sodium dodecyl sulfate polyacrylamide gels, geranyl diphosphate synthase purified to electrophoretic homogeneity migrated with a molecular mass of 66 [plus or minus] 2 kD. Michaelis constants for isopentenyl diphosphate and dimethylallyl diphosphate were 8.5 and 56.8 [mu]M, respectively. The enzyme required Mn2+ and Mg2+ as cofactors and its activity was enhanced by Triton X-100. Inorganic pyrophosphate, aminophenylethyl diphosphate, and geranyl diphosphate had inhibitory effects on the enzyme.     

The enthalpies of hydrolysis of acyclic, monocyclic, and glycoside cyclic phosphate diesters.

The enthalpies of hydrolysis of acyclic, monocyclic, and glycoside cyclic phosphate diesters have been measured by flow microcalorimetry using a phosphohydrolase isolated from Enterobacter aerogenes as catalyst. The values so obtained (kilocalories per mol) (at 25 degrees) for sodium salts are: diethyl phosphate, minus 1.8 plus or minus 0.5; ethylene phosphate, minus 6.4 plus or minus 0.2; trimethylene phosphate, minus 3.0 plus or minus 0.2; tetramethylene phosphate, minus 2.2 plus or minus 0.1; methyl beta-D-ribofuranoside cyclic 3:5-phosphate, minus 11.1 plus or minus 0.2; methyl alpha-D-glucopyranoside cyclic 4:6-phosphate, minus 6.3 plus or minus 0.1; and cyclic adenosine 3:5-monophosphate (5-ester bond), minus 11.1 plus or minus 0.4 (10-minus 3 M Mg-2+). The enthalpy of hydrolysis of the 3-ester bond of cyclic adenosine 3:5-monophosphate (10-minus 3 M Mg-2+) has been revised to minus 11.1 plus or minus 0.2 kcal/mol from the value of minus 13.2 plus or minus 0.4 kcal/mol reported previously (greengard, p., rudolph, s.a., and sturtevant, j. m. (1969) j. biol. Chem. 244, 4798). All these values pertain to the hydrolysis of singly charged diesters to form singly charged monoesters. The data for the acyclic and monocyclic phosphodiesters are in qualitative agreement with their hydrolytic reactivities. The enthalpies measured for the hydrolysis of the glycoside cyclic phosphates cannot now be explained on the basis of their structures or reactivities; perhaps a contribution to the enthalpies by solvation or a previously unrecognized geometric strain effect may be responsible for the large exothermic enthalpies of these cyclic phosphate diesters. Changes in the heat capacity, increment Cp, for some of the hydrolytic reactions were also measured.     

Essential Arginyl Residue at the Active Site of Pyrophosphate:Fructose 6-Phosphate 1-Phosphotransferase from Potato (Solanum tuberosum) Tuber

The aim of this work was to test the proposal that the active site of pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) contains an essential arginyl residue. Enzyme activity was inhibited equally in the glycolytic and gluconeogenic directions by arginine-modifying reagents. The second-order rate constants for 2,3-butanedione and phenylglyoxal were 13.1 [plus or minus] 0.45 and 55.3 [plus or minus] 1.3 M-1 min-1, respectively. The corresponding values for the kinetic order of inactivation by these modifying reagents were 0.84 [plus or minus] 0.049 for 2,3-butanedione and 0.89 [plus or minus] 0.052 for phenylglyoxal. The substrates, fructose 6-phosphate and pyrophosphate, and a range of substrate analogs protected the enzyme from inactivation by 2,3-butanedione. These data suggest that modification of no more than one arginyl residue at, or close to, the active site is required to inhibit the enzyme. This result supports the proposal that the active site of PFP in plants is equivalent to that of the bacterial ATP-phosphofructokinase (S.M. Carlisle, S.D. Blakeley, S.M. Hemmingsen, S.J. Trevanion, T. Hiyoshi, N.J. Kruger, and D.T. Dennis [1990] J Biol Chem 265: 18366-18371).     

Substrate-cofactor interactions for glycogen phosphorylase b: a binding study in the crystal with heptenitol and heptulose 2-phosphate

The structural relationships between substrate and pyridoxal phosphate in glycogen phosphorylase b (EC 2.4.1.1) have been studied by X-ray diffraction experiments at 3-A resolution. Recent work [Klein, H. W., Im, M. J., & Helmreich, E. J. M. (1984) in Chemical and Biological Aspects of Vitamin B6 Catalysis (Evangelopoulos, A. E., Ed.) pp 147-160, Liss, New York] has shown that phosphorylase in the presence of inorganic phosphate catalyzes the conversion of heptenitol to heptulose 2-phosphate. The latter compound is a dead-end product and a most potent inhibitor (Ki = 14 microM). The X-ray diffraction studies show that heptenitol binds at the catalytic site of phosphorylase in a position essentially identical with that observed for the glucopyranose moiety of glucose 1-phosphate. Incubation of a phosphorylase b crystal for 50 h in a solution containing the substrates heptenitol and inorganic phosphate and the activators AMP and maltohetaose resulted in the formation of a phosphorylated product bound at the active site. The structure of this product, as analyzed by a difference Fourier synthesis at 3 A, is consistent with that of heptulose 2-phosphate. Analysis of the surrounding soak solution by thin-layer chromatography showed that heptulose 2-phosphate was produced under these conditions. Heptulose 2-phosphate binds with its glucopyranose moiety in the same position as that for glucose 1-phosphate, but there is a marked difference in phosphate positions. The presence of the methyl group in the beta-configuration in heptulose 2-phosphate forces a change in the torsion angle O5-C1-O1-P from 117 degrees as observe in glucose 1-phosphate to -136 degrees in heptulose 2-phosphate. The "down" position of the phosphate (with respect to the crystallographic z axis) results in a change in the distance between the 5'-phosphorus atom of the pyridoxal phosphate and the phosphorus atom of the substrate from 6.8 (with glucose 1-phosphate) to 4.5 A (with heptulose 2-phosphate). The closest distance between the phosphate oxygen of the cofactor and a phosphate oxygen of heptulose 2-phosphate is 2.7 A, and it is assumed that there must be a hydrogen bond between them. These observations are consistent with the NMR experiments reported in the preceding paper in which sharing of a proton between heptulose 2-phosphate and pyridoxal 5'-phosphate is observed [Klein, H.W., Im, M. J., Palm, D., & Helmreich, E. J. M. (1984) Biochemistry (preceding paper in this issue)].(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of High-Affinity Slow Anion Channel Blockers and Evidence for Stomatal Regulation by Slow Anion Channels in Guard Cells

Slow anion channels in the plasma membrane of guard cells have been suggested to constitute an important control mechanism for long-term ion efflux, which produces stomatal closing. Identification of pharmacological blockers of these slow anion channels is instrumental for understanding plant anion channel function and structure. Patch clamp studies were performed on guard cell protoplasts to identify specific extracellular inhibitors of slow anion channels. Extracellular application of the anion channel blockers NPPB and IAA-94 produced a strong inhibition of slow anion channels in the physiological voltage range with half inhibition constants (K1/2) of 7 and 10 [mu]M, respectively. Single slow anion channels that had a high open probability at depolarized potentials were identified. Anion channels had a main conductance state of 33 [plus or minus] 8 pS and were inhibited by IAA-94. DIDS, which has been shown to be a potent blocker of rapid anion channels in guard cells (K1/2 = 0.2 [mu]M), blocked less than 20% of peak slow anion currents at extracellular or cytosolic concentrations of 100 [mu]M. The pharmacological properties of slow anion channels described here differ from those recently described for rapid anion channels in guard cells, fortifying the finding that two highly distinct types or modes of voltage- and second messenger-dependent anion channel currents coexist in the guard cell plasma membrane. Bioassays using anion channel blockers provide evidence that slow anion channel currents play a substantial role in the regulation of stomatal closing. Interestingly, slow anion channels may also function as a negative regulator during stomatal opening under the experimental conditions applied here. The identification of specific blockers of slow anion channels reported here permits detailed studies of cell biological functions, modulation, and structural components of slow anion channels in guard cells and other higher plant cells.     

Phosphoenolpyruvate carboxykinase in mouse pancreatic islets. ATP-induced changes in sensitivity to Mn2+ activation

The presence of high phosphoenolpyruvate carboxykinase (EC 4.1.1.32) activity in mouse islet cytosol has been demonstrated. The enzyme was activated by Mn2+ with a Ka of 100 X 10(-6) mol/l. The mean total activity of the Mn2+-stimulated phosphoenolpyruvate carboxykinase in islet cytosol estimated at 22 degrees C with saturating concentrations of the substrates oxaloacetate and ITP was 146 pmol/min per micrograms DNA. Km was calculated to be 6 X 10(-6) mol/l for oxaloacetate and 140 X 10(-6) mol/l for ITP. The islet phosphoenolpyruvate carboxykinase activity was not increased after starvation of the animals for 48 h. Preincubation of the cytosol at 4 degrees C with Fe2+, quinolinate, ATP, Pi, glucose 6-phosphate, fructose 1,6-bisphosphate, NAD+, NADH, oxaloacetate, ITP, cyclic AMP and Ca2+ had no effect on the enzyme activity. However, preincubation of the cytosol at 37 degrees C with ATP-Mg inhibited the Mn2+-stimulated phosphoenolpyruvate carboxykinase activity progressively with time and in a concentration-dependent manner. A similar but weaker inhibitory effect was observed with p[NH]ppA, whereas p[CH2]ppA, ADP, AMP, adenosine and Pi had no effect. It is tentatively suggested that ATP and p[NH]ppA either by adenylation or otherwise affect the interaction between islet phosphoenolpyruvate carboxykinase and the recently discovered Mr = 29000 protein modulator of the enzyme in such a way - perhaps by causing a dissociation between them - that phosphoenolpyruvate carboxykinase loses its sensitivity to Mn2+ activation.     

Simultaneous Measurement of Intracellular pH and K+ or NO3- in Barley Root Cells Using Triple-Barreled,Ion-Selective Microelectrodes

The manufacture and use of triple-barreled microelectrodes, which are capable of simultaneous in vivo measurement of intracellular pH and the activities of K+ or NO3- and cell membrane potential (Em), are described. Scanning electron micrographs showed that the three tips were aligned and that the overall tip diameter was approximately 0.8 [mu]m. When filled with 100 mM KCl, all three barrels simultaneously reported identical transmembrane potentials, showing that all three tips were located in the same subcellular compartment. Intracellular estimates of Em in barley (Hordeum vulgare L. cv Klaxon) root epidermal cells obtained with these triple-barreled microelectrodes were indistinguishable from those obtained using single- or double-barreled microelectrodes. Measurements made with triple-barreled K+ and pH-selective microelectrodes in root cells of 7-d-old barley plants grown in a nutrient solution containing 0.5 mM K+ yielded cytosolic and vacuolar populations having mean K+ activity values of 71.3 and 68.7 mM, respectively. The associated mean pH values ([plus or minus]SE) were 7.26 [plus or minus] 0.06 (cytosol) and 5.18 [plus or minus] 0.08 (vacuole). Analysis of whole-tissue digests confirmed the microelectrode measurements. Measurements made using triple-barreled pH- and nitrate-selective microelectrodes confirmed earlier double-barreled measurements of pH and nitrate in barley root epidermal cells growing in 10 mM nitrate.     

A new assay for the determination of phosphoribosylamine.

This report describes a new assay for glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) (EC 2.4.2.14) based on the formation of a stable compound between the ribose-5-phosphate moiety of phosphoribosylamine and [³⁵S]cysteine. The assay is comparable in sensitivity to the [¹⁴C]glutamine assay, and its potential in the evaluation of NH₃ utilization by this enzyme is documented. The stoichiometry of the enzymatic reaction and the analysis of the ribose-5-phosphate/cysteine compound demonstrate that 1 mol of phosphoribosylamine (or ribose-5-phosphate) combines with 1 mol of [³⁵S] cysteine to form this compound. Preliminary studies with a number of carbohydrates indicate that an aldopentose phosphate is most reactive with cysteine. The substitution of a phosphate, pyrophosphate, or purine ring at C-1 of the pentose markedly reduces its reactivity with cysteine.