Purification and characterization of phosphoribulokinase from wheat leaves

Homogeneous phosphoribulokinase (PRK; ATP: d-ribulose-5-phosphate 1-phosphotransferase, EC 2.7.1.19) was isolated from wheat leaves with a specific activity of 15 kat mg^-1 protein. The purification included ammonium sulfate cuts, isoelectric precipitation, and hydrophobic and affinity chromatography on pentylagarose and Blue Sepharose CL 6B, respectively. Gel filtration of the purified enzyme yielded a 83000 Da protein. Subunits of about 42000 Da were estimated from sodium dodecyl sulfate-polyacrylamide gels. Wheat leaf PRK was stable for at least four weeks when stored at 4°C. Saturation curves for ribulose 5-phosphate (Ru5P) and ATP followed Michaelis-Menten kinetics (K _m values: K _{m Ru5P}=50–80 M; K _{m ATP}=70 M). The saturation curve for MgCl₂ was sigmoidal (half-maximal velocity <0.5 mM). The affinity for Ru5P, ATP and Mg²⁺ was not affected by pH changes comparable to pH shifts in the stroma. In contrast to chloroplast fructose-bisphosphatase (Zimmermann et al. 1976, Eur. J. Biochem. 70, 361–367) the affinity for ligands remained unchanged in the dithiothreitol-activated and in the non-activated state. The activity of PRK was increasingly sensitive to inhibition by 3-phosphoglyceric acid with decreasing pH below pH 8.0.Abbreviations DTT dithiothreitol - EDTA ethylenediamine-tetraacetic acid - PRK phosphoribulokinase - Ru5P ribulose-5-phosphate - SDS-PAGE sodium dodecyl sulfate-polyacryl-amide gel electrophoresis     

Effects of potassium on the anion and cation contents of primary cultures of mouse astrocytes and neurons

Inastrocytes, as [K⁺]_o was increased from 1.2 to 10 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. As [K⁺]_o was increased from 10 to 60 mM, intracellular concentration of these three ions showed no significant change. When [K⁺]_o was increased from 60 to 122 mM, an increase in [K⁺]_i and [Cl^–]_i and a decrease in [Na⁺]_i were observed.Inneurons, as [K⁺]_o was increased from 1.2 to 2.8 mM, [Na⁺]_i and [Cl^–]_i were decreased, whereas [K⁺]_i was increased. As [K⁺]_o was increased from 2.8 to 30 mM, [K⁺]_i, [Na⁺]_i and [Cl^–]_i showed no significant change. When [K⁺]_o was increased from 30 to 122 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. Inastrocytes, pH_i increased when [K⁺]_o was increased. Inneurons, there was a biphasic change in pH_i. In lower [K⁺]_o (1.2–2.8 mM) pH_i decreased as [K⁺]_o increased, whereas in higher [K⁺]_o (2.8–122 mM) pH_i was directly related to [K⁺]_o. In bothastrocytes andneurons, changes in [K⁺]_o did not affect the extracellular water content, whereas the intracellular water content increased as the [K⁺]_o increased. Transmembrane potential (E_m) as measured with Tl-204 was inversely related to [K⁺]_o between 1.2 and 90 mM, a ten-fold increase in [K⁺]_o depolarized the astrocytes by about 56 mV and the neurons about 52 mV. The E_m values measured with Tl-204 were close to the potassium equilibrium potential (E_k) except those in neurons at lower [K⁺]_o. However, they were not equal to the chloride equilibrium potential (E_Cl) at [K⁺]_o lower than 30 mM in both astrocytes and neurons. Results of this study demonstrate that alteration of [K⁺]_o produced different changes in [K⁺]_i, [Na⁺]_i, [Cl^–]_i, and pH_i in astrocytes and neurons. The data show that astrocytes can adapt to alterations in [K⁺]_o, in such a way to maintain a more suitable environment for neurons.     

Cyclic AMP-dependent regulation of activities of synthetase and phosphodiesterase of 2′,5′-oligoadenylate in NIH 3T3 cells

Summary Dihydrofolate synthetase (EC 6.3.2.12) from N. gonorrhoeae was isolated and enzyme characteristics were determined. The purified enzyme was found quite stable when stored at –60 °C. About 50% of the enzyme activity wag destroyed within 6 weeks when kept at 4 °C. Maximum velocity was observed at pH 9.3. The enzyme required a monovalent cation, K⁺ or NH₄ ⁺ , and divalent cation, Mg²⁺ or Mn²⁺ for its function. ATP at 5 mM concentration gave maximum activity. K_m values for dihydropteroate and L-glutamate at pH 9.3 were 3.5 × 10^–5 M and 6.5 × 10^–4 M, respectively. Patterns of product inhibition by dihydrofolate were found to be non-competitive with respect to dihydropteroate, having a K_i value of 5.1 ± 0.8 × 10^–4 M, and competitive with respect to L-glutamate, having a K_i value of 6.2 × 10^–4 M.     

The thermodynamic efficiency of the Ca2+-Mg2+-ATPase is one hundred percent

The thermodynamic efficiency of the Ca²⁺-Mg²⁺-ATPase of skeletal sarcoplasmic reticulum has been evaluated by comparing the Ca²⁺ gradient established with the ATP/(ADP*P_i) ratio. The evaluation was made at an external Ca²⁺ level (4.7 × 10^–8 M) which is below theK _m value of 7 × 10^–8 M. The Mg-ATP and phosphate concentrations were held constant (0.1 mM) and the ADP concentration was varied. Maximal uptake to an internal free Ca²⁺ concentration of 17 mM was observed at infinite ATP/(ADP*P_i) ratio (absence of ADP). This corresponds to a [Ca²⁺]_i/[Ca²⁺]₀ gradient of 3.6 × 10⁵. A Ca²⁺ gradient one-half as large was observed at an ATP/(ADP*P_i) ratio of 3.5 × 10³ M^–1. The square of the Ca²⁺ gradient is shown to be proportional to the ATP/(ADP*P_i) ratio, for finite values of the latter. The proportionality constant is identical to the equilibrium constant for hydrolysis of ATP (9.02 × 10⁶ M) under these conditions (0.1 mM Mg²⁺, 30°C). The intrinsic thermodynamic efficiency of the pump is shown to be 100%, with a maximal uncertainty of 3%. The efficiency is lower under less optimal conditions, when the pump is inhibited and passive leak processes compete.Dedicated to Prof. Philip George, University of Pennsylvania, whose instruction, research, and example made this contribution possible.     

Methodology for assessing respiration and cellular incorporation of radiolabeled substrates by soil microbial communities

A method is described for determining biodegradation kinetics of both naturally occurring and xenobiotic compounds in surface and sub-surface soil samples. The method measures both respiration and uptake into cellular biomass of¹⁴C-labeled substrates. The estimation of biomass incorporation entailed removal of cells from soil particles by washing the soil with a polyvinyl-pyrrolidone/pyrophosphate solution and H₂O₂. After separation of the cells and the soil particles by centrifugation, the cells were trapped on membrane filters for liquid scintillation counting. Mass balances were easily obtained. The technique was used to measure metabolic activity in soil profiles, including unsaturated and saturated zones. First order rate constants (K₁) were in the range of 10^–3–10^–2 hour^–1 for amino acid metabolism and 10^–5–10^–4 hour^–1 for m-cresol metabolism. Saturation kinetics were observed for amino acids and m-cresol. m-Cresol K₁ values for uptake often exceeded those for respiration by greater than a factor of ten. V_max values were low (amino acids, 10¹–10² ng g^–1 hour^–1; m-cresol, 10^–1 ng g^–1 hour^–1), whereas K_m values were quite high (amino acids, 10³–10⁴ ng g^–1; m-cresol 10³–10⁵ ng g^–1). Saturation was not observed in many horizons even at 10⁵ ng g^–1 dry soil. Frequently, respiration obeyed saturation kinetics whereas uptake was first order. It is concluded that measuring only kinetics of respiration may lead to severe underestimations of biodegradation rates.     

Human and bovine brain cathepsin L and cathepsin H: Purification,physico-chemical properties,and specificity

Cathespin L (EC 3.4.22.15) and cathepsin H (EC 3.4.22.16) have been purified from brain cortex to apparent homogeneity by a simultaneous procedure involving acid extraction of homogenate at pH 4.2, ammonium sulfate fractionation (30–80%), chromatography on pepstatin-Sepharose, CM-Sephadex C-50, DEAE-Sephadex A-50, phenyl- and concanavalin A-Sepharose and isoelectric focusing. Cathepsin L and cathepsin H were assayed in the presence of dithiothreitol and Na₂EDTA (2 mM each) with Z-Phe-Arg-NHMec (pH 5.5) and Lys-NNa (pH 6.5) respectively. Cathepsin L consists of 2 polypeptide chains with M_r 25 000 and 5 000, M_r of cathepsin H is 28 000. Cathepsin L exists in brain tissue in two multiple forms with pI values 5.7 and 5.9, pI of cathepsin H is 6.8. Substrate specificity of these thiol proteinases was tested with proteins (pyridoxyl-hemoglobin, azocasein) and low M_r naphthylamide and methylcoumarylamide substrates: Lys-NNa, Arg-NNa, Dz-Arg-NNa, Z-Arg-Arg-NNaOMe, Z-Phe-Arg-NHMec, Z-Phe, Val-Arg-NHMec, Z-Gly-Gly-Arg-NHMec. Z-Phe-Arg-NHMec is the best substrate for cathepsin L (K_M=5 M, K_cat=21 s^–1), Arg-NNa—for cathepsin H (K_M=0.1 mM, K_cat=1.93 s^–1), being endoaminopeptidase cathepsin H also hydrolyses Bz-Arg-NNa (K_M=0.7 mM, K_cat=1.3 s^–1). Both proteinases are inhibited by traditional inhibitors of cysteine proteinases and E-64, but leupeptin turned to be more effective inhibitor of cathepsin L (K_i=2.4 nM) than of cathepsin H (K_i=9.2 M), the latter enzyme being sensitive to puromycin and benzethonium chloride as well. Z-Phe-Phe-CHN₂ and Z-Phe-Ala-CHN₂ are potent irreversible inhibitors of brain cathepsin L with K_2nd 150 000 and 137 000 M^–1 s^–1 respectively. Properties of the enzymes from human and bovine brain are similar.Special Issue Dedicated to Dr. Abel Lajtha.     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

Transport of neutral, cationic and anionic amino acids by systems B, b, XAG, and ASC in swine small intestine

Amino acid influx across the brush border membrane of the intact pig ileal epithelium was studied. It was examine whether in addition to system B, systems ASC and b^o,+ were involved in transport of bipolar amino acids. The kinetics of interactions between lysine and leucine demonstrates that system b^o,+ is present and accessible also to -glutamine. -aspartate (K_1/2 0.3 mM) and -glutamate (K_i 0.5 mM) share a high affinity transporter with a maximum rate of 1.3 μmol cm⁻² h⁻¹, while only -glutamate with a K_1/2 of 14.4 mM uses a low affinity transporter with a maximum rate of 2.7 μmol cm⁻² h⁻¹, system ASC, against which serine has a K_i of 1.6 mM. In the presence of 100 mM lysine, -glutamine (A), leucine (B), and methionine (C) fulfilled the criteria of the ABC test for transport by one and the same transporter. However, serine inhibits not only transport of -glutamate but also of glutamine (K_i 0.5 mM), and -glutamate inhibits part of the transport of glutamine. The test does, therefore, only indicate that the three bipolar amino acids have similar affinities for transport by systems B and ASC. Further study of the function of system B must be carried out under full inhibition by lysine and glutamate.     

Kinetic study of the cellobiase activity of Trichoderma reesei cellulase complex at high substrate concentrations

At high cellobiose concentrations, the cellobiase activity of a Trichoderma reesei cellulase preparation does not follow Michaelis–Menten kinetics and shows substrate inhibition. Several rate equations were fitted to the initial rate-cellobiose concentration data. The best fit is obtained for a rate equation corresponding to partial substrate inhibition of cellobiase. In this case, the K_m, V_max and K_I values obtained are 1.1 mM, 16 IU ml^–1 and 26 mM, respectively.     

A Novel Optical Intracellular Imaging Approach for Potassium Dynamics in Astrocytes

Astrocytes fulfill a central role in regulating K⁺ and glutamate, both released by neurons into the extracellular space during activity. Glial glutamate uptake is a secondary active process that involves the influx of three Na⁺ ions and one proton and the efflux of one K⁺ ion. Thus, intracellular K⁺ concentration ([K⁺]_i) is potentially influenced both by extracellular K⁺ concentration ([K⁺]_o) fluctuations and glutamate transport in astrocytes. We evaluated the impact of these K⁺ ion movements on [K⁺]_i in primary mouse astrocytes by microspectrofluorimetry. We established a new noninvasive and reliable approach to monitor and quantify [K⁺]_i using the recently developed K⁺ sensitive fluorescent indicator Asante Potassium Green-1 (APG-1). An in situ calibration procedure enabled us to estimate the resting [K⁺]_i at 133±1 mM. We first investigated the dependency of [K⁺]_i levels on [K⁺]_o. We found that [K⁺]_i followed [K⁺]_o changes nearly proportionally in the range 3–10 mM, which is consistent with previously reported microelectrode measurements of intracellular K⁺ concentration changes in astrocytes. We then found that glutamate superfusion caused a reversible drop of [K⁺]_i that depended on the glutamate concentration with an apparent EC₅₀ of 11.1±1.4 µM, corresponding to the affinity of astrocyte glutamate transporters. The amplitude of the [K⁺]_i drop was found to be 2.3±0.1 mM for 200 µM glutamate applications. Overall, this study shows that the fluorescent K⁺ indicator APG-1 is a powerful new tool for addressing important questions regarding fine [K⁺]_i regulation with excellent spatial resolution.     

A model for the regulation of brain adenylate cyclase by ionic equilibria

Multiple-equilibrium equations were solved to investigate the individual and separate effects of Mg²⁺, Mn²⁺, Ca²⁺, ATP^4–, and their complexes on the kinetics of brain adenylate cyclase. The effects of divalent metals and/or ATP^4– (in excess of their participation in complex formation) were determined and, from the corresponding apparent affinity values, the following kinetic constants were obtained:K _m(MgATP)=1.0 mM,K _i(ATP^4–)=0.27 mM,K _m(MnATP)=0.07 mM, andK _i(CaATP)=0.015 mM. MgATP, MnATP, ATP^4–, and CaATP were shown to compete for the active site of the enzyme. Hence, it is proposed that endogenous metabolites with a strong ligand activity for divalent metals, such as citrate and some amino acids, become integrated into a metabolite feedback control of the enzyme through the release of ATP^4– from MgATP. Ca²⁺ fluxes may participate in the endogenous regulation of adenylate cyclase by modifying the level of CaATP. The free divalent metals show an order of affinityK _0.5(Ca²⁺)=0.02 mM,K _0.5(Mn²⁺)=3.8 mM,K _0.5(Mg²⁺)=4.7 mM, and an order of activity Mn²⁺>Mg²⁺>Ca²⁺. The data indicate that Mn²⁺ and Mg²⁺ ions may compete for a regulatory site distinct from the active site and increaseV _m without changingK _m(MgATP),K _m(MnATP), orK _i(ATP^4–). The interactions of ATP^4– and CaATP, which act as competitive inhibitors of the reaction of the enzyme with the substrates MgATP and MnATP, and Mg²⁺ and Mn²⁺, which act as activators of the enzyme in the absence of hormones, are shown to follow the random rapid equilibrium BiBi group-transfer mechanism of Cleland with the stipulation that neither Mg²⁺ nor Mn²⁺, in excess of their respective participation in substrate formation, are obligatorily required for basal activity. ATP^4– and CaATP are involved in dead-end inhibition. For MgCl₂ saturation curves at constant total ATP concentration, the computer-generated curves based on the RARE BiBi model predict a change in the Hill cooperativityh from a basal value of 2.6, when Mg²⁺ is not obligatorily required, to 4.0 when the addition of hormones or neurotransmitters induces an obligatory requirement for Mg²⁺.Abbreviations used: Me, divalent metal; Me_T (Mg_T or Mn_T), total Me (Me²⁺ and its complexes); ATP_T, total ATP (ATP^4– and its complexes).     

Na+/H+ exchange in crayfish neurons: dependence on extracellular sodium and pH

Summary Intracellular pH (pH_i) regulation was studied in crayfish neurons with pH-, and Na⁺-sensitive microelectrodes. It was confirmed to involve both a HCO ₃ ^– -dependent and a HCO ₃ ^– -independent mechanism. The latter was identified as the amiloride-sensitive Na⁺/H⁺ exchange described in vertebrate cells. Its dependence on extracellular pH (pH_e) and Na⁺ concentration ([Na⁺]_e) was studied in CO₂-free external solutions at 20°C. The steady state pH_i and the rate constant (k) of the exponential pH_i recovery following an acid load were determined. At pH_e=7.5 and [Na⁺]_e=200 mM, the average steady state pH_i was 7.09±0.12 (as compared to 7.30±0.10 in the presence of 5 mM bicarbonate). The dependence of the rate constant of recovery on [Na⁺]_e could be described by Michaelis-Menten kinetics; at pH_e=7.5 the apparentK _m andK _max were 39 mM and 1.4 mmol·l^–1·min^–1, respectively. Decreasing pH_e reduced the rate of recovery, the variations ofk with pH_e conforming to a simple titration curve with an apparent pK of 7.05±0.21. These kinetic properties of the Na⁺/H⁺ exchange in crayfish neurons are similar to those described in vertebrate cells.Preliminary results were presented at the First International Congress of Comparative Physiology and Biochemistry (Liège, Belgium, 1984)     

Parallel control of the inward-rectifier K+ channel by cytosolic free Ca2+ and pH inVicia guard cells

The influence of cytosolic pH (pH_i) in controlling K⁺-channel activity and its interaction with cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K⁺-channel currents were recorded under voltage clamp while pH_i or [Ca²⁺]_i was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K⁺ concentration, current through inward-rectifying K⁺ channels (I_K,in) was evoked on stepping the membrane from a holding potential of –100 mV to voltages from –120 to –250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na⁺-acetate outside imposed acid loads, lowering pH_i from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pH_i was independent of the weak acid used, and indicated a H⁺-buffering capacity which rose from 90 mM H⁺/pH unit near 7.5 to 160 mM H⁺/pH unit near pH_i 7.0. With acid-going pH_i, (I_K,in) was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of –150 mV or the voltage-dependence for channel gating. Washout of the weak acid was followed by transient rise in pH_i lasting 3–5 min and was accompanied by a reduction in (I_K,in) before recovery of the initial resting pH_i and current amplitude. The pH_i-sensitivity of the current was consistent with a single, titratable site for H⁺ binding with a pK_a near 6.3. Acid pH_i loads also affected current through the outward-rectifying K⁺ channels (I_K,out) in a manner antiparallel to (I_K,in) The effect on I_K, out was also scalar, but showed an apparent pK_a of 7.4 and was best accommodated by a cooperative binding of two H⁺. Parallel measurements showed that Na⁺-butyrate loads were generally without significant effect on [Ca²⁺]_i, except when pH_i was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca²⁺]_i in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pH_i changes and K⁺-channel responses. The action on [Ca²⁺]_i coincided with a greater variability in (I_K,in) stimulation evident at pH_i values around 7.0 and below, and with negative displacements in the voltage-dependence of (I_K,in) gating. These results distinguish the actions of pH_i and [Ca²⁺]_i in modulating (I_K,in) they delimit the effect of pH_i to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pH_i as a second messenger capable of acting in parallel with, but independent of [Ca²⁺]_i in controlling the K⁺ channels.Abbreviations BCECF 2,7-bis (2-carboxyethyl)-5(6)-carboxy fluorescein - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - g_K ensemble (steady-state) K⁺-channel conductance - I_K,out, I_K,in outward-, inward-rectifying K⁺ channel (current) - IN current-voltage (relation) - Mes 2-(N-morpholinolethanesulfonic acid - pH_i cytosolic pH - V membrane potential     

Kinetics of glycerol uptake by the perfused rat liver Membrane transport, phosphorylation and effect on NAD redox level

The kinetics of glycerol uptake by the perfused rat liver were determined according to a model which includes membrane transport, intracellular phosphorylation and competitive inhibition of glycerol phosphorylation by l-glycerol 3-phosphate. The membrane transport obeys first-order kinetics at concentrations below 10 mM in the affluent medium. The K_m of the glycerol phosphorylation was was 10 μM and the K_i of the l-glycerol 3-phosphate inhibition was 50 μM. The maximum activity (V) was 3.70 μmoles/min per g liver wet wt. These results are similar to in vitro kinetics of the glycerol kinase, except that K_i was found to be somewhat lower in the intact organ. At low glycerol concentrations, gradient exists across the liver cell membrane.The increase in the lactate to pyruvate concentration ratio during glycerol metabolism is related to the actual concentration of l-glycerol 3-phosphate, not to the rate of glycerol uptake.     

Uptake of sulphate,taurine, cysteine and methionine by symbiotic and free-living dinoflagellates

Sulphate uptake by Amphidinium carterae, Amphidinium klebsii and Gymnodinium microadriaticum grown on artificial seawater medium with sulphate, cysteine, methionine or taurine as sulphur source occurred via an active transport system which conformed to Michaelis-Menten type saturation kinetics. Values for K _m ranged from 0.18–2.13 mM and V _max ranged from 0.2–24.2 nmol · 10⁵ cells^–1 · h^–1. K _m for symbiotic G. microadriaticum was 0.48 mM and V _max was 0.2 nmol · 10⁵ cells^–1 · h^–1. Sulphate uptake was slightly inhibited by chromate and selenate, but not by tungstate, molybdate, sulphite or thiosulphate. Cysteine and methionine (0.1 mM), but not taurine, inhibited sulphate uptake by symbiotic G. microadriaticum, but not by the two species of Amphidinium. Uptake was inhibited 45–97% under both light and dark conditions by carbonylcyanide 3-chlorophenylhydrazone (CCCP); under dark conditions sulphate uptake was 40–60% of that observed under light conditions and was little affected by 3-(3,4-dichlorophenyl) 1,1-dimethylurea (DCMU).The uptake of taurine, cysteine and methionine by A. carterae, A. klebsii, cultured and symbiotic G. microadriaticum conformed to Michaelis-Menten type saturation kinetics. K _m values of taurine uptake ranged from 1.9–10 mM; for cysteine uptake from 0.6–3.2 mM and methionine from 0.001–0.021 mM. Cysteine induced a taurine uptake system with a K _m of 0.3–0.7 mM. Cysteine and methionine uptake by all organisms was largely unaffected by darkness or by DCMU in light or darkness. CCCP significantly inhibited uptake of these amino acids. Thus energy for cysteine and methionine uptake was supplied mainly by respiration. Taurine uptake by A. carterae was independent of light but was inhibited by CCCP, whereas uptake by A. klebsii and symbiotic G. microadriaticum was partially dependent on photosynthetic energy. Taurine uptake by cultured G. microadriaticum was more dependent on photosynthetic energy and was more sensitive to CCCP. Cysteine inhibited uptake of methionine and taurine by cultured and symbiotic G. microadriaticum to a greater extent than in the Amphidinium species. Methionine did not greatly affect taurine uptake, but did inhibit cysteine uptake. Taurine did not affect the uptake of cysteine or methionine.     

Biosynthesis and function of trehalose inEctothiorhodospira halochloris

Trehalose-6-phosphate synthase, catalyzing the reaction between UDP-glucose and glucose 6-phosphate and forming trehalose 6-phosphate, was isolated and partially purified (30-fold) from the phototrophic, haloalkaliphilic bacteriumEctothiorhodospira halochloris. The activity is stabilized by 20mM MgCl₂, 50mM NaCe and 2M glycine betaine. The molecular weight was 63000.The enriched enzyme had a MgCl₂ optimum at 3–6mM, a pH optimum at 7.5 (in Tris-HCl buffer) and a temperature optimum at 50°C. The K_m-values were 1.5×10^–3M for UDP-glucose and 2×10^–3M for glucose 6-phosphate. The enzyme showed a salinity dependence with optimal concentrations between 100 and 300mM salt. Higher concentrations of salt resulted in a decrease in activity. In the presence of inhibitory salt concentrations the compatible solute glycine betaine had a protective effect with a maximum between 0.5 and 2.0M.     

Purification and characterization of D-glycerate-3-kinase from maize leaves

Glycerate kinase (GK; EC 2.7.1.31) from maize (Zea mays L.) leaves was purified by a sequence of ammonium-sulfate precipitations and chromatography on diethylaminoethyl-cellulose, hydroxyapatite, Sephadex G-75SF and dye ligand (Green A) columns. The purest preparation was almost 1300-fold enriched and had a specific activity of 68 mol · min^-1 · (mg protein) ^-1. The enzyme was a monomer of a relative molecular mass (M_r) of 44 kDa (kdalton) as determined by gel filtration, electrophoresis in dissociating conditions and by immunoblots. The enzyme was only weakly recognized by polyclonal antibodies against purified spinach GK, indicating substantial differences in molecular structure of the two proteins. Highly reducing conditions stabilized GK activity and were required for activation of crude leaf enzyme. The enzyme had a broad pH optimum of 6.8–8.5, and formed 3-phosphoglycerate and ADP as reaction products. Apparent K _ms for D-glycerate and Mg-ATP were 0.11 and 0.25 mM, respectively. The enzyme was strongly affected by a number of phosphoesters, especially by 3-phosphoglycerate (K _i= 0.36 mM), fructose bisphosphates and nucleoside bisphosphates. Inhibition by 3-phosphoglycerate was competitive to Mg-ATP and noncompetitive to D-glycerate. Pyruvate was found noncompetitive to D-glycerate (K _is=4 mM). The ratio of stromal concentration of Mg-ATP to phosphoesters, particularly to 3-phosphoglycerate, may be of importance in the regulation of GK during C₄-photosynthesis.Abbreviations DEAE diethylaminoethyl - kDa kdalton - GAP-DH glyceraldehyde phosphate dehydrogenase - GK glycerate kinase - LDH lactate dehydrogenase - 2-ME 2-mercaptoethanol - M_r relative molecular mass - PEP phosphoenolpyruvate - PGA(PK) phosphoglycerate (phosphokinase) - PK pyruvate kinase - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis     

Kinetics of Na+-, K+-ATPase Inhibition by an Endogenous Modulator (II-A)

We have previously reported the isolation by gel filtration and anionic exchange HPLC of two brain Na⁺, K⁺-ATPase inhibitors, II-A and II-E, and kinetics of enzyme interaction with the latter. In the present study we evaluated the kinetics of synaptosomal membrane Na⁺, K⁺-ATPase with II-A and found that inhibitory activity was independent of ATP (2–8 mM), Na⁺ (3.1–100 mM), or K⁺ (2.5–40 mM) concentration. Hanes-Woolf plots showed that II-A decreases V_max in all cases; K_M value decreased for ATP but remained unaltered for Na⁺ and K⁺, indicating respectively uncompetitive and noncompetitive interaction. However, II-A became a stimulator at 0.3 mM K⁺ concentration. It is postulated that brain endogenous factor II-A may behave as a sodium pump modulator at the synaptic region, an action which depends on K⁺ concentration.     

Effect of light intensity and ammonium-N on carotenogenesis ofTrentepohlia odorata andDunaliella bardawil

AxenicTrentepohlia odorata was cultured at three different NH₄Cl levels (3.5 × 10^–2, 3.5 × 10^–3, 3.5 × 10^–4 M) and three different light intensities (48, 76, 122 µmol m^–2 s^–1). Chloride had no effect on growth over this range of concentration. High light intensity and high NH₄Cl concentration enhanced the specific growth rate. The carotenoid content increased under a combination of high light intensity and low N concentration. WhenD. bardawil was exposed to the same combination of growth conditions, there was an increase in its carotenoid content. The light saturation and the light inhibition constants (K _s andK _i, respectively) for growth, and the saturation constant (K _m) for NH₄Cl were determined. TheK _s andK _i values were higher inT. odorata (66.7 and> 122 mol m^–2 s^–1, respectively) than inD. bardawil (5.1 and 14.7 µmol m^–2 s^–1, respectively). TheK _m value determined at 122 µmol m^–2 s^–1, however, was lower inT. odorata (0.048 µM) than inD. bardawil (0.062 µM).Author for correspondence     

Kinetics of Na+, K+-ATPase Inhibition by a Rat Brain Endogenous Factor (II-E)

Previous work from this laboratory led to the isolation by gel filtration and anionic exchange HPLC of a rat brain fraction named II-E, which highly inhibits synaptosomal membrane Na⁺, K⁺-ATPase activity. In this study we evaluated the kinetics of such inhibition and found that inhibitory potency was independent of Na⁺(1.56–200 mM), K⁺(1.25–40 mM), or ATP (1–8 mM) concentration. Hanes-Woolf plots indicated that II-E decreases Vmax but does not alter K_Mvalue, and suggested uncompetitive inhibition for Na⁺, K⁺or ATP. However, II-E became a stimulator at 0.5 mM ATP concentration. It is postulated that this brain factor may modulate ionic transport at synapses, thus participating in central neurotransmission.