Drought stress stimulates p-nitrophenyl phosphate hydrolysis rate of the plasma membrane H+-ATPase from wheat leaves

The influence of drought stress on the ATP and p-nitrophenyl phosphate (PNPP) hydrolysis activity by plasma membrane H⁺-ATPase was investigated using purified plasma membrane vesicles from wheat leaves by two-phase partitioning. Drought stress increased the ATPase activity, and the optimal pH was shifted from 6.5 to about 7.0. Drought stress also stimulated the PNPP hydrolysis rate. The K_m for PNPP hydrolysis was moved from 4.49 ± 0.33 mM to 3.64 ± 0.12 mM. In addition, the PNPP hydrolysis was more sensitive to vanadate under drought compared to the control. However, the inhibitory effect of hydroxylamine on the ATPase was not changed by the present drought stress. In addtion, drought stress also decreased the trypsin activation of PNPP hydrolysis by PM H⁺-ATPase. These results suggested that drought stress altered the catalytic mechanism of the plasma membrane H⁺-ATPase, and the stimulation of its activity by drought stress was mainly due to increase of the catalytic activity of its phosphatase domain. It is also suggested that drought stress might alter the structure or property of the C-terminal end of PM H⁺-ATPase, therefore increasing the catalytic activity of the phosphatase domain.     

Purification and properties of fructose diphosphate aldolase from Ascaris suum muscle

A fructose diphosphate aldolase has been isolated from ascarid muscle and crystallized by simple column chromatography and an ammonium sulfate fractionation procedure. It was found to be homogeneous on electrophoresis and Sephadex G-200 gel filtration. This enzyme has a fructose diphosphate/fructose 1-phosphate activity ratio close to 40 and specific activity for fructose diphosphate cleavage close to 11. K_m values of ascarid aldolase are 1 × 10⁻⁶m and 2 × 10⁻³m for fructose diphosphate and fructose 1-phosphate, respectively. The enzyme reveals a number of catalytic and molecular properties similar to those found for class I fructose diphosphate aldolases. It has C-terminal functional tyrosine residues, a molecular weight of 155,000, and is inactivated by NaBH₄ in presence of substrate. Data show the presence of two types of subunits in ascarid aldolase; the subunits have different electrophoretic mobilities but similar molecular weights of 40,000. Immunological studies indicate that the antibody-binding sites of the molecules of the rabbit muscle aldolase A or rabbit liver aldolase B are structurally different from those of ascarid aldolase. Hybridization studies show the formation of one middle hybrid form from a binary mixture of the subunits of ascarid and rabbit muscle aldolases. Hybridization between rabbit liver aldolase and ascarid aldolase was not observed. The results indicate that ascarid aldolase is structurally more related to the mammalian aldolase A than to the aldolase B.     

Regulation in the chemolithotrophthiobacillus neapolitanus: Fructose-1,6-diphosphatase

Summary Partially purified fructose diphosphatase from the obligate chemolithotroph,Thiobacillus neapolitanus has been characterized, and some of its regulatory properties described. The enzyme had a high effinity for its substrate, but was inhibited by substrate at concentrations above 1 mM. The enzyme had an absolute requirement for a divalent cation. In the absence of EDTA there was a single pH optimum in the alkaline range between 8.5 and 9.5; in the presence of EDTA there was considerable was activity at both neutral and alkaline pH. This diphosphatase was inhibited by AMP at 10^–4 M or greater-, the lower the pH, the greater the AMP inhibition. Treatment of the enzyme with 5×10^–5 Mpara hydroxy mercuribenzoate allowed retention of full catalytic activity while abolishing considerable AMP inhibition. Exposure of the enzyme to several concentrations of urea had no effect on the AMP inhibition. Homocystine (0.06 mM) and coenzyme A (0.1 mM) had no effect. At 1 mM, PEP caused 60% inhibition, 2, 3-diphosphoglyceric acid produced 26% inhibition, and pyruvate had no effect.     

Regulation of the structure and catalytic properties of plasma membrane H<Superscript>+</Superscript>-ATPase involved in adaptation of two reed ecotypes to their different habitats

The properties and kinetics of ATP and p-nitrophenyl phosphate (PNPP) hydrolysis activities of plasma membrane H⁺-ATPase from the two reed ecot ypes, swamp reed (SR) and dune reed (DR), were investigated. The pH optimum of the plasma membrane H⁺-ATPase in both reed ecotypes was similar but the sensitivity of the enzyme to the reaction medium pH seemed to be higher in DR than that in SR. Compared to SR, the DR exhibited a higher V_max value for ATP hydrolysis whereas the K_m value was almost similar in both reed ecotypes. The PNPP hydrolysis of the plasma membrane H⁺-ATPase was also studied in both reed ecotypes at increasing PNPP concentrations. K_m and V_max for PNPP hydrolysis showed great differences in the two reed ecotypes and in DR the K_m and V_max values were 2- and 10-fold, respectively, higher than those in SR. The ATP hydrolysis activity of the plasma membrane was markedly inhibited by hydroxylamine in both reed ecotypes, and the percentage inhibition of ATP hydrolysis rate seemed higher in DR than that in SR. In addition, the structure or property of the C-terminal end of the plasma membrane H⁺-ATPase were also different in the two reed ecotypes. These data suggest that different isoforms of the plasma membrane H⁺-ATPase might be developed and involved in the adaptation of the plant to the long-term drought-prone habitat.This research was supported by Natural Science Foundation of China (No. 30270238 & No. 30470274) and the National Key Basic Research Special Funds of China (G1999011705).     

Cyclic GMP stimulation and inhibition of cyclic AMP phosphodiesterase from thymic lymphocytes

A particulate preparation of cyclic AMP phosphodiesterase from rat thymic lymphocytes exhibited two apparent Km's at 0.9×10⁻⁶M and 8.0×10⁻⁶M. The enzyme with the higher Km was stimulated by cyclic GMP by a mechanism involving an increase in the Vmax of the enzyme with no change in the Km. Cyclic GMP competitively inhibited the enzyme with the low apparent Km which had a Ki for cyclic GMP of 4×10⁻⁵M. The modulation of cyclic AMP phosphodiesterase activity by cyclic GMP in the control of cyclic AMP-mediated lymphocyte proliferation is discussed.     

Purification and Regulatory Properties of Fructose 1,6-Diphosphatase from Hydrogenomonas eutropha

Fructose diphosphatase of Hydrogenomonas eutropha H 16, produced during autotrophic growth, was purified 247-fold from extracts of cells. The molecular weight of the enzyme was estimated to be 170,000. The enzyme showed a pH optimum of 8.5 in both crude extracts and purified preparation. The shape of the pH curve was not changed in the presence of ethylenediaminetetraacetic acid. The enzyme required Mg²⁺ for activity. The MgCl₂ saturation curve was sigmoidal and the degree of positive cooperativity increased at lower fructose diphosphate concentrations. Mn²⁺ can replace Mg²⁺, but maximal activity was lower than that observed with Mg²⁺ and the optimal concentration range was narrow. The fructose diphosphate curve was also sigmoidal. The purified enzyme also hydrolyzed sedoheptulose diphosphate but at a much lower rate than fructose diphosphate. The enzyme was not inhibited by adenosine 5′-monophosphate but was inhibited by ribulose 5-phosphate and adenosine 5′-triphosphate. Adenosine 5′-triphosphate did not affect the degree of cooperativity among the sites for fructose diphosphate. The inhibition by adenosine 5′-triphosphate was mixed and by ribulose 5-phosphate was noncompetitive. An attempt was made to correlate the properties of fructose diphosphatase from H. eutropha with its physiological role during autotrophic growth.     

Homotropic effect of CO 2 in ribulose-1,5-diphosphate carboxylase reaction

The concentration effect of aqueous CO₂ on the reaction velocity of spinach leaf ribulose-1,5-diphosphate carboxylase has been reevaluated. The homotropic effect of CO₂ in the enzyme reaction supports the previously reported allosteric nature of the enzyme in the CO₂-fixation process in chloroplasts. The concentration of CO₂ giving the half maximal reaction velocity, S_0.5, has been calculated to be 1.47 × 10⁻⁵M.     

A reinvestigation of the kinetic parameters of phosphoenolypyruvate carboxykinase

A new assay for phosphoenolpyruvate carboxykinase has been devised whose sensitivity is sufficient to detect the formation by crude hepatic extracts of 1 × 10⁻¹¹ moles of phosphoenolpyruvate. By the utilization of this assay it has been determined that the apparent Km of oxaloacetate is in the range of 1 to 5 × 10⁻⁶ M for the hepatic enzymes from rat and guinea pig. These values are approximately two orders of magnitude lower than all previous data and indicate that the enzyme is sensitive to modulations of oxaloacetate concentrations that occur physiologically.     

Properties and regulation of C-1-fructose-1,6-diphosphatase from spinach chloroplasts

Chloroplast fructose diphosphatase (EC 3.1.3.11) was purified according to the procedures of Racker and Schroeder [1] and Buchanan et al. [2] and the properties compared. Neither preparation contained fructose diphosphatase from the cytoplasm. The preparations had similar molecular weights, pH optima, affinities for fructose diphosphate and Mg²⁺ and were similarly activated by EDTA, dithiothreitol and cystamine.Mg²⁺, fructose diphosphate and dithiothreitol all activate chloroplast fructose diphosphatase more so at suboptimal pH values. The combined effects of these substances under estimated physiological conditions in the chloroplast stroma in the light and in darkness were consistent with almost full activity of the enzyme during illumination but no activity in the dark.     

Effects of freezing on plasma membrane H<Superscript>+</Superscript>-ATPase of the callus from <Emphasis Type="Italic">Chorispora bungeana</Emphasis>

The influence of freezing treatment on plasma membrane (PM) H⁺-ATPase was investigated using plasma membrane vesicles isolated from calluses from Chorispora bungeana Fisch. & C.A. Mey. by the discontinuous sucrose gradient centrifugation. Freezing treatment (−4 °C) for 5 d resulted in significant increases in the ATPase activity and the activity of p-nitrophenyl phosphate (PNPP) hydrolysis, decreases in the K_m for ATP hydrolysis and PNPP hydrolysis, and the shift of optimal pH from 6.5 to 7.0. Also, the activity PNPP hydrolysis was less sensitive to vanadate after freezing treatment compared to control, while the inhibition of ATP hydrolysis by hydroxylamine was more sensitive. In addition, freezing treatment also decreased the activation effects of trypsin on PNPP hydrolysis, but increased the activation effects of lysophosphatidylcholine on ATP hydrolysis. Taken together, these results suggested that PM H⁺-ATPase might play an important role during adaptation to freezing and enhancing the frost hardness in C. bungeana.     

Nucleoside diphosphatase from pig liver: purification and some properties

A procedure is described for purification of nucleoside diphosphatase from pig liver microsomes which avoids exposure of the enzyme to potentially denaturing conditions. The purest fractions obtained have specific activities of approximately 100 units/mg and appear to contain approximately 35% NDPase on a protein basis. Pig liver nucleoside diphosphatase resembles the enzyme obtained from other mammalian tissues in its substrate specificity and in its interaction with MgATP^2? as an allosteric modifier. However the molecular weight of the pig liver enzyme appears higher than that reported for other nucleoside diphosphatases, and activation by MgATP^2? is attributable to an increase in the maximal rate of nucleoside diphosphate hydrolysis rather than to a decrease in K_m. These differences in properties seem to be due to a species difference since similar properties were found with pig liver enzyme prepared by a different extraction procedure. The kinetic parameters which describe the reaction catalyzed by pig liver nucleoside diphosphatase are insensitive to changes in [H⁺]over the range pH 6.5–8.6. The intracellular location of nucleoside diphosphatase is microsomal in both pig and chicken liver.     

Evidence for a phosphoryl-enzyme intermediate in phosphate ester hydrolysis by purple acid phosphatase from bovine spleen

The possibility of the existence of a covalent enzyme-phosphoryl intermediate, E-PO3, during catalysis of phosphate ester hydrolysis by the purple acid phosphatase (PAP) from bovine spleen has been examined. Transphosphorylation experiments show that up to 22% of the phosphoryl group from p-nitrophenyl phosphate (PNPP) can be transferred to primary alcohols. Burst experiments at high pH (9.1 or 8.1 for reduced or oxidized PAP, respectively), where hydrolysis of a phosphoenzyme intermediate is expected to be rate-limiting, show clear evidence for stoichiometric bursts of p-nitrophenolate from PNPP. The formation of base-stable, acid-sensitive adducts between PAP and the 32PO3 group of [gamma-32P]ATP has been demonstrated. The pH dependence of the kinetics parameters for reduced PAP has been determined over the range pH 3-8; a feature with a pKa of approximately 6.75 that is attributable to the enzyme-substrate complex is observed. Taken together, the present results are consistent with a two-stem pseudo Uni Bi mechanism that utilizes a covalent enzyme-phosphoryl intermediate, possibly a phosphohistidine.     

Impact of ZnSO and ZnO Nanoparticles on Seed Germination and Seedling Growth of Lettuce

Micronutrient nanoparticles (NPs) are currently an option for chemical fertilization and biostimulation in crops. However, there is little information on the phytotoxic or biostimulatory effects of NPs at low concentrations of some elements, such as Zn. In this study, the effect of low concentrations of Zn oxide (ZnO) NPs on germination, growth variables, and nutritional attributes of lettuce (Lactuca sativa L.) was evaluated in comparison to Zn sulfate. Romaine lettuce seeds were treated with ZnSO₄^-- × 7H₂O and ZnO NPs at Zn molar concentrations of 1 × 10⁻³, 5 × 10⁻³, 1 × 10⁻⁴, 5 × 10⁻⁴, 1 × 10⁻⁵, 5 × 10⁻⁵, 1 × 10⁻⁶, and 5 × 10⁻⁶. The seeds treated with ZnSO₄⁻ at 5 × 10⁻⁶ registered the highest radicle length, 73% more than the control treatment. The seeds treated with ZnSO₄⁻ at 5 × 10⁻³ registered the lowest values, with 50% less than the control treatment. ZnO NPs at 5 × 10⁻⁶ significantly increased content of chlorophyll A and B and total phenolics. These results indicate the possible existence of a mechanism related to the intrinsic nanoparticle properties, especially at low concentrations.

     

The isolation of pig liver monoamine oxidase

Monoamine oxidase from pig liver has been isolated and purified approximately three hundred-fold. This enzyme has a molecular weight of 1,200,000, is highly polymeric, and contains subunits of molecular weight 146,000, as determined by Sephadex chromatography. The apparent K_m at 25°C is 1.28 × 10^?6 M at pH 9.0 (0.05 M glycine) and 1.74 × 10^?5 M at pH 7.2 (0.2 M phosphate) using benzylamine as a substrate. This enzyme contains approximately 8 copper(II) ions per 1,200,000 molecular weight.     

Measurement of the binding of antifibrinolytic amino acids to various plasminogens

A method for studying the binding of various antifibrinolytic amino acids to plasminogen has been devised. This method is based upon the ability of inhibitors of the streptokinase-induced conversion of plasminogen to plasmin to produce an alteration in the s_20,w⁰ of native plasminogen accompanying their binding to plasminogen. Typical examples of antifibrinolytic amino acids, e.g., 6-amino hexanoic acid, trans-4-aminomethyl cyclohexane-1-carboxylic acid, and l-lysine cause alterations in the s_20,w⁰ of streptokinase-insensitive plasminogens as well as streptokinase-sensitive plasminogens from 5.1–5.6 S to 4.1–4.7 S depending upon the particular plasminogen used. Titration of the s_20,w⁰ of human plasminogen (streptokinase-sensitive) using absorption optics in the analytical ultracentrifuge with the above three compounds led to dissociation constants of 4.5 ± 0.8 × 10⁻⁴m, 8.0 ± 0.8 × 10⁻⁵m, and 6.8 ± 0.8 × 10⁻²m, respectively. When duck plasminogen (streptokinase-insensitive) was used, dissociation constants of 5.6 ± 0.7 × 10⁻⁴m, 9.0 ± 0.8 × 10⁻⁵m, and 8.8 ± 0.7 × 10⁻²m, were obtained.     

Hymenolepis diminuta: further characterization of the membrane-bound acid phosphatase activity associated with the brush border membrane of the tapeworm's tegument

The acid phosphate activity (APA) associated with the isolated brush border membrane of the tapeworm, Hymenolepis diminuta, hydrolyzed p-nitrophenyl phosphate (PNPP), pyrophosphate (PPi), and beta-glycerophosphate (beta GP). Inhibition of PNPP hydrolysis at pH 4.0 was inhibited in a competitive manner by the following compounds (listed in order of decreasing affinity with their apparent inhibitor constants (Ki')): molybdate (0.031 mM); PPi (0.147 mM); NaF (0.150 mM); o-carboxyphenyl phosphate (0.261 mM); inorganic phosphate (0.770)); arsenate (3.45 mM); tartrate (22.1 mM); and beta GP (29.8 mM). Cu2+, formaldehyde, and arsenite at 10:1, 80:1, and 200:1 inhibitor to substrate ratios did not inhibit APA. The maximal rate of hydrolysis (Vmax) of each substrate was greater at pH 4.0 than 5.0. The apparent Michaelis constant (Km') for PNPP increased from 0.233 to 0.351 mM when the pH was raised from 4.0 to 5.0. The Km' for PPi decreased from 0.101 to 0.046 mM, while the Km' for beta GP changed from 2.04 to 2.22 mM under similar circumstances. APA and alkaline phosphatase activity increased as a function of temperature up to 45 degrees C.     

A "Reverse burst" active site titration procedure for human carbonic anhydrase B.

Between pH 7 and pH 10.5, p-nitrophenyl $\text{p}$-sulfamyl benzoate (PNP-SAB) binds very strongly to human carbonic anhydrase B (dissociation constant on the order of 10^?9 M or less at pH 7.5), but is not hydrolyzed by the enzyme. Because the binding is essentially stoichiometric under readily accessible conditions, this ester may be used as an active site titrant, by measuring the rapid hydrolysis of excess unbound PNP-SAB catalyzed by an added nucleophile (“reverse burst”).     

Purification and properties of a D-fructose 1,6-bisphosphatase from Saccharomyces cerevisiae.

Fructose 1,6-bisphosphatase (EC 3.1.3.11) from Saccharomyces cerevisiae has been purified to homogeneity. A molecular weight of 115,000 has been obtained by gel filtration. The enzyme appears to be a dimer with identical subunits. The apparent K_m for fructose bisphosphatase varies with the Mg²⁺ concentration of the enzyme, being 1 × 10^?6m at 10 mm Mg²⁺ and 1 × 10^?5m at 2 mm Mg²⁺. Other phosphorylated compounds are not significantly hydrolyzed by the enzyme. An optimum pH of 8.0 is exhibited by the enzyme. This optimum is not changed by addition of EDTA. AMP inhibits the enzyme with a K_i of 8.0 × 10^?5m at 25 °C. The inhibition is temperature dependent, the value of K_i increasing with raising temperature. 2-Deoxy-AMP is also inhibitory with a K_i value at 25 °C of 1.6 × 10^?4m. An ordered uni-bi mechanism has been deduced for the reaction with phosphate leaving the enzyme as the first product and the fructose 6-phosphate as the second one.     

Fructose 1,6-diphosphatase in striated muscle

1. The occurrence of fructose diphosphatase in muscle tissue was investigated with reference to the question whether lactate can be converted into glycogen in muscle, as postulated by Meyerhof (1930), fructose diphosphatase being one of the enzymes required for this conversion. 2. Fructose diphosphatase was found in skeletal muscle of man, dog, cat, rat, mouse, rabbit, guinea pig, cattle, sheep, pigeon, fowl and frog. Under the test conditions between 5 and 60 μmoles of substrate were split/g. fresh wt./hr. at 22°. 3. Like liver fructose diphosphatase, the muscle enzyme is inhibited by substrate concentrations above 0·1 mm, by AMP and by trace quantities of Zn²⁺, Fe²⁺ and Fe³⁺; it is `activated' by EDTA. Inhibitions by the above agents may account for the failure of previous authors to detect the enzyme. 4. Heart muscle of several vertebrate species and the smooth muscle of pigeon and fowl gizzard had no measurable activity. 5. The presence of fructose diphosphatase and the virtual absence of the enzyme systems converting pyruvate into phosphopyruvate means that lactate and pyruvate cannot be converted into glycogen in muscle, whereas the phosphorylated C₃ compounds can. The reconversion into carbohydrate of lactate (which readily diffuses out of muscle) occurs in liver and kidney only. The reconversion of phosphorylated C₃ intermediates (which cannot diffuse out of the tissue) can occur only within the muscle. 6. α-Glycerophosphate is probably the main intermediate requiring conversion into glycogen. The possible role of α-glycerophosphate formation in vertebrate muscle, already well established in insect muscle, is discussed.     

Isolation and characterization of a pyrophosphate-dependent phosphofructokinase from Propionibacterium shermanii.

A pyrophosphate-dependent phosphofructokinase (pyrophosphate; D-fructose-6-phosphate-1-phosphotransferase) has been purified and characterized from extracts of Propionibacterium shermanii. The enzyme catalyzes the transfer of phosphate from pyrophosphate to fructose 6-phosphate to yield fructose-1,6-P2 and phosphate. This unique enzymatic activity was observed initially in Entamoeba histolytica (Reeves, R.E., South, D.J., Blytt, H.G., and Warren, L. G. (1974) J. Biol. Chem. 249, 7734-7741). This is the third pyrophosphate-utilizing enzyme that these two diverse organisms have in common. The others are phosphoenolpyruvate carboxytransphosphorylase and pyruvate phosphate dikinase. The PPi-phosphofructokinase from P. shermanii is specific for fructose-6-P and fructose-1,6-P2, no other phosphorylated sugars were utilized. Phosphate could be replaced by arsenate. The Km values are: phosphate, 6.0 X 10(-4) M; fructose-1, 6-P2, 5.1 X 10(-5) M; pyrophosphate, 6.9 X 10(-5) M; and fructose-6-P, 1.0 X 10(-4) M. The S20w is 5.1 S. The molecular weight of the native enzyme is 95,000. Sodium dodecyl sulfate electrophoresis of the enzyme showed a single band migrating with an Rf corresponding to a molecular weight of 48,000. Extracts of P. shermanii have PPi-phosphofructokinase activity approximately 6 times greater than ATP-phosphofructokinase and 15 to 20 times greater than fructose diphosphatase activities. It is proposed that (a) PPi may replace ATP in the formation of fructose-1-6-P2 when the organism is grown on glucose and (b) when the organism is grown on lactate or glycerol the conversion of fructose-1,6-P2 to fructose-6-P during gluconeogenesis may occur by phosphorolysis rather than hydrolysis.