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.

     

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.     

Simultaneous determination of superoxide dismutase and catalase in biological materials by polarography.

The polarographic method of catalytic currents applied to a wave of oxygen permits the simultaneous assay of superoxide dismutase and catalase in biological materials with high speed and reproducibility and minimal manipulation of tissues. Washed red blood cells and tissue homogenates give rise to a strong polarographic maximum, apparently due to heme proteins, which interferes with the measurement. This maximum is suppressed by addition of approximately 0.2% plasma. Therefore, the determination of the two enzymes in red blood cells can be carried out by direct addition of whole blood to the polarographic solution. Thirty microliters of blood are enough for optimal determination of both enzymes. The method can determine superoxide dismutase and catalase at concentrations as low as 2 × 10⁻¹¹m and 5 × 10⁻¹⁰m, respectively, and shows a linear correlation between measured activity and enzyme levels. The average values of the two enzymes in human red blood cells was found by this method to be 2.6 × 10⁻⁶m for catalase and 1.8 × 10⁻⁶m for superoxide dismutase, which agree with previously reported values.     

Insulin-receptor interactions in liver cell membranes

The specific binding of ¹²⁵I-insulin to liver cell membranes is a saturable process with respect to insulin. Binding is displaced by low concentrations of native insulin but not by biologically inactive insulin derivatives or by other peptide hormones. The rate constants of association (3.5 × 10⁶ mole⁻¹ sec⁻¹) and of dissociation (2.7 × 10⁻⁴ sec⁻¹) of the insulin-membrane complex can be determined independently. The dissociation constant of the complex, determined from the rate constants and from equilibrium data, is about 7 × 10⁻¹¹M. Complex formation does not result in degradation of the insulin molecule. The binding interaction is a dissociable process involving a homogeneous membrane structure which is almost certainly the biologically significant receptor. The kinetic properties, and the effects of enzymic perturbations of the membrane, suggest that the insulin receptors of liver and of adipose tissue cells may be very similar structures.     

P-nitrophenyl phosphate as substrate for rabbit liver fructose diphosphatase

Purified rabbit liver fructose diphosphatase has been found to catalyze the hydrolysis of p-nitrophenyl phosphate, PNPP. It has been established that the hydrolysis of p-nitrophenyl phosphate is due to fructose diphosphatase through studies of the chromatographic properties of the enzyme, its temperature sensitivity, dependence on divalent cations and its inhibition by fructose diphosphate. The K_m for PNPP is 6 × 10⁻³M at pH 9.2, 5 × 10⁻⁴M at pH 7.5. This substrate should facilitate studies of the kinetics and mechanism of action of fructose diphosphatase and the comparison of this enzyme with other alkaline phosphatases.     

Columns for rapid chromatographic separation of small amounts of tracer-labeled transfer ribonucleic acids

A small reversed-phase chromatographic system for the separation of [³H] or [¹⁴C] aminoacyl-tRNA's is described. This system has the advantages of speed (chromatographic runs can be completed in 30 to 60 min), sensitivity (samples containing as little as 3.5 × 10⁻⁹ gm or 60 × 16⁻⁶A₂₆₀ unit of phenylalanyl-tRNA have been resolved as a chromatographic peak), and excellent resolution of isoaccepting tRNA's. The system has been applied to samples of E. coli tRNA's and calf liver tRNA's.     

Effect of phosphoenolpyruvate and oxaloacetate on ca uptake by isolated mung bean mitochondria

Phosphoenolpyruvate partially inhibits the accumulation of Ca²⁺ in isolated mung bean (Phaseolus aureus Roxb.) mitochondria. Succinate-supported Ca²⁺ uptake is twice as sensitive to phosphoenolpyruvate inhibition as is NADH- or malate/pyruvate-supported Ca²⁺ uptake. Pyruvate, atractylate, and ATP, but not ITP, reverse the phosphoenolpyruvate-induced inhibition. Oxaloacetic acid inhibits succinate-supported Ca²⁺ uptake completely while partially inhibiting NADH-supported Ca²⁺ uptake. The oxaloacetate inhibition of NADH-supported Ca²⁺ uptake is greater than that produced by phosphoenolpyruvate. It is suggested that inhibition of Ca²⁺ uptake is due to the conversion of phosphoenolpyruvate into oxaloacetate via phosphoenolpyruvate carboxykinase, with oxaloacetate responsible for the actual inhibition of Ca²⁺ uptake.     

A protein-binding assay for phallotoxins using muscle actin

The high affinity of phallotoxins to filamentous actin (K_D = 3.6 × 10⁻⁸m) has been used to assay small amounts of phallotoxins. The limit of detection was as low as 160 ng of phallotoxins. This is 50 times lower than the limit of all other procedures known so far. The assay was successfully applied to the analysis of phallotoxins in crude extracts of Amanita pholloides mushrooms.     

Analysis of complexes of metabolites with europium tetracycline using capillary electrophoresis coupled with laser-induced luminescence detection

Glycolysis and Krebs cycle intermediates were incubated with Eu³⁺-tetracycline and separated using capillary electrophoresis utilizing post-column laser-induced luminescence detection in a sheath flow cuvette. 3-phopshoglycerate, phosphoenolpyruvate, adenosine diphosphate, phosphate, citrate and oxaloacetate were detected at a concentration of 100 μM or lower. When all these detected metabolites were contained within the same sample it was found that they interfered with one another. Of all the metabolites, oxaloacetate showed the highest detectability. The system was found to yield a linear response for oxaloacetate from 50 nM to 10 μM. The injected volume of sample was 400 pL. This corresponds to 2 × 10^?17 mol of injected oxaloacetate from the 50 nM sample. As an application, the system was used to assay the enzyme aspartate aminotransferase, for whom oxaloacetate is a product. After a 1 h incubation period, 1.2 × 10^?13 M (3.3 μU/mL) enzyme was sufficient to form a detectable product signal. Extension of this incubation to 18 h permitted the detection of the activity of 1.2 × 10^?14 M (330 nU/mL) enzyme. This is the equivalent of 4.8 ymoles (2.9 molecules) of enzyme in the 400 pL injection volume. The enzyme’s catalytic rate was determined to be 240 s^?1 under the conditions used. In a second application, homogenates of Drosophila melanogaster were analyzed for metabolites, providing several peaks, including one which had the same retention time as citrate.     

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.     

A simple and accurate spectrophotometric assay for phosphoenolpyruvate carboxylase activity

The rate of phosphoenolpyruvate carboxylase activity measured through the conventional coupled assay with malate dehydrogenase is underestimated due to the instability of oxaloacetate, which undergoes partial decarboxylation into pyruvate in the presence of metal ions. The addition of lactate dehydrogenase to the conventional assay allows the reduction of pyruvate formed from oxaloacetate to lactate with the simultaneous oxidation of NADH. Then, the enzymic determination of substrate and products shows that the combined activities of malate dehydrogenase and lactate dehydrogenase account for all the phosphoenolpyruvate consumed. The net result of the improved assay is a higher V_max with no apparent effect on K_m. The free divalent cation concentration appears to be the major factor in the control of the rate of oxaloacetate decarboxylation.     

Enzymes concerned with beta-carboxylation in marine phytoplankter. Purification and properties of phosphoenolpyruvate carboxykinase.

The enzyme resonsible for β-carboxylation, with eventual incorporation of CO₂ into amino acids, has been studied in three diatoms and a dinoflagellate. The enzyme from Phaeodactylum tricornutum has been purified to homogeneity and has an absolute requirement for ADP and Mn²⁺. This enzyme is best described as phosphoenolpyruvate carboxykinase (carboxylating), but it differs considerably from enzymes with the same name, isolated from mammalian and bacterial sources, in that the reaction of phosphoenolpyruvate with bicarbonate lies strongly in the direction of formation of oxaloacetate and ATP. This conservation of the high-energy phosphate groups of phosphoenolpyruvate in ATP would be advantageous to diatoms living at low levels of light intensity in seawater.     

Gluconeogenesis from L-serine in rat liver.

Livers of fed and fasted rats were perfused $\text{in situ}$ in the presence and absence of 4.8 mM quinolinate, an $\text{in vivo}$ inhibitor of phosphoenolpyruvate carboxykinase. An assay of the hepatic activities of serine dehydratase and serine pyruvate transaminase and a comparison of the $\text{in vivo}$ incorporation of radioactivity from serine 3-¹⁴C and serine U-¹⁴C into blood glucose were also carried out in the above nutritional states. Our results demonstrate that gluconeogenesis from L-serine proceeds through two pathways. One, involving the reversal of the biosynthetic route of serine, bypasses conversion to pyruvate phosphoenolpyruvate and oxaloacetate and is not inhibited by quinolinate. This pathway appears to be the only one active in the fed state but produces a very insignificant amount of glucose. The other involves serine dehydratase mediated conversion of serine to pyruvate, is inhibited by quinolinate and becomes predominant during starvation.     

Temperature dependence of accuracy of DNA polymerase I from Geobacillus anatolicus

Klenow-like DNA polymerase I fragment from Geobacillus anatolicus (GF) was cloned and purified. The accuracy of GF was measured in vitro at three different temperatures under single turnover conditions as well as using a forward mutation assay. In pre-steady-state kinetic measurements, when temperature was raised from 22 °C to 50 °C, the rate (k_pol) for cognate dTTP and non-cognate dATP nucleotide incorporations increased six- and four-fold, respectively, whereas the K_d for both nucleotide incorporations changed only slightly. As a result, the error frequency was remained constant (∼4 × 10⁻⁴) over this temperature range. The accuracy of GF was also measured using a forward mutation assay during a single cycle of DNA synthesis of the lacZα complementation gene in M13mp2 DNA. In this assay, which scores various types of replication errors, mutant frequency of GF was 5 × 10⁻³ at 72 °C which is four-fold higher than that of 37 °C.     

Kinetic and functional properties of human mitochondrial phosphoenolpyruvate carboxykinase

The cytosolic form of phosphoenolpyruvate carboxykinase (PCK1) plays a regulatory role in gluconeogenesis and glyceroneogenesis. The role of the mitochondrial isoform (PCK2) remains unclear. We report the partial purification and kinetic and functional characterization of human PCK2. Kinetic properties of the enzyme are very similar to those of the cytosolic enzyme. PCK2 has an absolute requirement for Mn²⁺ ions for activity; Mg²⁺ ions reduce the K_m for Mn²⁺ by about 60 fold. Its specificity constant is 100 fold larger for oxaloacetate than for phosphoenolpyruvate suggesting that oxaloacetate phosphorylation is the favored reaction in vivo. The enzyme possesses weak pyruvate kinase-like activity (k_cat=2.7 s^?1). When overexpressed in HEK293T cells it enhances strongly glucose and lipid production showing that it can play, as the cytosolic isoenzyme, an active role in glyceroneogenesis and gluconeogenesis.     

Dissociation of hemoglobin into subunits. Monomer formation and the influence of ligands

The formation of monomer from several hemoglobins has been investigated by sedimentation equilibrium. The use of the split-beam photoelectric scanning absorption optical system has enabled observations to be made routinely down to 1 μg/ml. (6.2 × 10⁻⁸m-heme) with strict spectral control of the integrity of the hemoglobin molecule. The results show that the dissociation constant of dimer to monomer at neutral pH and moderate ionic strength is so small that monomer is present in reversible equilibrium with dimer only in fractions too small to be detectable. Any appreciable monomer formation is irreversible and accompanied by usually pronounced spectral changes. This irreversible monomer formation is probably a consequence of the presence of heavy-metal ions in solution and may be inhibited by 10⁻³m-EDTA. Hemoglobin ligands possessing chelating ability also inhibit monomer formation.     

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.     

A sensitive method for measuring oxygen consumption

A technique is presented whereby oxygen consumption rates of the order of 10⁻⁶ μl/hr can be measured, thus providing a means for studying the respiration rates of single cells, even slowly respiring ones. The technique is based on the principle of incubating cells in extremely small chambers containing highly concentrated hemoglobin solutions. The absorbance shift occurring when hemoglobin is transformed from its oxygenated to its deoxygenated form is recorded microspectrophotometrically. The results obtained by this technique seem to be well in accordance with those of the Warburg manometric method. The technique is convenient and easy to handle and the sensitivity can be varied within wide limits, permitting different types of materials to be studied. In experiments using yeast cells, respiration rates from 1.0 × 10⁻⁶ to 1.8 × 10⁻⁶ μl O₂/cell/hr were revealed.     

Highly purified intact chloroplasts from mesophyll protoplasts of the C4 plant Digitaria sanguinalis. Inhibition of phosphoglycerate reduction by orthophosphate and by phosphoenolpyruvate

Purified mesophyll protoplasts from the C₄ plant Digitaria sanguinalis were used to prepare intact mesophyll chloroplasts with low cytoplasmic contamination. The procedure involved breakage of protoplasts, differential centrifugation, partition in a dextran-polyethylene glycol two-phase system, and Percoll density gradient centrifugation. The final chloroplast preparation contained about 80% intact chloroplasts with a phosphoenolpyruvate carboxylase contamination of 0.2–1% of the original protoplast activity, corresponding to 1–6 μmol ¹⁴CO₂ fixed/mg Chl h. The purified chloroplasts showed substrate-dependent oxygen evolution in the range of 40–150 μmol substrate reduced/mg Chl h, with phosphoglycerate or oxaloacetate as substrate. Both reactions were stimulated 1.5 fold by pyruvate and further by addition of the other substrate. These measurements indicated that phosphoglycerate reduction was limited by substrate transport across the chloroplast envelope. Without added substrate, the chloroplasts consumed oxygen via pseudo-cyclic electron transport in the light. Also this reaction was stimulated by pyruvate. Phosphoglycerate-dependent oxygen evolution was inhibited by P_i and by phosphoenolpyruvate to about the same extent with purified chloroplasts, but only by P_i with protoplast extracts. This suggests that phosphoglycerate, P_i and phosphoenolpyruvate share a common carrier, similar to the P_i-translocator in C₃ chloroplasts, and that the lack of inhibition obtained with phosphoenolpyruvate and unpurified chloroplasts is artefactual, possibly due to oxaloacetate formation from added phosphoenolpyruvate and concomitant stimulation of oxygen evolution by oxaloacetate reduction. Furthermore, the results suggest that phosphoenolpyruvate is transported with a K_m similar to that of P_i in C₄ mesophyll chloroplasts.