Nitrate-induced polypeptides in membranes from corn seedling roots

The polypeptide composition of the membranes from corn (Zeamays L.) seedling roots upon nitrate induction was determinedby two-dimensional gel electrophoresis and silver-staining.The synthesis of five polypeptides (49, 48, 35, 33, and 32 kDa)in the tono-plast fraction and four polypeptides (50, 49, 38,and 33 kDa) in the plasma membrane fraction was induced by both2.5 mM Ca(NO₃)₂ and 5 mM KNO₃. Extensive washing of the membraneswith salt and NaOH demonstrated that three induced polypeptides(49, 48, and 35 kDa) in the tonoplast fraction and two inducedpolypeptides (49 and 33 kDa) in the plasma membrane fractionwere integral proteins. After incubation of seedlings in N-freemedium for 4 d, the 49 and 32 kDa polypeptides in the tonoplastfraction had disappeared. By the sixth day in N-free medium,the 35 kDa polypeptide had disappeared from the tonoplast fraction.The 50 kDa polypeptide of the plasma membrane fraction was nolonger detectable in seedlings incubated for 6 d in N-free medium.The size of the spots corresponding to the 33 kDa polypeptidesof both membrane fractions and to the 49 kDa polypeptide ofthe plasma membrane fraction was reduced following incubationof seedlings in N-free medium. The changes in nitrate-inducedpolypeptides in both membrane fractions following transfer toN-free medium correlated with a reduced capacity to take upnitrate in the treated seedlings. The results support the conclusionthat the nitrate-induced polypeptides may be involved in nitratetransport across the tonoplast and plasma membrane. Key words: Nitrate transport, induction, membrane peptides     

Nitrate uptake and induction of nitrate reductase in excised corn roots

The characteristics of nitrate uptake and induction of nitrate reductase were studied in excised roots of corn (Zea mays L.). Upon initial exposure to nitrate, the low initial rate of nitrate uptake gradually increased until a steady uptake rate was achieved in 1 to 2 hours depending on the NO(3) (-) concentration. The pattern was observed over a wide range (0.2-5 mm) of nitrate concentrations and was independent of the accompanying cation.The nitrate uptake pattern as a function of increasing external nitrate concentrations (0.2-50 mm) followed saturation type kinetics. The reciprocal plot of the data was not linear but hyperbolic, indicating that more than one Km for nitrate uptake can be resolved from the data. This suggests the existence of either one carrier system with changing kinetic constants or the existence of dual uptake systems. The pattern of induction of nitrate reductase was coincident with the pattern of nitrate uptake as a function of time and increasing nitrate concentrations. The rate of induction of nitrate reductase was regulated by the rate of nitrate flux.Washing the roots for 2 hours enhances nitrate uptake by 2.5-fold over the nonwashed tissue. The presence of nitrate in the washing solution leads to further (3.5-fold over control) increases in the rate of nitrate uptake supporting the contention that nitrate plays a specific role in the induction of the inducible nitrate carrier independent of the washing effect.     

Correlated induction of nitrate uptake and membrane polypeptides in corn roots

Induction of corn (Zea mays L.) seedling root membrane polypeptides was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis in relation to induction of nitrate uptake. When nitrate uptake was studied using freshly harvested roots from 4-day old corn seedlings, a steady state rate of uptake was achieved after a lag of 2 to 3 hours. The plasma membrane fraction from freshly harvested roots (uninduced) and roots pretreated in 5 millimolar nitrate for 2.5 or 5 hours (induced) showed no differences in the major polypeptides with Coomassie blue staining. Autoradiography of the ³⁵S-methionine labeled proteins, however, showed four polypeptides with approximate molecular masses of 165, 95, 70, and 40 kilodaltons as being induced by both 2.5 and 5-hour pretreatment in 5 millimolar nitrate. All four polypeptides appeared to be integral membrane proteins as shown by Triton X-114 (octylphenoxypolyethoxyethanol) washing of the membrane vesicles. Autoradiography of the two-dimensional gels revealed that several additional low molecular weight proteins were induced. A 5-hour pretreatment in 5 millimolar chloride also induced several of the low molecular weight polypeptides, although a polypeptide of about 30 kilodaltons and a group of polypeptides around 40 kilodaltons appeared to be specifically induced by nitrate. The results are discussed in relation to the possibility that some of the polypeptides induced by nitrate treatment may be directly involved in nitrate transport through the plasma membrane.     

Essential arginine residues in tryptophanase from Escherichia coli.

Tryptophanase from Escherichia coli B/1t7-A is inactivated by the arginine-specific reagent, phenylglyoxal, in potassium phosphate buffer at pH 7.8 AND 25 degrees. Apo- and holoenzyme are inactivated at the same rate, and inactivation of both is correlated with modification of 2 arginine residues/tryptophanase monomer. Substrate analogs having a carboxyl group protect the holoenzyme against both inactivation and arginine modification but have no effect on the inactivation or modification of the apoenzyme. Phenylglyoxal-modified apotryptophanase retains the capacity to bind the coenzyme, pyridoxal-P, but the spectrum of this reconstituted species differs from that of native holotryptophanase. Neither this reconstituted species nor the phenyglyoxal-modified holoenzyme shows the 500 nm absorption characteristic of the native enzyme when substrates are added. These results demonstrate a requirement for specific arginine residues for substrate binding and are discussed in the context of the known conformational and spectal forms of tryptophanase with regard to a possible role for arginine residues in formation of a catalytically effective enzyme-pyridoxal-P complex.     

Characterization of tonoplast polypeptides isolated from corn seedling roots

Tonoplast vesicles were isolated by discontinuous sucrose gradient centrifugation in the presence of Mg²⁺ from 5 day old corn (Zea mays L., Golden Cross Bantam) seedling roots. Marker enzyme assays indicated only a low degree of cross-contamination of tonoplast vesicles at the 10/23% (weight/weight) interface by other membrane components. Severalfold enrichment of tonoplast ATPase and pyrophosphatase was indicated in tonoplast fractions by dot blot studies with antibodies against an oat tonoplast ATPase and a mung bean tonoplast pyrophosphatase. Comparison of two-dimensional electrophoretic gels of tonoplast and microsomal membrane polypeptides revealed approximately 68 polypeptides to be specific to tonoplast by silver staining. Immunoblot analysis with antibodies against a tonoplast holoenzyme ATPase from oat roots revealed the presence of the 72, 60, and 41 kilodalton polypeptides in isolated tonoplast vesicles from corn roots. Affinity blotting with concanavalin A and secondary antibodies indicated the degree of glycosylation of tonoplast polypeptides, where 21 of 68 tonoplast-specific polypeptides contained detectable carbohydrate moieties. Salt and NaOH washes removed 38 of the tonoplast-specific polypeptides, indicating a peripheral association with the membrane. Thirteen of the peripheral polypeptides and eight of the integral polypeptides were identified as glycoproteins. This information on the polypeptide composition of the tonoplast of root cells will aid in gaining insight into the role of this membrane in controlling vacuolar functions.     

Essential arginine residues of creatine kinase from beef heart mitochondria

Creatine kinase from beef heart mitochondria is inactivated by 2,3-butanedione. The kinetics of inactivation of the mitochondrial enzyme is biphasic with a bend at a point corresponding to 50% inactivation. The inactivation rate constants of the first fast and the second slow phases of the reaction differ by one order of magnitude, thus suggesting the existence of two types of arginine residues, i.e. "fast" and "slow" ones, with different reactivities. The inactivation rate constant of the slow phase is very close to that for cytoplasmic creatine kinase. At saturating concentrations MgATP and MgADP afford complete protection of the slow phase of inactivation. It is assumed that the "slow" arginine is involved in the binding of metal-nucleotide substrates in the enzyme active center.     

Essential arginine residues in D-glyceraldehyde-3-phosphate dehydrogenase.

Two arginyl residues per subunit of yeast D-glyceraldehyde-3-phoshphate dehydrogenase were modified by treatment with butanedione without significant changes in the compostion of other amino acid residues. The modified enzyme displays no dehydrogenase activity. It retains the capacity for interacting with the coenzyme NAD, but binds it less firmly than does the native enzyme. The molar absorbance of the enzyme-NAD complex is markedly reduced and the reactivity of the active-center SH groups is changed in the modified enzyme. The native and modified enzymes show identical fluorescence spectra, absorbance and CD spectra.     

Essential arginine residues in human liver arylsulfatase A.

Human liver arylsulfatase A was treated with arginine-specific reagents (diones), resulting in a loss of enzyme activitity with apparent first-order kinetics. Sulfite and borate—competitive inhibitors of the enzyme—provided complete protection from inactivation by phenylglyoxal. Sulfite and substrate each likewise protected against enzyme inactivation by 2,3-butanedione. A plot of pseudo-first-order rate constants of enzyme inactivation versus 2,3-butanedione concentrations suggests that an essential arginine residue is modified with a loss in function of the binding site or of the active site of the protein. Chemical analysis of the butanedione-treated sulfatase indicates that complete enzyme inactivation corresponds to a modification of only about 2 of the 20 arginine residues per enzyme subunit. Taken together, all of the results strongly suggest that arginine residues are essential for the activity of arylsulfatase A. An incidental discovery in this work is that borate ion is a competitive inhibitor of human arylsulfatase A with a K_i of 2.5 × 10^?4 M.     

Synthesis and turnover of nitrate reductase in corn roots

The induction and reinduction of nitrate reductase in root tip or mature root sections show essentially a similar pattern: a lag, a period of rapid increase in enzyme activity and finally a period of relatively minor change. Both inductions are sensitive to 6-methylpurine and cycloheximide. Kinetic studies with 6-methylpurine suggest that the half-life of the messenger RNA for nitrate reductase in both sections is about 20 minutes. The rate of decay of nitrate reductase activity induced by transfer to a nitrate-free medium is slower in root tips (t½ = 3 hours) than in mature root sections (t½ = 2 hours). The enzyme from mature root sections is also less stable to mild heat treatments (27 C; 40 C) than the enzyme from root tip sections. The results indicate that factors regulating enzyme turnover show important changes as root cells mature and may be significant in determining steady state levels of the enzyme.     

Multiphasic uptake of phosphate by corn roots

Abstract The concentration dependence of phosphate uptake was studied using root sections of corn (Zea mays L. cv. Ganga 5). Detailed and wide-range (57 concentrations in the range 1 μmol m^?3-75 mol m^?3), precise (average SEM < 2.5%, n= 6) and reproducible (similar patterns in three independent experiments and for 5, 10, 15, 20, 25 and 30°C) data revealed six (or seven) concentration-dependent phases separated by ‘jumps’ or sharp breaks. These transitions were independent of temperature and occurred over relatively narrow concentration ranges (0.0001–0.0004, 0.08–0.31, 1.0–3.5, (7.5–10), 18–20 and 57–59 mol m^?3). The intermediate phases obeyed Michaelis-Menten kinetics, whereas sigmoidal kinetics were observed at lower concentrations. Uptake within each of the two highest phases increased more rapidly with increasing external phosphate concentration than predicted from Michaelis-Menten kinetics but also saturated more rapidly. The latter finding is not consistent with free diffusion across the plasmalemma at high external phosphate concentrations. Kinetic models yielding continuous isotherms, e.g. the sum of one or two Michaelis-Menten terms and a diffusion term, cannot account for the data.     

On the mechanism of nitrate uptake by plant roots

Summary A study has been made on the influx and outflux of nitrogen compounds by the excised roots of barley, wheat and peas. A two way movement of nitrogen compounds was found to occur between root and external medium. Factors such as initial N content in the roots, species of plant and external concentration of N highly affect the extent to which this two-way movement proceeds. Further investigations are needed for more understanding of the nitrogen balance between plant roots and external medium.     

pH control in nitrate uptake studies with excised roots

Summary In the investigation of NO ₃ ^– uptake by excised roots, pH of salt solutions can be effectively controlled by periodic additions of a cation exchange resin, Amberlite IR-120, in the H⁺ form. This method of pH control provides for the addition of H⁺ to systems without an accompanying anion which might influence the absorption of NO ₃ ^– . The resin appeared to provide a favorable buffering capacity during an extended period and the method was especially useful for NO ₃ ^– uptake studies conducted at high root-to-solution ratios.Contribution from the Plant Nutrition Laboratory, Department of Botany, The University of Michigan, paper number 39. This work was supported by research grant GM-07339-02 from the National Institutes of Health, U. S. Public Health Service.     

Effect of glucose on the induction of nitrate reductase in corn roots

In Zea mays L., addition of glucose to the induction medium has no effect on the induction of nitrate reductase during the initial 3 hours either in root tips (0-10 mm) or mature root sections (25-35 mm). With longer times, higher levels of enzyme activity are recovered from both root segments when glucose is present in the incubation medium. The induction in root tips is saturated by 10 mm NO(3) (-). Higher concentrations of NO(3) (-) are required for saturation in mature root sections. The response to glucose is seen over a wide range of external NO(3) (-) concentrations.Nitrate reductase activity is lost rapidly when nitrate is withdrawn from the induction medium. Additions of glucose do not prevent this loss. Additions of glucose have no effect on total uptake of NO(3) (-) by the root segments but they increase the anaerobic NO(2) (-) production in both root tips and mature root segments. This latter measurement is considered to be an estimate of an active NO(3) (-) pool in the cytoplasm. Thus the results show that glucose alters the distribution of NO(3) (-) within the root sections. This may be an important factor in controlling the in vivo stability of the enzyme or its rate of synthesis.     

Variations in sodium uptake along primary roots of corn seedlings

Entry of Na⁺ into segments of the apical 8-centimeter portion of corn (Zea mays) roots was investigated and analyzed for each centimeter segment separately. Influence of temperature in the 0 C to 30 C range was well described by the Arrhenius equation [U = A exp (-Ea/RT)]. Values of A and Ea differed for each segment, tending to lessen with increasing distance from root apex. Time course of Na⁺ entry was followed up to 70 minutes. Time relations of the process fit well the expression U = m [1 - exp (-nt)]. Calculated maximal uptake capacity (m) diminished with increasing distance from the apex. The data presented indicate that sodium uptake mechanisms vary qualitatively and quantitatively along corn roots. Thus, the use of entire roots for characterization of uptake mechanisms should be reassessed.     

Inhibition of phosphate uptake in corn roots by aluminum-fluoride complexes

F forms stable complexes with Al at conditions found in the soil. Fluoroaluminate complexes (AlF(x)) have been widely described as effective analogs of inorganic phosphate (Pi) in Pi-binding sites of several proteins. In this work, we explored the possibility that the phytotoxicity of AlF(x) reflects their activity as Pi analogs. For this purpose, (32)P-labeled phosphate uptake by excised roots and plasma membrane H(+)-ATPase activity were investigated in an Al-tolerant variety of maize (Zea mays L. var. dwarf hybrid), either treated or not with AlF(x). In vitro, AlF(x) competitively inhibited the rate of root phosphate uptake as well as the H(+)-ATPase activity. Conversely, pretreatment of seedlings with AlF(x) in vivo promoted no effect on the H(+)-ATPase activity, whereas a biphasic effect on Pi uptake by roots was observed. Although the initial rate of phosphate uptake by roots was inhibited by AlF(x) pretreatment, this situation changed over the following minutes as the rate of uptake increased and a pronounced stimulation in subsequent (32)Pi uptake was observed. This kinetic behavior suggests a reversible and competitive inhibition of the phosphate transporter by fluoroaluminates. The stimulation of root (32)Pi uptake induced by AlF(x) pretreatment was tentatively interpreted as a phosphate starvation response. This report places AlF(3) and AlF(4)(-) among Al-phytotoxic species and suggests a mechanism of action where the accumulation of Pi-mimicking fluoroaluminates in the soil may affect the phosphate absorption by plants. The biochemical, physiological, and environmental significance of these findings is discussed.     

Arylsulphatase activity of corn (Zea mays L.) seedling roots

The crude lysosomal fraction of corn seedling root tips contains an arylsulphatase (E.C. 3.1.6.1) which hydrolysed p-nitrophenyl sulphate at pH 8.0 but had no activity towards p-nitrocatechol sulphate. The Km value for p-nitrophenyl sulphate was 1.24 mM. The hydrolysis of p-nitrophenyl sulphate was linear up to 2 h and the rate was proportional to the amount of enzyme added. The enzyme was strongly inhibited by cyanide, fluoride and phosphate ions and did not resemble the arylsulphatases of bacterial and animal origin.     

Ammonium and amino acids as regulators of nitrate reductase in corn roots

When amino acids or ammonia are added to plant systems, the effects on the development of nitrate-dependent nitrate reductase activity are variable. In addition, amino acids added singly or as casein hydrolysate may not support a normal growth. A physiologically correct mixture of amino acids, one similar in composition to amino acids released by the endosperm, has been shown to support normal growth and protein synthesis in corn (Zea mays) embryos. In this investigation, we have used the mixture of corn amino acids to determine whether amino acids have an effect on the appearance or disappearance of nitrate reductase activity. The results show that these amino acids partially inhibit the induction of nitrate reductase in corn roots. The effect is more pronounced in mature root than in root tip sections. When glutamine and asparagine are included along with the "corn amino acid mixture," the inhibition is more severe. Amino acids or amino acid analogues added singly to the induction medium have a similar effect: i.e. when the induction of nitrate reductase is inhibited in the root tips (lysine, canavanine, azaserine, azetidine-2-carboxylic acid, dl-4-azaleucine, asparagine, and glutamine), that inhibition is more severe in mature root sections. Arginine enhanced the recovery of nitrate reductase in root tips but inhibited it in mature root sections. The effect of the amino acids is apparently on some phase of the induction processes (i.e. the uptake or distribution of nitrate or a direct effect on the synthesis of the enzyme) and not on the turnover of the enzyme.     

Properties of potassium uptake by seedling roots of grape cultivars

Uptake rates of (⁸⁶Rb)K⁺ by seedling roots of six cultivars were measured and compared with K⁺ content of the root, K⁺ leakage, H⁺ efflux, and K⁺-ATPase activity of a partially purified plasmalemma fraction.Different cultivars showed significantly different rates of (⁸⁶Rb)K⁺ uptake. The uptake rates of the first (0–5 min) period did not correlate with K⁺ content of the seedling roots.The rates of uptake in the 10 to 30 min period, supposed to be active, were negatively correlated with K⁺ content of the root. Roots consistently leaked K⁺ during the first 5 min. This leakage was positively correlated with the endogenous K⁺ content of the tissue.H⁺ efflux was significantly different among the cultivars and correlated with the K⁺-ATPase activity of a microsomal fraction partially purified on discontinuous (18/34%) sucrose gradient. The relationships among transport parameters are discussed.Part of the present research was carried out at the Department of Agricultural Biotechnology of University of Padova, Italy.Part of the present research was carried out at the Department of Agricultural Biotechnology of University of Padova, Italy.     

Anion uptake in maize roots: Interactions between chlorate and nitrate

Effects of nitrate, chloride and chlorate ions upon nitrate and chlorate uptake by roots of maize ( Zea mays L., cv. B73) seedlings were examined. Net nitrate uptake, ³⁶ClO₃⁻ influx and ³⁶Cl⁻ influx (the latter two in a background of 0.5 m M KNO₃) displayed similar pH profiles with optima at pH 5.5 and below. External, non-labeled chloride had little effect on the accumulation of ³⁶ClO₃⁻ (both in 5 h and 20 min uptake assays), while nitrate and chlorate had almost identical, marked inhibitory effects. Nitrate pretreatment caused an apparent induction of both ³⁶ClO₃⁻ and ¹⁵NO₃⁻ uptake activities. After 5 h of treatment in nitrate, the uptake activities of chloride- and chlorate-pretreated plants increased to that of nitrate-pretreated plants. During 6 h exposure to chlorate, ³⁶ClO₃⁻ uptake activity of nitrate-pretreated plants decreased to that of chlorate- and chloride-pretreated plants. The results support the existence of a shared nitrate/chlorate transport system in maize roots which is not inhibited by external chloride, and which is induced by nitrate, but not by chlorate or chloride. The suggestion is made that selection of chlorate-resistant mutants of maize can identify nitrate uptake as well as nitrate reductase mutants.