Contribution of Malate and Amino Acid Metabolism to Cytoplasmic pH Regulation in Hypoxic Maize Root Tips Studied Using Nuclear Magnetic Resonance Spectroscopy

³¹P-, ¹³C-, and ¹⁵N-nuclear magnetic resonance spectroscopy were used to determine the roles of malate, succinate, Ala, Asp, Glu, Gln, and γ-aminobutyrate (GABA) in the energy metabolism and regulation of cytoplasmic pH in hypoxic maize (Zea mays L.) root tips. Nitrogen status was manipulated by perfusing root tips with ammonium sulfate prior to hypoxia; this pretreatment led to enhanced synthesis of Ala early in hypoxia, and of GABA at later times. We show that: (a) the ability to regulate cytoplasmic pH during hypoxia is not significantly affected by enhanced Ala synthesis. (b) Independent of nitrogen status, decarboxylation of Glu to GABA is greatest after several hours of hypoxia, as metabolism collapses. (c) Early in hypoxia, cytoplasmic malate is in part decarboxylated to pyruvate (leading to Ala, lactate, and ethanol), and in part converted to succinate. It appears that activation of malic enzyme serves to limit cytoplasmic acidosis early in hypoxia. (d) Ala synthesis in hypoxic root tips under these conditions is due to transfer of nitrogen ultimately derived from Asp and Gln, present in oxygenated tissue. We describe the relative contributions of glycolysis and malate decarboxylation in providing Ala carbons. (e) Succinate accumulation during hypoxia can be attributed to metabolism of Asp and malate; this flux to succinate is energetically negligible. There is no detectable net flux from Glc to succinate during hypoxia. The significance of the above metabolic reactions relative to ethanol and lactate production, and to flooding tolerance, is discussed. The regulation of the patterns of metabolism during hypoxia is considered with respect to cytoplasmic pH and redox state.     

Metabolic Response of Maize Roots to Hyperosmotic Shock : An in VivoP Nuclear Magnetic Resonance Study

³¹P nuclear magnetic resonance spectroscopy was used to study the response of maize (Zea mays L.) root tips to hyperosmotic shock. The aim was to identify changes in metabolism that might be relevant to the perception of low soil water potential and the subsequent adaptation of the tissue to these conditions. Osmotic shock was found to result in two different types of response: changes in metabolite levels and changes in intracellular pH. The most notable metabolic changes, which were produced by all the osmotica tested, were increases in phosphocholine and vacuolar phosphate, with a transient increase in cytoplasmic phosphate. It was observed that treatment with ionic and nonionic osmotica produced different effects on the concentrations of bioenergetically important metabolites. It is postulated that these changes are the result of hydrolysis of phosphatidylcholine and other membrane phospholipids, due to differential activation of specific membrane-associated phospholipases by changes in the surface tension of the plasmalemma. These events may be important in the detection of osmotic shock and subsequent acclimatization. A cytoplasmic alkalinization was also observed during hyperosmotic treatment, and this response, which is consistent with the activation of the plasmalemma H⁺-ATPase, together with the other metabolic changes, may suggest the existence of a complex and integrated mechanism of osmoregulation.     

Sugar-Starvation-Induced Changes of Carbon Metabolism in Excised Maize Root Tips

Excised maize (Zea mays L.) root tips were used to study the early metabolic effects of glucose (Glc) starvation. Root tips were prelabeled with [1-13C]Glc so that carbohydrates and metabolic intermediates were close to steady-state labeling, but lipids and proteins were scarcely labeled. They were then incubated in a sugar-deprived medium for carbon starvation. Changes in the level of soluble sugars, the respiratory quotient, and the 13C enrichment of intermediates, as measured by 13C and 1H nuclear magnetic resonance, were studied to detect changes in carbon fluxes through glycolysis and the tricarboxylic acid cycle. Labeling of glutamate carbons revealed two major changes in carbon input into the tricarboxylic acid cycle: (a) the phosphoenolpyruvate carboxylase flux stopped early after the start of Glc starvation, and (b) the contribution of glycolysis as the source of acetyl-coenzyme A for respiration decreased progressively, indicating an increasing contribution of the catabolism of protein amino acids, fatty acids, or both. The enrichment of glutamate carbons gave no evidence for proteolysis in the early steps of starvation, indicating that the catabolism of proteins was delayed compared with that of fatty acids. Labeling of carbohydrates showed that sucrose turnover continues during sugar starvation, but gave no indication for any significant flux through gluconeogenesis.     

Ethanol Reassimilation and Ethanol Tolerance in Pichia stipitis CBS 6054 as Studied by C Nuclear Magnetic Resonance Spectroscopy

Ethanol reassimilation in Pichia stipitis CBS 6054 was studied by using continuous cultures, and the oxidation of [1-C]ethanol was monitored by in vivo and in vitro C nuclear magnetic resonance spectroscopy. Acetate was formed when ethanol was reassimilated. The ATP/ADP ratio and the carbon dioxide production decreased, whereas the malate dehydrogenase activity increased, in ethanol-reassimilating cells. The results are discussed in terms of the low ethanol tolerance in P. stipitis compared with that in Saccharomyces cerevisiae (S. W. Brown, S. G. Oliver, D. E. F. Harrison, and R. C. Righelato, Eur. J. Appl. Microbiol. Biotechnol. 11:151-155, 1981).     

Diffusion of Exchangeable Water in Cortical Bone Studied by Nuclear Magnetic Resonance

The rate-limiting step in the delivery of nutrients to osteocytes and the removal of cellular waste products is likely diffusion. The transport of osteoid water across the mineralized matrix of bone was studied by proton nuclear magnetic resonance spectroscopy and imaging by measuring the diffusion fluxes of tissue water in cortical bone specimens from the midshaft of rabbit tibiae immersed in deuterium oxide. From the diffusion coefficient (D_a = (7.8 ± 1.5) × 10⁻⁷ cm²/s) measured at 40°C (close to physiological temperature), it can be inferred that diffusive transport of small molecules from the bone vascular system to the osteocytes occurs within minutes. The activation energy for water diffusion, calculated from D_a measured at four different temperatures, suggests that the interactions between water molecules and matrix pores present significant energy barriers to diffusion. The spatially resolved profile of D_a perpendicular to the cortical surface of the tibia, obtained using a finite difference model, indicates that diffusion rates are higher close to the endosteal and periosteal surfaces, decreasing toward the center of the cortex. Finally, the data reveal a water component (∼30%) diffusing four orders of magnitude more slowly, which is ascribed to water tightly bound to the organic matrix and mineral phase.     

Observation of Cytoplasmic and Vacuolar Malate in Maize Root Tips by C-NMR Spectroscopy

The accumulation of malate by maize (Zea mays L.) root tips perfused with KH¹³CO₃ was followed by ¹³C nuclear magnetic resonance spectroscopy. In vivo nuclear magnetic resonance spectra contained distinct signals from two pools of malate in maize root tips, one at a pH ~5.3 (assigned to the vacuole) and one at a pH > 6.5 (assigned to the cytoplasm). The ratio of cytoplasmic to vacuolar malate was lower in 12 millimeter long root tips than in 2 millimeter root tips. The relatively broad width of the signals from C1- and C4-labeled vacuolar malate indicated heterogeneity in vacuolar pH. During the 3 hour KH¹³CO₃ treatment, ¹³C-malate accumulated first primarily in the cytoplasm, increasing to a fairly constant level of ~6 millimolar by 1 hour. After a lag, vacuolar malate increased throughout the experiment.     

Ethanol Reassimilation and Ethanol Tolerance in Pichia stipitis CBS 6054 as Studied by 13C Nuclear Magnetic Resonance Spectroscopy

Ethanol reassimilation in Pichia stipitis CBS 6054 was studied by using continuous cultures, and the oxidation of [1-¹³C]ethanol was monitored by in vivo and in vitro ¹³C nuclear magnetic resonance spectroscopy. Acetate was formed when ethanol was reassimilated. The ATP/ADP ratio and the carbon dioxide production decreased, whereas the malate dehydrogenase activity increased, in ethanol-reassimilating cells. The results are discussed in terms of the low ethanol tolerance in P. stipitis compared with that in Saccharomyces cerevisiae (S. W. Brown, S. G. Oliver, D. E. F. Harrison, and R. C. Righelato, Eur. J. Appl. Microbiol. Biotechnol. 11:151-155, 1981).     

Accumulation of Oxygen Gas in Excised Maize Root on Aging in Water, and Water Uptake by the Root

Transfer of excised maize root from wet sawdust to water causeda considerable reduction in the exudation rate of the root.After 1-day aging in water, the exudation rate increased about8-fold and the exudation continued for 3 days. Osmotic pressureof the exudate from the root decreased with time after excisionreaching almost zero in 2 days in spite of a high exudationrate. Concentrations of sugars, acids, Ca²⁺ and Mg²⁺ in theexudate decreased with the decrease of osmotic pressure, whilethe decrease in K⁺ concentration delayed and P₁ concentrationincreased. The gas content of the root, especially of O₂, increased duringaging in water. The accumulated O₂ gas may promote water uptake,because degasification of the root by evacuation induced a decreaseof water uptake. Also, the longitudinal gradient of the O₂ contentin the root coincides with the gradient of water uptake intensity. (Received February 7, 1982; Accepted July 2, 1983)     

Specificity and Reversibility of the Bicarbonate Effect on Hexose Uptake by Maize Root Tips

The inhibition of hexose uptake by bicarbonate ions was investigatedin detail in order to test the specificity and reversibilityof the effect and to compare it with those of other electrolytes.The degree of inhibition was similar at pH 7.0 and pH 8.0. AtpH 4.5 no influence of a high concentration of CO₂ on 3-O-methylglucoseuptake was found. Therefore, the inhibition of hexose uptakeby bicarbonate cannot be explained by consequences of CO₂ influx.The inhibition of sugar absorption by calcium and potassiumions was similar to that exerted by bicarbonate in so far asit was observed at higher pH only. The inhibition exerted bysodium salts of different monovalent weak acids was limitedto lower pH and needed some time to become established or reversed.The bicarbonate effect was independent of time and reversiblewithout a lag phase. Sodium salts of strong mineral monovalentacids did not differ significantly in their effect on sugaruptake. Bicarbonate inhibited phosphate uptake in a similarmanner to hexose uptake but strongly stimulated the absorptionof potassium. The bicarbonate effect is assumed to result froma change in the degree of coupling of secondary active transportto the proton pump. Key words: Inhibition, Transport coupling, pH, Proton pump     

Classification of Modern Resins by Solid State Nuclear Magnetic Resonance Spectroscopy

The carbon-13 nuclear magnetic resonance (NMR) spectra of modern resins have been used to characterize their botanical sources. Resins from four of the principal diterpenoid resinproducing genera, Agathis, Hymenaea, Pinus, and Araucaria, give distinct NMR spectra under conditions of normal and interrupted decoupling. The genus Wollemia gives the same spectra as Agathis. Samples from the triterpenoid resin-producing family Burseraceae and of gum resins were recorded for comparison. Resin samples from unknown or uncertain sources then were examined and assigned to their botanical sources.     

Effects of Hydroxyurea and Deoxyadenosine on the Synthesis of Deoxycytidine Phosphate and on the Uptake of Nucleosides in Excised Root Tips of Vicia faba

The effects of hydroxyurea and deoxyadenosine on the synthesis of deoxycytidine phosphate was studied by measuring the incorporation of [¹⁴C]-cytidine into acid soluble deoxycytidine phosphate in root tips of Vicia faba. Hydroxyurea and deoxyadenosine both markedly depressed the incorporation of [¹⁴C]-cytidine. Deoxyadenosine had the additional effect of inhibiting the uptake of [¹⁴C]-cytidine. Furthermore, millimolar concentrations of deoxyadenosine inhibited the uptake of micromolar concentrations of adenosine, thymidine, and deoxycytidine. The incorporation of [¹⁴C]-cytidine into RNA was only slightly affected by hydroxyurea. Deoxyadenosine inhibited the incorporation into RNA to about the same extent as the uptake of [¹⁴C]-cytidine. It is suggested that hydroxyurea reduced the incorporation of radioactive cytidine into deoxycytidine phosphate mainly by interfering with ribonucleotide reduction. The depression of [¹⁴C]-cytidine incorporation into deoxycytidine phosphate in the presence of deoxyadenosine is believed to be the result of an inhibition of both ribonucleotide reduction and [¹⁴C]-cytidine uptake.     

Characterisation of Soybean and Wheat Seeds by Nuclear Magnetic Resonance Spectroscopy

The effects of equilibration under different air relative humidities (RH, 1 – 90 %) and temperatures (35 and 45 °C) on soybean (Glycine max) and wheat (Triticum aestivum) seeds were studied using different techniques. Seed moisture content, electrical conductivity (EC) of seed leachate and per cent seed germination were measured following standard procedures, and compared with nuclear magnetic resonance spin-spin relaxation time (T₂) measurements. Moisture contents of soybean and wheat seeds, following the reverse sigmoidal trend, were greater at 35 than at 45 °C at any particular RH. Changes in T₂ were related to the changes in germination percentage and leachate EC of both soybean and wheat seeds. Equilibrating soybean seeds at RH 11 % decreased germination percentage with corresponding decrease in T₂. On the contrary, EC of seed leachate increased. In wheat seeds equilibrated at 45 °C, T² was maximal at RH 5.5 %. T₂ declined in seeds equilibrated at high RH (> 80 %) together with low germination percentage.     

Soluble Sugars, Respiration, and Energy Charge during Aging of Excised Maize Root Tips

Oxygen uptake and energy charge were monitored during aging of excised maize root tips and related to the soluble sugar content and exogenous sugar supply.     

局部供磷对玉米根系生长、磷吸收速率的影响

玉米幼苗种子根局部供磷可明显改变根系的形态。供磷区侧根生长增加,无磷区侧极生长减少。供磷区1次、2次侧根长度与2次侧根数量明显增加;而1次侧根数量则不增加。供磷区缩小时,根系生长加快,单位根区磷吸收速率增加,但单位根重磷吸收速率的增加不很明显。磷局部供应植株主要通过供磷区根系的生长来增加磷的吸收,以满足植株对磷的需求。局部供磷植株中转运到供磷根区的光合产物明显多于无磷根区。     

Proton Nuclear Magnetic Resonance Spectroscopy of Rabbit Brain Homogenate

Abstract: Proton nuclear magnetic resonance (¹H NMR) spectroscopy in conjunction with the inversion-recovery spin-echo pulse sequence was used to obtain spectra from rabbit brain homogenate. The instrumental parameters required for the acquisition of spectra together with the assignment of major peaks are given. The rationale and prospectus for the use of this technique for the study of neurochemistry is outlined.     

Xenobiotic Monitoring in Plants by F and H Nuclear Magnetic Resonance Imaging and Spectroscopy: Uptake of Trifluoroacetic Acid in Lycopersicon esculentum

¹⁹F and ¹H nuclear magnetic resonance imaging and spectroscopy have been used to monitor the uptake of trifluoroacetic acid in stems and leaves of Lycopersicon esculentum. The movement and location of a xenobiotic have been demonstrated in vivo by a noninvasive technique.     

Nuclear Magnetic Resonance Spectroscopy for the Study of B-Cell Epitopes

High-resolution nuclear magnetic resonance (NMR) spectroscopy is a structural technique that is finding increasing use in the study of antibody–antigen interactions. In this review we describe how the dynamic structural parameters obtained from NMR spectroscopy can further our understanding of B-cell epitopes and their function. Specific applications of NMR spectroscopy to examine the residues on peptides and proteins that contact the antibody combining site are also described. These include “footprinting” techniques using H–D exchange–COSY NMR spectroscopy, which are particularly useful for epitope mapping of protein antigens. For smaller systems, such as Fab–or Fv–peptide complexes, nuclear magnetization transfer difference NMR spectroscopy, transferred nuclear Overhauser effect spectroscopy, double-quantum-filtered NOE spectroscopy, and isotope editing techniques have been applied. The interpretation and limitations of the data obtained from these procedures and anticipated improvements in these applications in the future are discussed.     

Flocculation Effects on Bound Water in Sludges as Measured by Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance relaxation times (T₁ and T₂) were measured for flocculated and unflocculated samples of activated sludge. The weight of water and solids in the sludge samples was found and related to T₁ to find the relative percentage of bound water. The results suggest that the amount of bound water increases as the samples become more unflocculated. The values of T₁ and T₂ also indicate that unflocculated individual particles are characterized by loose packing of shorter molecules and that the addition of larger molecules may induce flocculation.     

Compartmentation of Nucleotides in Corn Root Tips Studied by P-NMR and HPLC

Corn (Zea mays L.) root tips were subjected to different conditions so that nucleotide levels varied over a wide range. Levels of nucleotides in corn root tips were measured using ³¹P nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography. Results indicate: (a) Similar amounts of NTP and sugar nucleotides were observed by in vivo NMR and in extracts. In contrast, a significant amount of NDP observed in root tip extracts was not detected by in vivo NMR. Thus, for a given sample, [NTP]/[NDP] ratios determined in vivo by ³¹P-NMR are always higher than ratios observed in extracts, deviating by ~4-fold at the highest ratios. The NMR-invisible pool of NDP appeared quite metabolically inert, barely changing in size as total cell NDP changed. We conclude that NDP in corn root tips is compartmented with respect to NMR visibility, and that it is the NMR-visible pool which responds dynamically to metabolic state. The NMR-invisible NDP could either be immobilized (and so have broad, undetectable NMR signals), or be complexed with species that cause the chemical shift of NDP to change (so it does not contribute to the NMR signal of free NDP), or both. (b) ³¹P-NMR cannot distinguish between bases (A, U, C, and G) of nucleotides. HPLC analysis of root tip extracts showed that the relative amount of each base in the NTP and NDP pools was quite constant in the different samples. (c) In extracts, for each of the nonadenylate nucleotides, [NTP]/[NDP] was linearly proportional to [ATP]/[ADP], indicating near equilibrium in the nucleoside diphosphokinase (NDPK) reaction. However, the apparent equilibrium constants for the phosphorylation of GDP and UDP by ATP were significantly lower than 1, the true equilibrium constant for the NDPK reaction. Thus, for a given sample, [ATP]/[ADP] ~ [CTP]/[CDP] > [UTP]/[UDP] > [GTP]/[GDP]. This result suggests that the different NDPs in corn root tips do not have equal access to NDPK.     

核磁共振波谱法在蛋白质三维结构解析中的应用

蛋白质特定的三维结构与其生物功能密切相关,因此,研究蛋白质的三维结构有助于揭示其生物功能机制。将核磁共振（NMR）波谱法应用于研究溶液状态下蛋白质的三维结构,能够更加准确地揭示蛋白质结构与生物功能之间的关系。本文综述了NMR解析蛋白质三维结构的理论和技术方法,以及NMR结合其他生物物理手段,并辅以分子建模计算法研究蛋白质三维结构的研究进展和最新方法,为精准解析蛋白质的三维结构提供思路及策略。