Ammonium molybdate-stannous chloride determination of orthophosphate in the presence of triton X-100

A colorimetric method for the determination of orthophosphate in the presence of Triton X-100 and the extent of their mutual interference is presented. Effects of albumin and trichloroacetic acid on the reaction are also examined. The method is based on the very sensitive reaction developed for determination of orthophosphate by complex formation with ammonium molybdate followed by reduction with stannous chloride. The method allows determination of 0.005 μmol of orthophosphate in the presence of up to 0.5% Triton X-100 and as little as 0.3% ($\text{v}\text{v}$) Triton X-100 in the absence of phosphate.     

Photosynthesis in Isolated Chloroplasts of the Crassulacean Acid Metabolism Plant Sedum praealtum

Intact chloroplasts were isolated from protoplasts of the Crassulacean acid metabolism plant Sedum praealtum D.C. Typical rates of CO₂ fixation or CO₂-dependent O₂ evolution ranged from 20 to 30 micromoles per milligram chlorophyll per hour and could be stimulated 30 to 50% by several Calvin cycle intermediates. The pH optimum for CO₂ fixation was 7.0 to 7.6 with considerable activity as low as pH 6.4. Low concentrations of orthophosphate (Pi) (optimum 0.4 millimolar) stimulated photosynthesis while high concentrations (5 millimolar) caused some inhibition. Both CO₂ fixation and CO₂-dependent O₂ evolution exhibited a relatively long lag phase (4 to 6 minutes) which remained constant between 0.4 to 5 millimolar Pi. The lag phase could be decreased by addition of dihydroxyacetone-phosphate or ribose 5-phosphate. Further results are presented which suggest these chloroplasts have a functional phosphate translocator.     

Control of glycolysis by orthophosphate and adenosine triphosphate in soluble extracts of germinating pea seeds

Summary Control of aerobic glycolysis by adenosine triphosphate and orthophosphate has been studied in cell-free extracts of germinating pea seeds. Orthophosphate accelerates glycolysis under all conditions studied. At high concentrations of magnesium ion ATP accelerates glycolysis, whereas at lower magnesium concentrations ATP severely inhibits glycolysis. The inhibitory effect of ATP is markedly relieved by orthophosphate. Metabolite analyses suggest an important regulatory role of phosphofructokinase and show that low ratios of F-6-P: FDP accompany the appearance of a high rate of glycolysis, and vice versa. Thus, ATP raises the F-6-P: FDP ratio at low magnesium levels, while P_i lowers this ratio. At high Mg²⁺ (where ATP accelerates glycolysis), ATP causes a low F-6-P: FDP ratio to appear. At low Mg²⁺ concentration, orthophosphate accelerates glycolysis by activation of phosphofructokinase; at high magnesium concentration, the chief effect of orthophosphate is its long-known role in facilitating the oxidation of triose phosphate.     

Characterization of the Formation and Distribution of Photosynthetic Products by Sedum praealtum Chloroplasts

Photoassimilation of ¹⁴CO₂ by intact chloroplasts from the Crassulacean acid metabolism plant Sedum praealtum was investigated. The main water-soluble, photosynthetic products were dihydroxyacetone phosphate (DHAP), glycerate 3-phosphate (PGA), and a neutral saccharide fraction. Only a minor amount of glycolate was produced. A portion of neutral saccharide synthesis was shown to result from extrachloroplastic contamination, and the nature of this contamination was investigated with light and electron microscopy. The amount of photoassimilated carbon partitioned into starch increased at both very low and high concentrations of orthophosphate. High concentrations of exogenous PGA also stimulated starch synthesis.

DHAP and PGA were the preferred forms of carbon exported to the medium, although indirect evidence suported hexose monophosphate export. The export of PGA and DHAP to the medium was stimulated by high exogenous orthophosphate, but depletion of chloroplastic reductive pentose phosphate intermediates did not occur. As a result only a relatively small inhibition in the rate of CO₂ assimilation occurred.

The rate of photoassimilation was stimulated by exogenous PGA, ribose 5-phosphate, fructose 1,6-bisphosphate, fructose 6-phosphate, and glucose 6-phosphate. Inhibition occurred with phosphoenolpyruvate and high concentrations of PGA and ribose 5-phosphate. PGA inhibition did not result from depletion of chloroplastic orthophosphate or from inhibition of ribulose 1,5-bisphosphate carboxylase. Exogenous PGA and phosphoenolpyruvate were shown to interact with the orthophosphate translocator.

     

Reduced osmotic potential effects on photosynthesis : identification of stromal acidification as a mediating factor

Addition of sorbitol, which facilitated reductions in reaction medium osmotic potential from standard (0.33 molar sorbitol, −10 bars) isotonic conditions to a stress level of 0.67 molar sorbitol (−20 bars), inhibited the photosynthetic capacity of isolated spinach (Spinacia oleracea) chloroplasts. This inhibition, which ranged from 64 to 74% under otherwise standard reaction conditions, was dependent on reaction medium inorganic phosphate concentration, with the phosphate optimum for photosynthesis reduced to 0.05 millimolar at the low osmotic potential stress treatment from a value of 0.25 millimolar under control conditions.

Stromal alkalating agents such as NH₄Cl (0.75 millimolar) and KCl (35 millimolar) were also found to affect the degree of low osmotic potential inhibition of photosynthesis. Both agents doubled the rate of NaHCO₃-supported O₂ evolution under the stress treatment, while hardly affecting the control rate at optimal concentrations. These agents also reduced the length of the lag phase of photosynthetic O₂ evolution under the stress treatment to a much greater degree. The rate-enhancement effect of these agents under the stress treatment was reversed by sodium acetate, which is known to facilitate stromal acidification.

The reaction medium pH optimum for photosynthesis under the stress treatment was higher than under control conditions. In the presence of optimal NH₄Cl, this shift was no longer evident.

Internal pH measurements indicated that the stress treatment caused a 0.43 and 0.24 unit reduction in the stromal and intrathylakoid pH, respectively, under illumination. This osmotically induced acidification was not evident in the dark. The presence of 0.75 millimolar NH₄Cl partially reversed the osmotically induced reduction in the illuminated stromal pH. It was concluded that stromal acidification is a mediating mechanism of the most severe site of low osmotic potential inhibition of the photosynthetic process.

     

Toxic effects of fatty acids on yeast cells: possible mechanisms of action.

As shown in a previous paper, threshold concentrations of lower and intermediate fatty acids inhibit the uptake of inorganic phosphate, growth, and cell division in yeast cells. This demonstrates that, apart from these effects, the acids cause an increase in the respiration quotient (RQ), inhibition of CO2 fixation, production of ethanol at the expense of anabolic processes, and inhibition of active amino acid transport in the yeast Candida utilis. On the other hand, the threshold concentrations have no effect on intracellular pH. The inhibition of the inorganic phosphate uptake cannot be the sole primary mode of action of fatty acids since the omission of inorganic phosphate in the incubation medium brings about an inhibition of anabolic processes that is lower than that brought about by fatty acids since the omission of inorganic phosphate in the incubation medium brings about an inhibition of anabolic processes that is lower than that brought by fatty acids at concentrations still premitting some phosphate uptake. Although 2,4-dinitrophenol and caproic acid at low concentrations cause an analogous decrease in biomass yield, their combination does not bring about any marked increase in the effect. Considering the physicochemical properties of fatty acids and their preferential action on energy-requiring processes, one of the key sites of action can be assumed to be the mitochondrial membrane. Fatty acids might inhibit the transport of anions, especially phosphate, across the membrane, and disturb the membrane potential by affecting the transport protons. The physiocochemical properties of fatty acids may also give rise to their binding to other intracellular membranes and to a subsequent interference with the function of the corresponding organelles.     

Effect of potassium chloride on the uptake and storage of phosphate by Saccharomyces mellis

The inorganic and polyphosphate pools of Saccharomyces mellis, grown in a medium containing excess phosphate, remain associated with the cells when the cells are suspended in a saline medium. If the cells are incubated in a medium containing 2 m KCl, the cells are altered in some manner which permits most of the orthophosphate and approximately one-third of the polyphosphate to be subsequently eluted by osmotic shock. At lower salt concentrations, beta-mercaptoethanol enhances this salt effect but is inactive by itself in this respect. At equivalent ionic strengths, the sodium salt of ethylenediaminetetraacetic acid behaves exactly like KCl or any other monovalent ionic compound in altering the cell to susceptibility to osmotic shock. No special effect of this anion at either high or low concentration could be detected. Resting cells are refractory to being altered in this manner by salts if an energy source, such as glucose, is included in the reaction mixture. Cells which are depleted of phosphate reserves will immediately incorporate phosphate when suspended in a medium containing inorganic phosphate and an energy source. These cells exhibit the phenomenon of "überkompensation." In resting cells, the inclusion of KCl in the reaction mixture prevents the conversion of orthophosphate into polyphosphate and, also, gradually decreases the ability of the organism even to assimilate orthophosphate. This effect is reversible, however, since the cells will incorporate phosphate in a normal manner if the cells are transferred to a non-salinized medium, or if a nitrogen source is included in the salinized reaction mixture so that the cells are now in a medium adequate for growth.     

Influence of Varying CO(2) and Orthophosphate Concentrations on Rates of Photosynthesis, and Synthesis of Glycolate and Dihydroxyacetone Phosphate by Wheat Chloroplasts

Intact chloroplasts of wheat (Triticum aestivum) were isolated from mesophyll protoplasts. With decreasing concentrations of bicarbonate from 10 to 0.3 millimolar (pH 8.0), the optimal concentration of orthophosphate (Pi) for photosynthetic O₂ evolution decreased from a value of 0.1 to 0.2 millimolar to 0 to 0.025 millimolar. The extremely low Pi optimum for photosynthesis at the low bicarbonate levels of 0.3 millimolar was increased by lowering the O₂ concentration from 253 (21% gas phase) to 72 micromolar (6% gas phase). The relative amount of glycolate and dihydroxyacetone phosphate (DHAP) synthesized under high and low levels of bicarbonate and varying levels of Pi was determined. At low levels of bicarbonate, glycolate was the main product, whereas at high bicarbonate levels, DHAP was the main product. Most of the DHAP and glycolate was found in the extrachloroplastic fraction.     

Concentration and Extinction Coefficient Determination for Oligonucleotides and Analogs Using a General Phosphate Analysis

A general phosphate analysis (GPA) is developed which assays the concentration of nucleic acid oligomers and their analogs based on stoichiometric phosphorus in the sequence. The method involves complete digestion of the oligomer sample to orthophosphate using acid at high temperature and subsequent colorimetric analysis by phosphomolybdate complex formation. GPA is applied to oligomers having phosphodiester, methylphosphonate, and phosphorothioate backbone linkages. Given the absorption spectra of oligomers having these backbones, extinction coefficients are obtained and compared to other quantitative and predictive methods. In addition to sequences having the usual nucleoside residues found in naturally occurring nucleic acids, oligomers having base analog residues can be readily quantified by GPA.     

Volutin Granules in Zoogloea ramigera

Zoogloea ramigera, a gram-negative bacterium found in activated sludge, formed volutin granules when excess orthophosphate was added to a phosphate-starved culture. These volutin granules were stainable by hydrogen sulfide after lead acetate treatment and extractable by N-perchloric acid but were not adsorbed by activated charcoal. They appeared to consist of inorganic polyphosphate. Optimum granule formation in the arginine broth required 10 g of glucose, 3 mg of phosphate, and 1 to 20 mg of magnesium per liter of medium. At an Mg(2+) concentration of 1 mg/liter, very large granules appeared which often appeared to fill the cell. An excess of glucose, orthophosphate, or magnesium reduced granule formation. In the absence of sulfate, moderate granulation occurred in arginine broth before the addition of excess orthophosphate; granulation did not increase after the addition of phosphate.     

Modification of Rhodospirillum rubrum ribulose bisphosphate carboxylase with pyridoxal phosphate. 1. Identification of a lysyl residue at the active site.

Ribulose 1,5-bisphosphate carboxylase isolated from Rhodospirillum rubrum was strongly inhibited by low concentrations of pyridoxal 5'-phosphate. Activity was protected by the substrate ribulose bisphosphate and to a lesser extent by other phosphorylated compounds. Pyridoxal phosphate inhibition was enhanced in the presence of magnesium and bicarbonate, but not in the presence of either compound alone. Concomitant with inhibition of enzyme activity, pyridoxal phosphate forms a Schiff base with the enzyme which is reversible upon dialysis and reducible with sodium borohydride. Subsequent to reduction of the Schiff base with tritiated sodium borohydride, tritiated N6-pyridoxyllysine could be identified in the acid hydrolysate of the enzyme. Only small amounts of this compound were present when the reduction was performed in the presence of carboxyribitol bisphosphate, an analogue of the intermediate formed during the carboxylation reaction. Therefore, it is concluded that pyridoxal phosphate modifies a lysyl residue close to or at the active site of ribulose bisphosphate carboxylase.     

Parachloromercuribenzenesulfonic Acid : a potential tool for differential labeling of the sucrose transporter

Vicia faba leaf discs without epidermis were pretreated with parachloromercuribenzenesulfonic acid (PCMBS), rinsed and incubated on [¹⁴C]sucrose (1 or 40 millimolar). Those sucrose concentrations were chosen as representative of the apparent uptake system 1 (1 millimolar) and system 2 (40 millimolar) previously characterized. Pretreatment with 0.5 millimolar PCMBS for 20 minutes inhibited system 1 and system 2 by about 70%.

Addition of unlabeled sucrose during PCMBS-pretreatment protected the carrier(s) from the inhibition, whereas glucose, fructose, and sucrose analogs were unable to afford protection. At 1 millimolar [¹⁴C]sucrose, the protection resulted in a small but consistent reduction of normal inhibition (from 63 to 45%) for sucrose concentrations of 50 millimolar and more during pretreatment. Contrarily, at 40 millimolar [¹⁴C]sucrose, the protection increased linearly with the sucrose concentration in the pretreatment medium, and complete prevention of inhibition was reached for 250 millimolar sucrose.

The protection was not due to exchange diffusion and was located in the veins. Michaelian kinetics indicated that PCMBS and sucrose compete with each other at the active site of the carrier.

Among 14 compounds tested (sugars, amino-acids, hormones, ³²P), sucrose uptake was by far the most sensitive to PCMBS. Sucrose preferentially protected its carrier(s) from inhibition. Treatment with 20 millimolar cysteine or 20 millimolar dithioerythreitol reversed inhibition by PCMBS pretreatment.

     

Effect of magnesium and ATP on ATPase of sugarcane vacuoles

Kinetic analysis of the Mg²⁺-dependence of tonoplast ATPase from suspension-cultured cells of sugarcane showed that the enzyme activity increased with increasing magnesium concentrations till 1–3 mM and then decreased consideably for higher concentrations. This kinetic could be explained by the assumption that MgATP^2- is the substrate of ATPase: MgATP^2- concentration increases with increasing concentration of magnesium till, at high concentrations of magnesium, Mg₂ATP is formed. No evidence for a direct role of Mg²⁺ as activator or inhibitor was found. These data corroborate previous findings that MgATP^2- is the sole substrate of the vacuolar ATPase of sugarcane (Thom and Komor 1984). High concentrations of ATP seemed to inhibit the ATPase. This result, however, could be traced back to interference of ATP with the Fiske-Subbarow method of phosphate determination. After adjustment of the test conditions, inhibition by ATP was no longer found. Reported data for ATPases of other plant materials, showing inhibition of enzyme activity with high magnesium or ATP concentrations, might be explicable in a similar way.Abbreviation Mes 2-(N-morpholino)ethane+Sulfonic acid     

The Regulatory Properties of Purified Phaseolus aureus Sucrose Synthetase

Phaseolus aureus sucrose synthetase, purified to homogeneity, was assayed in the presence of a variety of biological compounds to test for possible regulatory effectors. The oxidized form of nicotinamide adenine dinucleotide phosphate, as well as indoleacetic acid, gibberellic acid, and pyrophosphate were found to activate the forward reaction (sucrose degradation) and inhibit the reverse reaction (sucrose synthesis). The reduced form of nicotinamide adenine dinucleotide phosphate antagonizes the effect of the oxidized form. Fructose 1-phosphate and divalent cations inhibit the forward and activate the reverse reaction. Pyrophosphate and fructose 1-phosphate are effective only in the presence of magnesium chloride. Uridine triphosphate inhibits both the forward and reverse reactions. All effectors except gibberellic acid are active only in the millimolar range of concentrations; maximal stimulation for any effector is approximately 2-fold. The effects of combinations of effectors are roughly additive. Using pyrophosphate in the presence of magnesium chloride as an effector, results of kinetic studies offer a model by which an effector can activate an enzymatic reaction in one direction and inhibit in the reverse direction.     

Influence of end-products inhibition and nutrient limitations on the growth of Lactococcus lactis subsp. lactis

Lactococcus lactis was grown in a simple synthetic medium with glucose as substrate, enabling the precise quantification of each nutrient's contribution to growth. As expected, for the growth of lactic acid bacteria, the growth rate decreased progressively during the cultivation after a short period of exponential growth. End-products of fermentation, predominantly lactate and in minor amounts formate, acetate and ethanol, accumulated within the medium. Growth of the bacterium in fresh media supplemented with these end-products showed that the concentrations attained in the fermentor had no significant influence on the growth rate. As regards nutrients, vitamins and magnesium were never limiting during the culture. On the other hand, amino acid concentrations decreased, some of them being totally consumed and exhausted from the medium before growth ceased. However, growth in reconstituted media constructed with the amino acid concentrations remaining at different times of cultivation showed that amino acid depletion could not account for the observed growth decrease. Batch culture supernatant fluid was used as cultivation medium. Growth rates observed in supernatant cultures supplemented with various nutrients, compared to non-supplemented supernatant, showed that no addition improved growth. Finally, it was concluded that in the experimental conditions used in this study, growth inhibition was predominantly due to phenomena other than lactate inhibition and nutritional limitations, and hence associated with unidentified compounds produced in the fermentation.     

DEGRADATION OF PYRUVATE BY MICROCOCCUS LACTILYTICUS I. : General Properties of the Formate-Exchange Reaction.

McCormick, N. G. (University of Washington, Seattle), E. J. Ordal, and H. R. Whiteley. Degradation of pyruvate by Micrococcus lactilyticus. I. General properties of the formate-exchange reaction (J. Bacteriol. 83:887-898. 1962.-At an alkaline pH, extracts of Micrococcus lactilyticus(2) catalyze the phosphoroclastic degradation of pyruvate to formate and acetyl phosphate and the rapid exchange of formate into the carboxyl group of pyruvate. At an acid pH, hydrogen, carbon dioxide, and acetyl phosphate are produced, and carbon dioxide is exchanged into the carboxyl group of pyruvate. A concentration of approximately 1 m phosphate is required for the phosphoroclastic reaction and formate exchange; the production of carbon dioxide and hydrogen is greatly inhibited by high concentrations of phosphate. Formate exchange requires a divalent metal ion and is stimulated by reducing agents and an atmosphere of hydrogen. Inhibition by p-chloromercuribenzoate, Zn(++), Cd(++), and arsenite indicates that sulfhydryl groups on the enzyme are involved in the reaction; the inhibition by arsenite and Cd(++) may be relieved by 2,3-dimercaptopropanol, suggesting that vicinal dithiols may be required. Inhibition by hypophosphite may reflect a competition with formate for a site on the enzyme.At an alkaline pH, alpha-ketobutyrate is degraded to propionate and formate, whereas alpha-ketoglutarate is fermented to succinate, propionate, carbon dioxide, hydrogen, and formate. Formate is exchanged into the carboxyl groups of alpha-ketobutyrate and alpha-ketoglutarate under these conditions. Only traces of alpha-ketovalerate and alpha-ketoisovalerate are fermented at an alkaline pH and the exchange of formate into these compounds is very low.The addition of viologen dyes under the conditions used for formate exchange causes a reduction of pyruvate, alpha-ketobutyrate, alpha-ketovalerate, and alpha-ketoisovalerate to the corresponding alpha-hydroxy acids.     

Hydroponic growth and the nondestructive assay for dinitrogen fixation

Hydroponic growth medium must be well buffered if it is to support sustained plant growth. Although 1.0 millimolar phosphate is commonly used as a buffer for hydroponic growth media, at that concentration it is generally toxic to a soybean plant that derives its nitrogen solely from dinitrogen fixation. On the other hand, we show that 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid, pK_a 6.1, has excellent buffering capacity, and it neither interferes with nor contributes nutritionally to soybean plant growth. Furthermore, it neither impedes nodulation nor the assay of dinitrogen fixation. Hence, soybean plants grown hydroponically on a medium supplemented with 1.0 to 2.0 millimolar 2-(N-morpholino)ethanesulfonic acid and 0.1 millimolar phosphate achieve an excellent rate of growth and, in the absence of added fixed nitrogen, attain a very high rate of dinitrogen fixation. Combining the concept of hydroponic growth and the sensitive acetylene reduction technique, we have devised a simple, rapid, reproducible assay procedure whereby the rate of dinitrogen fixation by individual plants can be measured throughout the lifetime of those plants. The rate of dinitrogen fixation as measured by the nondestructive acetylene reduction procedure is shown to be approximately equal to the rate of total plant nitrogen accumulation as measured by Kjeldahl analysis. Because of the simplicity of the procedure, one investigator can readily assay 50 plants individually per day.     

Comparison of the Coomassie brilliant blue, bicinchoninic acid and Lowry quantitation assays, using non-glycosylated and glycosylated proteins.

The concentrations of several non-glycosylated and glycosylated recombinant and native proteins were determined by three widely used colorimetric methods: Coomassie brilliant blue, bicinchoninic acid and Lowry, and, for comparison, by amino acid composition analysis. The colorimetric methods gave results differing from the values derived from the amino acid analysis, in some cases by up to 60%. For the non-glycosylated recombinant proteins, the results were in relatively good agreement with each other and with the values determined on the basis of the amino acid analysis. The Coomassie blue method was strongly dependent on the hydrophobicity of the individual protein. The bicinchoninic acid method gave results closest to those of the amino acid analysis. For the glycosylated proteins, both recombinant and native, the Coomassie blue assay gave values lower, whereas the two other methods gave values higher than those determined on the basis of the amino acid analysis. The concentration of a recombinant interferon gamma receptor produced in two differently glycosylated forms was underestimated by the Coomassie blue assay and overestimated by the bicinchoninic acid and Lowry methods, while for the non-glycosylated form of the same protein, the three colorimetric methods delivered comparable values. The results suggest a potential interference of protein glycosylation with the colorimetric assays.     

Growth characteristics of a new methylomonad.

A methylomonad culture was isolated from pond water and examined as a potential source of single-cell protein. A medium containing magnesium sulfate, ammonium hydroxide, sodium phosphate, tap water, and methanol supported the growth of the isolate. Optimal growth conditions in batch cultures for the organism were: temperature, 30 to 33 degrees C; pH 7.1; and phosphate concentration, 0.015 M. The minimum doubling time obtained was 1.6 h. The specific growth rate in batch culture was dependent on the methanol concentration, reaching a maximum around 0.2% (wt/vol). Growth inhibition was apparent above 0.3% (wt/vol), and growth was completely inhibited above 4.6% (wt/vol) methanol. Although the inhibitory effect of formaldehyde on the specific growth rate was much greater than that of formate, the organism utilized formaldehyde, but not formate, as a sole carbon and energy source in batch cultures. The isolate was identified primarily by its inability to utilize any carbon source other than methanol and formaldehyde for growth. Although it is capable of rapid growth on methanol, the organism showed a very weak catalase activity. The amino acid content of the cells compared favorably with the reference levels for the essential amino acids specific by the Food and Agricultural Organization of the United Nations.     

Growth characteristics of a new methylomonad.

A methylomonad culture was isolated from pond water and examined as a potential source of single-cell protein. A medium containing magnesium sulfate, ammonium hydroxide, sodium phosphate, tap water, and methanol supported the growth of the isolate. Optimal growth conditions in batch cultures for the organism were: temperature, 30 to 33 degrees C; pH 7.1; and phosphate concentration, 0.015 M. The minimum doubling time obtained was 1.6 h. The specific growth rate in batch culture was dependent on the methanol concentration, reaching a maximum around 0.2% (wt/vol). Growth inhibition was apparent above 0.3% (wt/vol), and growth was completely inhibited above 4.6% (wt/vol) methanol. Although the inhibitory effect of formaldehyde on the specific growth rate was much greater than that of formate, the organism utilized formaldehyde, but not formate, as a sole carbon and energy source in batch cultures. The isolate was identified primarily by its inability to utilize any carbon source other than methanol and formaldehyde for growth. Although it is capable of rapid growth on methanol, the organism showed a very weak catalase activity. The amino acid content of the cells compared favorably with the reference levels for the essential amino acids specific by the Food and Agricultural Organization of the United Nations.