An enzymatic cycling method using pyruvate orthophosphate dikinase and firefly luciferase for the simultaneous determination of ATP and AMP (RNA)

A novel bioluminescent enzymatic cycling assay for ATP and AMP with concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK) was developed. In this system, AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. This resulted in constant luminescence once the stable phase had been reached. Background luminescence of the reagent was reduced with adenosine phosphate deaminase by degrading ATP and AMP in the reagent. The maximum recycling ratio calculated from the integrated luminescence value was 2.64 cycles/min. The measurable ranges for ATP and AMP were equal and were between 4 x 10(-13) and 4 x 10(-17) mol/assay. The amount of yeast RNA could be estimated in the range of 1 x 10(-8) to 1 x 10(-12) g/assay by estimating the amount of AMP resulting from the degradation of RNA with nuclease P1. Various food samples were subjected to measurement of the amount of ATP + AMP + RNA to provide an index for hygiene monitoring. For beef extract, sensitivity was improved by more than 20 million compared to the previous methods relying only on the amount of ATP as an index.     

Hydrolytic and alcoholytic dephosphorylation of nucleotides by acid phosphatase in the presence of ethanol

The effect of ethanol on the activity of acid phosphatase from wheat germ was studied, by using ribonucleoside monophosphates as the enzyme substrates. The nucleotides were effectively degraded to the corresponding nucleosides in the presence of ethanol at all concentrations tested, including a 96% (v/v) solution. However, the nucleotide dephosphorylation was accompanied by the liberation of orthophosphate only when the concentration of ethanol in the assay mixture did not exceed 15%. No inorganic phosphate was liberated when ethanol was present at higher concentrations. Instead, monoethyl phosphate was formed in quantities expected for orthophosphate. The results are explained in terms of phosphatase-catalysed alcoholysis.     

High-performance liquid chromatographic determination of pyrophosphate in the presence of a 20,000-fold excess of orthophosphate.

An HPLC method was based on anion-exchange separation of pyrophosphate (diphosphate) and orthophosphate and postcolumn spectrophotometric detection at 140 degrees C with a molybdenum(V)-molybdenum(VI) reagent. The reagent was easy to prepare, stable for at least 6 months at room temperature, and ready for the determination of pyrophosphate and orthophosphate by the so-called heteropoly blue method without use of any reducing agent. A photodiode-array detector for HPLC indicated the spectral characteristics of the heteropoply blue complex that was detectable at 330-800 nm. The HPLC method had a wide dynamic range from 3 x 10(-7) to 5 x 10(-4) M for both pyrophosphate and orthophosphate with a relative standard deviation of measurement of 10 approximately 2%. Pyrophosphate of 5 x 10(-7) and 5 x 10(-6) M, respectively, could be determined in the presence of a 20,000-fold excess of orthophosphate; 0.01 and 0.1 M.     

The use of a layering technique for enhancing stability of lyophilized reagents.

An enzyme-mediated assay has been developed for the measurement of salicylate using salicylate monooxygenase purified from Pseudomonas cepacia ATCC 29351. Two assay formulations were produced, based on either a multiple-reagent or a single-reagent formulation, to allow sufficient flexibility for automated use. The multiple-reagent formulation was especially suited to diagnostic laboratories performing infrequent manual salicylate estimation where stability of the reconstituted reagent is of paramount importance. This was achieved by preparing the enzyme and color reagents in separate vials, so keeping the enzyme at a stable pH. For more frequent assay use where a reconstituted reagent shelf life was less important, the single-reagent system offers advantages of convenience. However, the working reagent required a pH of 10.0 upon reconstitution. Although the enzyme was sufficiently active at this pH to give a reliable assay, its storage stability was poor at pH 10.0, preventing lyophilization of the reagent at a pH suitable for immediate use on reconstitution. This incompatibility was overcome by use of a layering technique. The enzyme was separated from the buffering solution in the same vial by freezing the buffering solution and then overlayering with the enzyme reagent prior to a second freezing cycle and subsequent freeze drying.     

Lysosomal enzymes in extracellular digestion in the unicellular eukaryote Tetrahymena

The ciliate Tetrahymena thermophila was starved for orthophosphate in a synthetic medium at pH 7.5. These cells did not utilize phosphorylcholine, final concentration 1 mM, as a phosphate source for cell growth and multiplication. If the phosphorylcholine solution, however, was incubated for 24 h at pH 5.5 with extracellular, "spent" medium from a culture in early stationary phase of growth, then it promoted culture growth readily at pH 7.5. It was shown that the spent medium in the same concentration did not stimulate growth in itself. It is concluded that extracellular digestion of phosphorylcholine enabled the cells to grow and multiply in a nutrient medium having organic phosphate compounds as the only phosphate source. It is argued that the phosphatases in the spent medium are of lysosomal origin.     

Enumeration of bacterial cell numbers by amplified firefly bioluminescence without cultivation

We recently developed a novel bioluminescent enzymatic cycling assay for ATP and AMP with the concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK), where AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. Background luminescence derived from contaminating ATP and AMP in the reagent was reduced using adenosine phosphate deaminase which degrades ATP, ADP, and AMP, resulting in constant and highly amplified bioluminescence with low background luminescence. To detect bacterial cells without cultivation, we applied the above bioluminescent enzymatic cycling reagent to rapid microbe detection system. ATP spots (0.31-5.0 amol/spot) at the level of a single bacterial cell were detected with 5 min signal integration, signifying that integrated luminescence was amplified 43 times in comparison to traditional ATP bioluminescence. Consequently, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Lactobacillus brevis in beer were detected without cultivation. Significant correlation was observed between the number of signal spots obtained using this novel system and the colony-forming units observed with the conventional colony-counting method (R(2)=0.973).     

A mathematical model of the Calvin photosynthesis cycle

1. A mathematical model is presented for photosynthetic carbohydrate formation in C3 plants under conditions of light and carbon dioxide saturation. The model considers reactions of the Calvin cycle with triose phosphate export and starch production as main output processes, and treats concentrations of NADPH, NAD+, CO2, and H+ as fixed parameters of the system. Using equilibrium approximations for all reaction steps close to equilibrium steady-state and transient-state relationships are derived which may be used for calculation of reaction fluxes and concentrations of the 13 carbohydrate cycle intermediates, glucose 6-phosphate, glucose 1-phosphate, ATP, ADP, and inorganic (ortho)phosphate. 2. Predictions of the model were examined with the assumption that photosynthate export from the chloroplast occurs to a medium containing orthophosphate as the only exchangeable metabolite. The results indicate that the Calvin cycle may operate in a single dynamically stable steady state when the external concentration of orthophosphate does not exceed 1.9 mM. At higher concentrations of the external metabolite, the reaction system exhibits overload breakdown; the excessive rate of photosynthate export deprives the system of cycle intermediates such that the cycle activity progressively approaches zero. 3. Reactant concentrations calculated for the stable steady state that may obtain are in satisfactory agreement with those observed experimentally, and the model accounts with surprising accuracy for experimentally observed effects of external orthophosphate on the steady-state cycle activity and rate of starch production. 4. Control analyses are reported which show that most of the non-equilibrium enzymes in the system have a strong regulatory influence on the steady-state level of all of the cycle intermediates. Substrate concentration control coefficients for cycle enzymes may be positive, such that an increase in activity of an enzyme may raise the steady-state concentration of the substrate is consumes. 5. Under optimal external conditions (0.15-0.5 mM orthophosphate), reaction flux in the Calvin cycle is controlled mainly by ATP synthetase and sedoheptulose bisphosphatase; the cycle activity approaches the maximum velocity that can be supported by the latter enzyme. At lower concentrations of external orthophosphate the cycle activity is controlled almost exclusively by the phosphate translocator.(ABSTRACT TRUNCATED AT 400 WORDS)     

New assay for enzymatic phosphate release: application to aspartate transcarbamylase and other enzymes

The colorimetric method for phosphate determination described in the preceding paper is adapted for the assay of orthophosphate liberated in the aspartate transcarbamylase reaction. The method provides for simple, accurate, and sensitive measurement of enzyme activity. The assay uses ammonium molybdate and zinc acetate to form a colored complex with the enzymatically released phosphate; mild conditions which minimize the nonenzymatic background degradation of the substrate, carbamoyl phosphate, are used. Since the assay procedure is relatively rapid, it is especially attractive in situations where results are desired immediately. The method can be used for the assay of any enzyme which releases inorganic phosphate, even in the presence of labile organophosphate compounds.     

A model for formulation of protein assay.

The principle of the protein assay using the reaction of an alkaline copper-protein complex with the Folin-Ciocalteu phenol reagent has been investigated. In contrast to the long-established Lowry method, a stable and rapid protein assay is developed without a buffering agent in alkaline copper solution. In the absence of a buffering agent, the reaction pH drops relatively rapidly and moves the reaction toward a more stable pH. When the reaction of alkaline copper-protein complex with Folin-Ciocalteu reagent is started at around pH 11.7, the reaction color absorbance reaches a plateau in approximately 10 min and remains stable to allow a reliable measurement of the absorbance. In the absence of the buffering agent sodium carbonate, the alkaline copper solution is also stable for months. The principle of the protein assay is presented as a model that can be used to formulate protein assays of desired specification.     

THE IDENTIFICATION AND PURIFICATION OF A CELL-SURFACE ALKALINE PHOSPHATASE FROM THE DINOFLAGELLATE PROROCENTRUM MINIMUM (DINOPHYCEAE)1

Two cell-surface proteins were identtjied in the dinoflagellate Prorocentrum minimum (Pavillard) Schilkr strain CCMP 1329 that are evident in phosphate-limited cultures, but not in nitrate-limited cultures or cultures growing exponentially in complete media. These proteins were detected by labeling cell-surface proteins with the biotinylating reagent succinimidyl 6-(biotinamido) hexanoate. One protein, of appoximately 200,000 daltons was purified using differential centrifugation, detergent extraction, and gel filtration chromatography. This purified protein was able to hydrolyze orthophosphate groups from p-nitrophenylphosphate at pH 8, indicating it is an alkaline phosphatase, although it is larger than other alkaline phosphatases isolated to date tom most microorganisms. This protein may be induced to help P. minimum cleave orthophosphate groups from organic forms of phosphate in marine environments. Ultimately, this protein could represent a unique antigen for developing an antibody probe for examining the relationships between phosphate stress and bloom formation in P. minimum, and perhaps other dinoflagellates, in the field.     

PHOSPHATE UPTAKE BY SYNECHOCOCCUS LEOPOLIENSIS (CYANOPHYCEAE): ENHANCEMENT BY CALCIUM ION1

The influence of metallic, cations (added at 10 μM-1 mM) on the uptake of orthophosphate from 0.2–10 μM solution by Synechococcus leopoliensis (Racib.) Komarek was investigated. All cations tested except Mg²⁺ and Zn²⁺ stimulated phosphate uptake. The most pronounced stimulation of phosphate uptake was caused by Ca²⁺·Ca²⁺ markedly decreased the half-saturation concentration for orthophosphate uptake, apparently by acting upon the metabolic processes of phosphate transport into the cell. Phosphate did not influence Ca²⁺ fluxes across the cell-surface.     

Determination of inorganic phosphate in the presence of Triton X-100

Two methods have been developed for the rapid and accurate estimation of orthophosphate in the presence of Triton X-100. The first is unaffected by up to 2.0–2.5% (v/v) of the detergent in the assay samples, while the second method is essentially unaffected by Triton X-100 and is also suitable for use in the presence of acid labile organic phosphate. Both are proportional in the range 0.05–1.0 μmole of orthophosphate in the assay.     

Some effects of inorganic phosphate on O2 evolution by isolated chloroplasts

1. After an initial lag, isolated spinach chloroplasts evolved O₂ in illuminated reaction mixtures containing bicarbonate but no added phosphate. This evolution soon ceased but could be restarted by the addition of phosphate.

2. The phosphate requirement could be met by orthophosphate, inorganic pyrophosphate, ATP or ADP but not by AMP. Approx. 3 molecules of O₂ were evolved for each molecule of orthophosphate added and approx. 6 for each molecule of pyrophosphate.

3. With CO₂ as the sole added substrate the extent of the initial lag in O₂ evolution was not greatly affected by small quantities of added orthophosphate but as the concentration of orthophosphate was increased there was a progressive increase in the lag and a progressive decrease in the maximum rate. Pyrophosphate failed to produce these effects at a 100 times the concentration and in the presence of pyrophosphate the orthophosphate inhibition was less severe. There was little or no orthophosphate inhibition in the presence of substrate quantities of 3-phosphoglycerate or ribose 5-phosphate and CO₂.

4. There was also a requirement for phosphate by chloroplasts evolving O₂ in the presence of 3-phosphoglycerate or ribose 5-phosphate plus CO₂. In the presence of endogenous phosphate only, added ribose 5-phosphate suppressed the O₂ evolution which normally followed the addition of 3-phosphoglycerate.

5. The results provide direct support for the proposed phosphate requirement of the photosynthetic carbon cycle and are discussed in this context. They also imply that orthophosphate, ribose 5-phosphate and 3-phosphoglycerate can penetrate the intact chloroplast envelope with considerable rapidity.     

Removal capacity of water plant alum sludge for phosphorus in aqueous solutions

Abstract

Alum sludge, which is a waste product from a potable water treatment process, was tested as an inexpensive alternate adsorbent for phosphorus in wastewater. The sludge was composed dominantly of sand size aggregates, and could remain stable in aqueous media. The majority of reactive Al in alum sludge was present as an amorphous phase, and seemed to be the major absorbent for P. The batch sorption test showed that the removal of P was influenced by the solubility of Al, Fe and organic carbon depending on pH condition. The acidic condition favored the removal of P, and there was a side effect in the P removal process such as dissolution of Al and organic C at acidic (pH < 4) and alkaline (pH < 8) conditions. The pH range from 4 to 6 was effective for all inorganic and organic phosphates with a low solubility of Al and organic C. The maximum adsorption capacity of alum sludge was calculated as 25,000 mg/kg for orthophosphate, and followed the order: orthophosphate > pyrophosphate > triphosphate > organic phosphate (adenosin). From the column test with a 30 mg/L orthophosphate solution at a flow rate of 3.0 ml/min, the alum sludge removed P to less than 1.0 mg/L over 250 pore volumes at initial pH 4, and 200 pore volumes at initial pH 5, respectively.     

A new method of separating inorganic orthophosphate from phosphoric esters and anhydrides by an immobilized catalyst column.

A column of polyvinylpolypyrrolidone packed in a 1-ml Tuberculin syringe is used as a stationary phase for affinity chromatography of phosphomolybdate. When a mixture of inorganic orthophosphate, phosphoric esters, and phosphoric anhydrides is introduced into such a column in the presence of molybdate (2–3%, pH 3–5), inorganic orthophosphate adsorbs specifically to the column material as phosphomolybdate, while other phosphate compounds, which do not react with molybdate, drain through. Mild centrifugation (8–50g) is used to hasten elution to minimize the hydrolysis of acid-labile phosphates. The method described allows separation of radioactive phosphate compounds from a small amount of solution (0.2–1.0 ml) without either organic solvent extraction or transfer of sample, which may cause error and/or contamination. With 3% molybdate, pH 3.0, 98.5 ± 0.6% of ATP is recovered, while 0.05 ± 0.01% of inorganic orthophosphate is eluted in the effluent. Retained inorganic orthophosphate can be eluted later by 0.5 m ammonium hydroxide with a recovery of 98.2 ± 0.9%. Unlike other methods of separating phosphomolybdate, this one is virtually insensitive to the presence of reducing reagents.     

Orthophosphate determination in the presence of high concentrations of ATP

A method to measure orthophosphate which contaminates samples of ATP was developed. Concentrations of orthophosphate as low as 0.4% of the ATP concentration were determined using a zinc-molybdate reagent [D. A. Bencini, J. R. Wild, and G. A. O'Donovan, Anal. Biochem. 132, 254-258 (1983)] and continuous spectrophotometric monitoring of chromophore formation. Since the rate of ATP hydrolysis was pseudo-first order and was slow compared to the rate of chromophore formation, the initial concentration of phosphate could be readily determined by extrapolation to zero time. The method is rapid and reproducible, and requires a single, stable reagent.     

A simple assay for 3-deoxy-d-manno-octulosonate cytidylyltransferase and its use as a pathway screen

This article describes the adaptation of a simple colorimetric assay for inorganic pyrophosphate to the enzyme 3-deoxy-d-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase, KdsB, EC 2.7.7.38), a key enzyme in the biosynthesis of lipopolysaccharide (LPS) in Gram-negative organisms. This assay is particularly useful because it can be combined with the malachite green (MG) assay for inorganic phosphate to form an assay system capable of determining inorganic phosphate and inorganic pyrophosphate in the same solution (the MG/EK (eikonogen reagent) assay). This assay system has the potential for simultaneous screening of the 3-deoxy-d-manno-octulosonate (KDO) biosynthesis pathway. We tested this potential using two enzymes, KdsB and KdsC, involved in the biosynthesis and use of the key bacterial 8-carbon sugar, KDO.     

Identification of histidine-119 as the target in the site-specific inactivation of ribonuclease A by ferrate ion.

Ferrate ion, a powerful oxidant which is an analog of orthophosphate ion, has previously shown some promise as a site-specific probe of enzymes which interact with phosphate compounds. In order to explore the general applicability of this reagent, it has been tested against ribonuclease A, an enzyme whose structure and active center have been well described. Treatment with a molar ratio of ferrate to enzyme of less than 20 leads to a loss of 87% of the activity. The known competitive inhibitors, 2'-cytidylic acid, inorganic pyrophosphate, and orthophosphate all protect the enzyme from inactivation. Inactivation is accompanied by a loss of the capacity to bind 2'-cytidylic acid. Ferrate inactivation at pH 5.0 is accompanied by the modification of only one amino acid. The amino acid which was identified by amino acid and sequence analyses of peptide fragments obtained by cyanogen bromide treatment and selective proteolysis proved to be histidine-119, whose essential role at the active center has long been established.     

Seasonal variation in the phosphatase activity of waters and sewage sludges

Summary The alkaline phosphatase activity of water from eight locations differing in orthophosphate concentration has been determined over a period from late Autumn to late Summer. Evidence for induction/repression effects was conjectural, but cellular activity was highest in the environment of lowest orthophosphate concentration. Environments were sampled on a number of occasions and pH/phosphatase activity profiles constructed. The pH of maximum activity of low phosphate environments was in the acid region, that of high phosphate environments was in the alkaline region. There was little difference in the character and distribution of constitutive phosphatases in representative bacterial cultures from a high phosphate and a low phosphate environment. It seems likely that the phosphatase activity of a water at a particular time will be influenced by its nutrient and physico-chemical status as well as its ambient orthophosphate concentration.     

A simple assay for 3-deoxy-d-manno-octulosonate cytidylyltransferase and its use as a pathway screen

This article describes the adaptation of a simple colorimetric assay for inorganic pyrophosphate to the enzyme 3-deoxy-d-manno-octulosonate cytidylyltransferase (CMP–KDO synthetase, KdsB, EC 2.7.7.38), a key enzyme in the biosynthesis of lipopolysaccharide (LPS) in Gram-negative organisms. This assay is particularly useful because it can be combined with the malachite green (MG) assay for inorganic phosphate to form an assay system capable of determining inorganic phosphate and inorganic pyrophosphate in the same solution (the MG/EK (eikonogen reagent) assay). This assay system has the potential for simultaneous screening of the 3-deoxy-d-manno-octulosonate (KDO) biosynthesis pathway. We tested this potential using two enzymes, KdsB and KdsC, involved in the biosynthesis and use of the key bacterial 8-carbon sugar, KDO.