A sensitive method for measuring pyrophosphate in the presence of a 10,000-fold excess of orthophosphate using inorganic pyrophosphatase

A simple method for measuring PP_i at concentrations down to 2 μm has been devised using the ability of inorganic pyrophosphatase to be inactivated by fluoride in the presence of PP_i. Orthophosphate (20 mm) and a number of other compounds did not interfere with the assay. The applicability of the method for direct measurement of PP_i in urine is demonstrated.     

The determination of inorganic orthophosphate in biological systems

A simple and rapid method is described for determining P_i by spectrophotometric measurement of a soluble complex of phosphomolybdic acid and Cirrasol ALN-WF, a non-ionic detergent formerly known as Lubrol W. The measured complex has a molar extinction coefficient of 4.59 · 10³ at 390 nm and little interference is found with relatively high concentrations of chelating agents, salts, and other compounds which interfere with most other P_i assays. Linearity is observed in the range 0–1.2 μmoles P_i and developed assay samples are stable for 8 h at 20 °C or 24 h at 4 °C. The method is suitable for use in the presence of moderate concentrations of protein or ATP.After suitable modification the assay can be used at pH 4.0. Sensitivity is reduced at this pH (ε_{M, 390nm} = 2.79 · 10³) but linearity is maintained up to 1 μmole P_i and the coloured complex is stable for 4 h at 20 °C. The pH-4 procedure is suitable for measurement of P_i in the presence of very labile phosphate esters such as creatine phosphate.The phosphomolybdic acid-Cirrasol complex can be reduced at ambient temperature in both the above systems. A blue complex results with ε_{M, 820nm} of 9.9 · 10³ at pH 4.0, and 1.8 · 10⁴ under more acidic conditions.     

A method for the concentration and for the colorimetric determination of nanomoles of inorganic pyrophosphate

A generally applicable, inexpensive, and sensitive method for the determination of inorganic pyrophosphate (PP_i) was developed. PP_i was quantitatively separable from solution even in nanomolar concentrations by filtration through a membrane filter in the presence of CaCl₂ and KF. The separated PP_i was dissolved by immersing the filter in 0.5 n H₂SO₄. Inorganic phosphate (P_i) was removed by precipitating it as a phosphomolybdate-triethylamine complex and the PP_i was measured as a green pyrophosphomolybdate in the presence of 2-mercaptoethanol. Nucleotides and phosphate esters do not react. PP_i can be accurately assayed even when there is a 10⁴-fold excess of P_i. Trimetaphosphate, tripolyphosphate, and tetrapolyphosphate also give this green color, but the rate of the color formation is 50 times slower than that with PP_i. Thus this interference of the polyphosphates can be eliminated or the polyphosphates can be assayed simultaneously with the PP_i in the same sample.     

Modifications in the enzymatic assay for inorganic phosphate.

A recent publication from this laboratory (1) described an enzymatic assay for inorganic phosphate (P_i) which eliminates the need for standards and, through mild reaction conditions, avoids the hydrolysis of labile organic phosphates. Those features are advantageous, particularly for P_i measurements in biological samples. However, subsequent inquiries from other laboratories and our own experience indicated that the assay, as described (1), does not perform well. Specifically, it was found that the assay range was 10-fold narrower than that reported, completion times were 3- to 5-fold longer, and the reaction with P_i standards was only 90–95% complete.Because of these deficiencies we have systematically evaluated every aspect of the assay and have found that the difficulties are eliminated and the assay is improved and simplified by the following changes; (i) Triethanolamine is used in place of Tris as the assay buffer; (ii) triose phosphate isomerase is eliminated and the levels of other enzymes are adjusted to obtain optimum reaction conditions; (iii) ammonium sulfate is removed from the analytical enzymes. The modified procedure described below is more convenient, is linear up to P_i concentrations of 0.1 μmol/ml in the assay, gives complete reaction of P_i standards and quantitative recovery of P_i added to biological extracts, and comes to stable endpoints in 30 min or less (depending on the amount of P_i in the assay).     

Colorimetric determination of inorganic pyrophosphate by a manual or automated method.

A microprocedure for the colorimetric determination of inorganic pyrophosphate (PP_i) in the presence or absence of orthophosphate (P_i) has been developed. PP_i is estimated quantitatively as the amount of chromophore formed with molybdate reagent, 1-amino-2-naphthol-4-sulfonic acid in bisulfite and thiol reagent (monothioglycerol or 2-mercaptoethanol). The latter is obligatory for color formation. P_i is estimated without thiol reagent. The two chromophores differ in absorption spectra, the greatest difference being at 580 nm. For both, color develops fully by 10 min and is stable up to 1 hr. Just less than 0.4 μm PP_i can be detemined. The extinction coefficients are 2.70 × 10⁴ and 8.76 × 10³ for PP_i and P_i, respectively, both with thiol reagent present, and 2.77 × 10³ for P_i with no thiol reagent.A ten-fold excess of P_i does not interfere with the determination of PP_i and in fact can be estimated in the same mixture. A 15-fold excess, however, diminishes the accuracy of PP_i estimations. Trichloroacetic acid and sodium fluoride inhibi color formation, but this inhibition is overcome by the addition of sodium acetate buffer, pH 4.0. Nucleoside triphosphates and adenosine 3′:5′-cyclic monophosphate are stable in the reaction mixture.The method was tested in assays of Escherichia coli DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6). Progress curves measured by either the rate of PP_i formation or the rate of synthesis of labeled RNA were very similar. Product PP_i formed by as little as 0.6 unit of RNA polymerase in a 225-μl incubation medium could be measured.An automated version of the method was devised which allows accurate determination of PP_i down to 1 μm (without range expander attachment) at a sampling rate of 20–40 tubes/hr.     

High-resolution preparative gel electrophoresis: separation and recovery of functional messenger RNA species

Certain open-chain polyols were shown to interfere with the determination of phosphorus of the Lowry-Lopez method by forming a complex with Mo₇O₂₄^6?. The ability to interfere with the assay increased with increasing chain length of the polyols: Ethylene glycol and glycerol did not react at all; i-erythritol reacted to a small extent, but hexitols and perseitol formed stronger complexes. Depending on the polyol, interference occurred even at 0.2 mm (hexitols) or 2 mm (xylitol) concentrations. At these concentrations the polyols interfered only to a small extent with the phosphorus assays based on the use of Triton X-100 and molybdate. The complex formation was exploited in the development of a colorimetric polyol assay.     

A new, convenient method for the rapid analysis of inorganic pyrophosphate.

A new method for the rapid analysis of inorganic pyrophosphate (PP_i) which utilizes the enzyme ATP sulfurylase is described. All components of the assay system are commercially available and inexpensive. The assay is linear over the range of 0.5–50.0 nmol of PP_i and is not affected by inorganic phosphate. ATP and PP_i can both be analyzed using this method.     

Yeast inorganic pyrophosphatase substrate recognition

Monodentate Co(NH₃)₅PP_i was determined not to be a substrate for yeast inorganic pyrophosphatase while P¹,P²-bidentate Co(NH₃)₄PP_i was turned over by the enzyme at a rate of 7.5 min^?1. A kinetic analysis of the substrate activities of the P¹,P²-bidentate complexes, Co(en)₂PP_i, Cr(NH₃)₄PP_i, Cr(H₂O)(NH₃)₃PP_i, Cr(H₂O)₂(NH₃)₂PP_i, and Cr(H₂O)₄PP_i was carried out in order to access the potential role of the metal-water ligands in productive binding. While substitution of the H₂O ligands with NH₃ ligands had a minimal affect on the K_m for Mg²⁺, the binding affinity of the complexes decreased with an increasing $\text{NH}{}_3\text{H}{}_2\text{O}$ ligand ratio as did the turnover number of the corresponding central complexes. The Co(en)₂PP_i complex was hydrolyzed at a rate approximately 0.6% of that for the Co(NH₃)₄PP_i complex. The substrate activities of β,γ-bidentate Co(NH₃)₄PPP_i and α,β,γ-tridentate Co(NH₃)₃PPP with pyrophosphatase were also tested. While both complexes were shown to bind tightly to the Mg²⁺-activated enzyme neither was hydrolyzed. On the other hand, in the presence of the Zn²⁺-activated enzyme the tridentate complex was turned over at a rate of 0.17 min^?1 while the bidentate complex remained inert to hydrolysis.     

Effects of inorganic phosphate on the photosynthetic carbon reduction cycle in extracts from the stroma of pea chloroplasts

1. Turnover of the photosynthetic carbon reduction cycle has been demon-strated in chlorophyll-free reaction mixtures containing chloroplast stromal extract, as evidenced by the fixation of CO₂ following addition of small amounts of 3-phosphoglycerate.2. The activity of the photosynthetic carbon reduction cycle in this system is inhibited by inorganic phosphate (P_i), with activity reduced to 50% by about 6.5 mM P_i. P_i also increased the lag period which elapsed before a steady rate of CO₂ fixation was obtained.3. The effect of P_i on the rate of 3-phosphoglycerate reduction following the addition of substrate amounts of some cycle intermediates was investigated. Substantial inhibition was observed with fructose 1,6-bisphosphate, sedoheptulose 1,7-bisphosphate and erythrose 4-phosphate as substrates. P_i also affected the activity of ribulose-bisphosphate carboxylase, with stimulation at P_i concentrations below 2.5 mM and inhibition at higher concentrations.4. The results showed that the sedoheptulose bisphosphatase reaction is inhibited more strongly by P_i than the fructose bisphosphatase reaction.5. It is concluded that the previously established inhibitory effects of P_i on photosynthesis by intact isolated chloroplasts may be partly due to these inhibitory effects of P_i on the reactions of the photosynthetic carbon reduction cycle.     

A requirement for chelation in obtaining functional chloroplasts of sunflower and wheat.

In studying conditions for obtaining photosynthetically functional chloroplasts from mesophyll protoplasts of sunflower and wheat, a strong requirement for chelation was found. The concentration of chelator, either EDTA or pyrophosphate (PP_i), required for maximum activation depended on the pH, the concentration of orthophosphate (P_i) in the assay, and the chelator used. Studies with EDTA indicate that including the chelator in the isolation, resuspension, and assay media, in the absence of divalent cations, was most effective. Increased concentration of EDTA from 1 to 10 mm broadened the pH response curve for photosynthesis, inasmuch as a higher concentration of chelator was required for activation of photosynthesis at lower pH.Either EDTA, PP_i, or citrate could activate photosynthesis of sunflower chloroplasts isolated and assayed at pH 8.4. At pH 7.6, PP_i and EDTA were equally effective at low P_i concentrations but PP_i was particularly effective in shortening the induction period at high concentrations of P_i (2.5 mm) in the assay medium. Including 1 mm 3-phosphoglycerate in the assay medium with or without P_i could not replace the need for chelation. However, 3-phosphoglycerate + EDTA in the assay medium with 0.5 mm P_i, pH 7.6, gave a short induction period and rates of photosynthesis similar to those with 10 mm PP_i. The results suggest that PP_i can have a dual effect at the lower pH through chelation and inhibition of the phosphate transporter.Photosynthesis by sunflower chloroplasts isolated and assayed at pH 8.4 with 0.2 mm EDTA (+ 0.5 mm P_i in the assays) was severely inhibited by 2 mM CaCl₂, MgCl₂, or MnCl₂. Wheat chloroplasts isolated and assayed at pH 8.4 without chelation, and assayed with 0.2 mm P_i, had low rates of photosynthesis (25 μmol O₂ evolved mg^?1 chlorophyll h^?1) which were strongly inhibited by 2 to 4 mm MgCl₂, MnCl₂, or CaCl₂. With inclusion of EDTA and P_i at optimum levels, isolated chloroplasts of sunflower and wheat have high rates of photosynthesis and PP_i or divalent cations are not of benefit.     

The effect of sodium on inorganic phosphate- and p-nitrophenyl phosphate-facilitated ouabain binding to (Na+ + K+)-activated ATPase

The effect of the hydrolysis product P_i and the artificial substrate p-nitrophenyl phosphate (p-nitrophenyl-P) on ouabain binding to (Na⁺ + K⁺)-activated ATPase was investigated.The hypothesis that (Mg²⁺ + p-nitrophenyl-P)-supported ouabain binding might be due to P_i release and thus (Mg²⁺ + P_i)-supported could not be confirmed.The enzyme · ouabain complexes obtained with different substrates were characterized according to their dissociation rates after removal of the ligands facilitating binding. The character of the enzyme · ouabain complex is determined primarily by the monovalent ion present during ouabain binding, but, qualitatively at least, it is immaterial whether binding was obtained with p-nitrophenyl phosphate or P_i.The presence or absence of Na⁺ during binding has a special influence upon the character of the enzyme · ouabain complex. Without Na⁺ and in the presence of Tris ions the complex obtained with (Mg²⁺ + P_i) and that obtained with (Mg²⁺ + p-nitrophenyl-P) behaved in a nearly identical manner, both exhibiting a slow decay. High Na⁺ concentration diminished the level of P_i-supported ouabain binding, having almost no effect on p-nitrophenyl phosphate-supported binding. Both enzyme · ouabain complexes, however, now resembled the form obtained with (Na⁺ + ATP), as judged from their dissociation rates and the K⁺ sensitivity of their decay. The complexes obtained at a high Na⁺ concentration underwent a very fast decay which could be slowed considerably after adding a low concentration of K⁺ to the resuspension medium. The most stable enzyme · ouabain complex was obtained in the presence of Tris ions only, irrespective of whether p-nitrophenyl phosphate or P_i facilitated complex formation. The presence of K⁺ gave rise to a complex whose dissociation rate was intermediate between those of the complexes obtained in the presence of Tris and a high Na⁺ concentration.It is proposed that the different ouabain dissociation rates reflect different reactive state of the enzyme. The resemblance between the observations obtained in phosphorylation and ouabain binding experiments is pointed out.     

Regulation of C4 photosynthesis: mechanism of activation and inactivation of extracted pyruvate, inorganic phosphate dikinase in relation to dark/light regulation

Requirements for activation of inactive pyruvate, inorganic phosphate (P_i) dikinase extracted from darkened maize leaves were examined. Incubation with P_i plus dithiothreitol resulted in a rapid recovery of activity comparable to that in illuminated leaves. However, contrary to previous findings, most of this activity (60–95%) was recovered by adding P_i alone. There was no activation with dithiothreitol alone. Dependency on dithiothreitol, in addition to P_i was minimal at about pH 7.5 but was substantial at higher pH. Anaerobic conditions did not enhance P_i-dependent activation. Active enzyme, isolated from illuminated leaves, was inactivated by incubating with ADP and this occurred in the presence of dithiothreitol. ATP and AMP were not effective but ATP may be a corequirment for ADP-dependent inactivation. Enzyme inactivated by ADP required P_i for reactivation. We conclude that interconversion of dithiol and disulfide forms of the enzyme is not critical for the dark/light regulation of pyruvate, P_i dikinase. The primary mechanism apparently involves an ADP-induced transformation to an inactive form which undergoes a P_i-mediated reactivation.     

Limiting Factors in Photosynthesis: V. Photochemical Energy Supply Colimits Photosynthesis at Low Values of Intercellular CO(2) Concentration

Although there is now some agreement with the view that the supply of photochemical energy may influence photosynthetic rate (P) at high CO₂ pressures, it is less clear whether this limitation extends to P at low CO₂. This was investigated by measuring P per area as a function of the intercellular CO₂ concentration (C_i) at different levels of photochemical energy supply. Changes in the latter were obtained experimentally by varying the level of irradiance to normal (Fe-sufficient) leaves of Beta vulgaris L. cv F58-554H1, and by varying photosynthetic electron transport capacity using leaves from Fe-deficient and Fe-sufficient plants. P and C_i were determined for attached sugar beet leaves using open flow gas exchange. The results suggest that P/area was colimited by the supply of photochemical energy at very low as well as high values of C_i. Using the procedure developed by Perchorowicz et al. (Plant Physiol 1982 69:1165-1168), we investigated the effect of irradiance on ribulose bisphosphate carboxylase (RuBPCase) activation. The ratio of initial extractable activity to total inducible RuBPCase activity increased from 0.25 to 0.90 as leaf irradiance increased from 100 to 1500 microeinsteins photosynthetically active radiation per square meter per second. These data suggest that colimitation by photochemical energy supply at low C_i may be mediated via effects on RuBPCase activation.     

The effect of toxins on inorganic phosphate release during actin polymerization

During the polymerization of actin, hydrolysis of bound ATP occurs in two consecutive steps: chemical cleavage of the high-energy nucleotide and slow release of the γ-phosphate. In this study the effect of phalloidin and jasplakinolide on the kinetics of P _i release was monitored during the formation of actin filaments. An enzyme-linked assay based spectrophotometric technique was used to follow the liberation of inorganic phosphate. It was verified that jasplakinolide reduced the P _i release in the same way as phalloidin. It was not possible to demonstrate long-range allosteric effects of the toxins by release of P _i from F-actin. The products of ATP hydrolysis were released by denaturation of the actin filaments. HPLC analysis of the samples revealed that the ATP in the toxin-bound region was completely hydrolysed into ADP and P _i . The effect of both toxins can be sufficiently explained by local and mechanical blockade of P _i dissociation.     

Butylated hydroxytoluene and inorganic phosphate plus Ca2+ increase mitochondrial permeability via mutually exclusive mechanisms

Mitochondria undergo a permeability transition (PT), i.e., become nonselectively permeable to small solutes, in response to a wide range of conditions/compounds. In general, opening of the permeability transition pore (PTP) is Ca²⁺- and P_i-dependent and is blocked by cyclosporin A (CsA), trifluoperazine (TFP), ADP, and butylated hydroxytoluene (BHT). Gudz and coworkers have reported [7th European Bioenergetics Conference, EBEC Short Reports (1992)7, 125], however, that, under some conditions, BHT increases mitochondrial permeability via a process that may not share all of these characteristics. Specifically, they determined that the BHT-induced permeability transition was independent of Ca²⁺ and was insensitive to CsA. We have used mitochondrial swelling to compare in greater detail the changes in permeability induced by BHT and by Ca²⁺ plus P_i with the following results. (1) The dependence of permeability on BHT concentration is triphasic: there is a threshold BHT concentration (ca. 60 nmol BHT/ mg mitochondrial protein) below which no increase occurs; BHT enhances permeability in an intermediate concentration range; and at high BHT concentrations (> 120 nmol/mg) permeability is again reduced. (2) The effects of BHT depend on the ratio of BHT to mitochondrial protein. (3) Concentrations of BHT too low to induce swelling block the PT induced by Ca²⁺ and P_i. (4) The dependence of the Ca²⁺-triggered PT on P_i concentration is biphasic. Below a threshold of 50–100 M, no swelling occurs. Above this threshold swelling increases rapidly. (5) P_i levels too low to support the Ca²⁺-induced PT inhibit BHT-induced swelling. (6) Swelling induced by BHT can bestimulated by agents and treatments that block the PT induced by Ca²⁺ plus P_i. These data suggest that BHT and Ca²⁺ plus P_i, increase mitochondrial permeability via two mutually exclusive mechanisms.     

The pathway of inorganic-phosphate efflux from isolated liver mitochondria during adenosine triphosphate hydrolysis

1. The distribution of P_i between mitochondria and suspending medium during uncoupler-stimulated hydrolysis of ATP by rat liver mitochondria [Tyler (1969) Biochem. J. 111, 665–678] has been reinvestigated, by using either mersalyl or N-ethylmaleimide as inhibitors of P_i transport and either buffered sucrose/EDTA or LiCl/EGTA solutions as suspending medium. More than 75% of the total P_i liberated was retained in mitochondria treated with either inhibitor at all ATP concentrations tested (0.2–2.5mm). With low ATP concentrations and mersalyl-treated mitochondria incubated in sucrose/EDTA, virtually all the P_i liberated was retained in the mitochondria. 2. Larger amounts of P_i appeared in the suspending medium during ATPase activity, despite the presence of N-ethylmaleimide, when LiCl/EGTA was used as suspending medium compared with sucrose/EDTA. Two sources of this P_i were identified: (a) a slow efflux of P_i from mitochondria to suspending medium despite the presence of N-ethylmaleimide; (b) a slow ATPase activity insensitive to carboxyatractyloside, which was stimulated by added Mg²⁺, partially inhibited by oligomycin or efrapeptin and strongly inhibited by EDTA. 3. It is concluded that liver mitochondria preparations contain two distinct forms of ATPase activity. The major activity is associated with coupled mitochondria of controlled permeability to adenine nucleotides and P_i and is stimulated strongly by uncoupling agents. The minor activity is associated with mitochondria freely permeable to adenine nucleotides and P_i, is unaffected by uncoupling agents and is activated by endogenous or added Mg²⁺. 4. When mitochondria treated with mersalyl were incubated in buffered sucrose solution, almost all the P_i liberated was recovered in the suspending medium, unless inhibitors of P_i-induced large-amplitude swelling such as EDTA, EGTA, antimycin, rotenone, nupercaine or Mg²⁺ were added. Thus the loss of the specific permeability properties of the mitochondrial inner membrane associated with large-amplitude swelling also influences the extent of P_i retention during ATPase activity. 5. The results confirm the previous conclusion (Tyler, 1969) that the P_i transporter provides the sole pathway for P_i efflux during uncoupler-stimulated ATP hydrolysis by mitochondria. It is concluded that more recent hypotheses concerning the influence of Mg²⁺ on mersalyl inhibition of the P_i transporter [Siliprandi, Toninello, Zoccaroto & Bindoli (1975) FEBS Lett. 51, 15–17] and a postulated role of the adenine nucleotide exchange carrier in P_i efflux [Reynafarje & Lehninger (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4788–4792] are erroneous and should be discarded.