Organic phosphate probes of the avian renal phosphate secretory mechanism

Parathyroid hormone (PTH) stimulates net renal inorganic phosphate (Pi) secretion in domestic fowl (Gallus domesticus). Recent evidence indicates that secreted Pi is derived from a highly sequestered, presumably organic, phosphate pool. A modified Sperber technique was used to survey the response of domestic fowl to unilateral renal portal infusions of organic phosphate compounds that had been implicated in previous studies of Pi secretion. None of the organic phosphate compounds produced a significant unilateral Pi secretory effect. It is concluded that these compounds neither directly stimulate Pi secretion in normal or parathyroidectomized birds, nor are they rate-limiting for the Pi secretory mechanism in birds infused with PTH.     

The influence of radioactive phosphate level on the absorption of phosphate by plants and on the determination of labile soil phosphate

Summary Previous work has suggested that the presence of P³² in fertilizers inhibits the uptake of the applied phosphate from the soil by plants, and also that if the applied phosphate is not incorporated uniformly in the soil there will be preferential uptake from regions of low specific activity. This made it desirable to determine the effect of P³²-level on phosphate uptake and the determination ofL-values in pot experiments in which the labelled phosphate source is added as discrete particles of the phosphate form of an anion-exchange resin.Increasing the level of P³² from 0.05 to 1.25 mo per gram of phosphorus in the added phosphate did not have a significant effect on the fresh weight, dry weight or total phosphorus uptake of the ryegrass crop. The measuredL-value showed a significant increase, about 15 per cent for a five-fold increase in P³² level, on each of the four soil types used, as would be expected if P³² depressed the uptake of labelled fertilizer phosphate.Although a significant effect of P³² was observed this does not invalidate a comparison of soils with respect toL-value.     

Measurement of phosphate concentration in the presence of very labile phosphate esters

There are a number of methods available for the measurement of phosphate ion concentration, which may be used when moderately labile phosphate esters such as ATP are present in low concentration. However, the highly acidic conditions usually employed make these unsuitable when very labile esters such as phosphocreatine are present. A method in which the phosphomolybdate complex is developed under mildly acidic conditions, using high molybdate concentrations to counteract the reduced assay sensitivity at high pH, is described. The assay is linear in the range 5-300 microM phosphate, and micromolar concentrations of phosphate can be reliably measured in the presence of millimolar phosphocreatine.     

The effect of phosphate addition on the solubility of phosphate in soil

Summary The effect of phosphate addition on the phosphate potential of suspensions of three contrasting soils was investigated. The results showed that even shaking the soil suspension for 14 days was insufficient to reach equilibrium in the soil: solution system. Increasing the phosphate additions to the soils resulted in decreasing phosphate potentials. Furthermore, on addition of phosphate to the soils, the solubility data did not conform to those anticipated on the basis of phosphate minerals expected to be present in the soils. The results indicated that the phosphate concentration in the soil-solution was controlled by an adsorption type of mechanism.     

Inorganic phosphate determination in the presence of a labile organic phosphate: assay for carbamyl phosphate phosphatase activity

A periodate-resorcinol method for bound sialic acid using the Technicon Autoanalyzer II is presented. It has a sensitivity similar to the manual method, is linear between 5 and 65 nmol/ml, requires less than 0.2 ml of sample, and can be run at the rate of 70 samples/h. Little cross-reaction with common matrix and cell components was found. The method is compatible with many commonly used volatile and nonvolatile chromatographic buffers. The use of a bound sialic acid standard such as N-acetylneuraminyl lactose is recommended.     

Effects of phosphate buffer capacity of soil on the phosphate requirements of plants

Summary Plant-uptake and yield data for ryegrass in a greenhouse experiment are used to estimate the theoretical fertilizer phosphate requirement (Pf) of 24 Sherborne soils. Pf is shown to be a function of three parameters: (i) quantity of P required by the plant (Pr) for optimum yield; (ii) quantity of soil P (Qr) required to maintain a non-limiting soil solution concentration (Ir); (iii) quantity of labile soil P (Q). Because of its large effect on Qr and Ir, the phosphate buffer capacity has an important effect on Pf. However Pf cannot be directly related to phosphate buffer capacity if Q is ignored. On soils of similar Q, increasing buffer capacity will always have a positive effect on Pf, but on soils of the same I, it may have a positive or negative effect on Pf. Consequently, Pf can only be simply, but inversely, related to Q or I on a group of soils of similar phosphate buffer capacity. re]19750513     

The roles of phosphate and the phosphate carrier in the mitochondrial permeability transition pore

Phosphate activation of the mitochondrial permeability transition pore (MPTP) opening is well-documented and could involve the phosphate carrier (PiC) that we have proposed is the pore's cyclophilin-D binding component. However, others have reported that following CyP-D ablation Pi inhibits MPTP opening while cyclosporine-A (CsA) inhibits MPTP opening only when Pi is present. Here we demonstrate that Pi activates MPTP opening under all energised and de-energised conditions tested while CsA inhibits pore opening whether or not Pi is present. Using siRNA in HeLa cells we could reduce PiC expression by 65-80% but this inhibited neither mitochondrial calcium accumulation nor MPTP opening.     

Synthesis of oxalyl phosphate and processing of the acyl phosphate by phosphoenolpyruvate-dependent enzymes

The mixed anhydride of oxalic and phosphoric acids, oxalyl phosphate, has been prepared by reaction of oxalyl chloride and inorganic phosphate in aqueous solution. The product was purified by anion exchange chromatography and characterized by 31P and 13C NMR. This acyl phosphate has a half-life of 51 h at pH 5.0 and 4 degrees C. Oxalyl phosphate, an analogue of phosphoenolpyruvate, is a slow substrate for pyruvate kinase, undergoing an enzyme-dependent phosphotransfer reaction to produce ATP from ADP. Oxalyl phosphate substitutes for phosphoenolpyruvate in the reaction catalyzed by pyruvate, phosphate dikinase. The acyl phosphate reacts with the free enzyme to give the phosphorylated form of the enzyme. Removal of the potent product inhibitor, oxalate, from the reaction mixtures by gel filtration chromatography permitted further reaction of the phosphorylated enzyme with pyrophosphate and AMP to give ATP and Pi in a single turnover assay. Oxalyl phosphate also served as a phospho group donor in a partial reaction catalyzed by phosphoenolpyruvate carboxykinase wherein GDP is phosphorylated at the expense of oxalyl phosphate.     

Characterization of a phosphate binding domain on the alpha-subunit of chloroplast ATP synthase using the photoaffinity phosphate analogue 4-azido-2-nitrophenyl phosphate

The photoaffinity phosphate analogue 4-azido-2 nitrophenyl phosphate (ANPP) was shown previously (Pougeois, R., Lauquin, G. J.-M., and Vignais, P. V. (1983) Biochemistry 22, 1241-1245) to bind covalently and specifically to a single catalytic site on one of the three beta-subunits of the isolated chloroplast coupling factor 1 (CF(1)). Modification by ANPP strongly inhibited ATP hydrolysis activity. In this study, we examined labeling of membrane-bound CF(1) by ANPP by exposing thylakoid membranes to increasing concentrations of the reagent. ANPP exhibited saturable binding to two sites on CF(1), one on the beta-subunit and one on the alpha-subunit. Labeling by ANPP resulted in the complete inhibition of both ATP synthesis and ATP hydrolysis by the membrane-bound enzyme. Labeling of both sites by ANPP was reduced by more than 80% in the presence of P(i) (> or = 10 mM) and ATP (> or = 0.5 mM). ADP was less effective in competing with ANPP for binding, giving a maximum of approximately 35% inhibition at concentrations > or = 2 mM. ANPP-labeled tryptic peptides of the alpha-subunit were isolated and sequenced. The majority of the probe was contained in three peptides corresponding to residues Gln(173) to Arg(216), Gly(217) to Arg(253), and His(256) to Arg(272) of the alpha-subunit. In the mitochondrial F(1) (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), all three analogous peptides are located within the nucleotide binding pocket and within close proximity to the gamma-phosphate binding site. The data indicate, however, that the azidophenyl group of bound ANPP is oriented at approximately 180 degrees in the opposite direction to the adenine binding site with reference to the phosphate binding site on the alpha-subunit. The study has confirmed that ANPP is a bona fide phosphate analogue and suggests that it specifically targets the gamma-phosphate binding site within the nucleotide binding pockets on the alpha- and beta-subunits of CF(1). The study also indicates that in the resting state of the chloroplast F(1)-F(0) complex both the alpha- and beta-subunits are structurally asymmetric.     

Cereal phosphate transporters associated with the mycorrhizal pathway of phosphate uptake into roots

A very large number of plant species are capable of forming symbiotic associations with arbuscular mycorrhizal (AM) fungi. The roots of these plants are potentially capable of absorbing P from the soil solution both directly through root epidermis and root hairs, and via the AM fungal pathway that delivers P to the root cortex. A large number of phosphate (P) transporters have been identified in plants; tissue expression patterns and kinetic information supports the roles of some of these in the direct root uptake pathways. Recent work has identified additional P transporters in several unrelated species that are strongly induced, sometimes specifically, in AM roots. The primary aim of the work described in this paper was to determine how mycorrhizal colonisation by different species of AM fungi influenced the expression of members of the Pht1 gene families in the cereals Hordeum vulgare (barley), Triticum aestivum (wheat) and Zea mays (maize). RT-PCR and in-situ hybridisation, showed that the transporters HORvu;Pht1;8 (AY187023), TRIae;Pht1;myc (AJ830009) and ZEAma;Pht1;6 (AJ830010), had increased expression in roots colonised by the AM fungi Glomus intraradices,Glomus sp. WFVAM23 and Scutellospora calospora. These findings add to the increasing body of evidence indicating that plants that form AM associations with members of the Glomeromycota have evolved phosphate transporters that are either specifically or preferentially involved in scavenging phosphate from the apoplast between intracellular AM structures and root cortical cells. Operation of mycorrhiza-inducible P transporters in the AM P uptake pathway appears, at least partially, to replace uptake via different P transporters located in root epidermis and root hairs. Electronic Supplementary Material Supplementary material is available for this article at     

Photoaffinity labeling of the mitochondrial phosphate carrier by 4-azido-2-nitrophenyl phosphate

The effect of 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive analogue of phosphate, on the phosphate carrier of pig-heart mitochondria has been investigated. In the dark, ANPP inhibits the transport of phosphate in a competitive manner with a Ki of 3.2 mM. Upon photoirradiation with visible light, [32P]ANPP binds covalently to the phosphate carrier and the inhibition becomes irreversible. Both the inhibition of phosphate transport and the incorporation of [32P]ANPP into the phosphate carrier depend on the concentration of the inhibitor and the pH of the medium. Incubation of the mitochondria with phosphate during illumination in the presence of ANPP protects the carrier against inactivation and decreases the amount of radioactivity which is found to be associated with the purified protein. By extrapolation it is calculated that at 100% inactivation of the phosphate carrier 0.35 mol of reagent are bound per mol of 33 kDa carrier protein. It is concluded that ANPP can be used for photoaffinity labeling of the mitochondrial phosphate carrier at the substrate-binding site.     

Direct demonstration of carbamoyl phosphate formation on the C-terminal domain of carbamoyl phosphate synthetase

Carbamoyl phosphate synthetase synchronizes the utilization of two ATP molecules at duplicated ATP-grasp folds to catalyze carbamoyl phosphate formation. To define the dedicated functional role played by each of the two ATP sites, we have carried out pulse/labeling studies using the synthetases from Aquifex aeolicus and Methanococcus jannaschii, hyperthermophilic organisms that encode the two ATP-grasp folds on separate subunits. These studies allowed us to differentially label each active site with [gamma-(32)P]ATP and determine the fate of the labeled gamma-phosphate in the synthetase reaction. Our results provide the first direct demonstration that enzyme-catalyzed transfer of phosphate from ATP to carbamate occurs on the more C-terminal of the two ATP-grasp folds. These findings rule out one mechanism proposed for carbamoyl phosphate synthetase, where one ATP acts as a molecular switch, and provide additional support for a sequential reaction mechanism where the gamma-phosphate groups of both ATP molecules are transferred to reactants. CP synthesis by subunit C in our single turnover pulse/chase assays did not require subunit N, but subunit N was required for detectable CP synthesis in the traditional continuous assay. These findings suggest that cross-talk between domain N and C is required for product release from subunit C.     

Biogenesis of the chloroplast phosphate translocator

Calcium-dependent proteolysis of several polypeptides from rat brain and synaptosomal cytosol was observed including proteolysis of polypeptides of M_r 340 000 and 300 000. These latter polypeptides comigrated with high-M_r microtubule-associated proteins of microtubule preparations from brain or synaptosomal cytosol. Calcium influx into intact synaptosomes due to depolarisation with high potassium or veratridine or treatment with the ionophore A23187 did not result in Ca²⁺-dependent proteolysis of any polypeptides. This may be due to the low calcium sensitivity of the protease since no proteolysis of the M_r 340 000 and 300 000 polypeptides was seen in synaptosomal cytosal at < 10 μM free Ca²⁺.