Inhibitory Effect Of Phosphate Research Articles

Visualisation of Phosphate in Subcalicoblastic Extracellular Calcifying Medium and on a Skeleton of Coral by Using a Novel Probe, Fluorescein-4-Isothiocyanate-Labelled Alendronic Acid

The overload of nutrients of anthropogenic origin, including phosphate, onto coastal waters has been reported to have detrimental effects on corals. However, to the best of our knowledge, the phosphate concentration threshold for inhibiting coral calcification is unclear owing to a lack of information on the molecular mechanisms involved in the inhibitory effect of phosphate. Therefore, in this study, we prepared a new phosphate analogue, fluorescein-4-isothiocyanate (FITC)-labelled alendronic acid (FITC-AA), from commercially available reagents and used it as a novel probe to demonstrate its transfer pathway from ambient seawater into Acropora digitifera. When the juveniles at 1 d post-settlement were treated with FITC-AA in a laboratory tank, this phosphate analogue was found in the subcalicoblastic extracellular calcifying medium (SCM) and was absorbed on the basal plate in the juveniles within a few minutes. When the juveniles bear zooxanthellae at 3months post-settlement, FITC-AA was observed on the corallite walls within a few minutes after adding ambient seawater. We concluded that FITC-AA in ambient seawater was transferred via a paracellular pathway to SCM and then absorbed on the coral CaCO3 skeletons because FITC-AA with a high polarity group cannot permeate through cell membranes.

Adsorption of humic acids to lake sediments: Compositional fractionation, inhibitory effect of phosphate, and implications for lake eutrophication

Humic acid (HA) and phosphate interactions play a vital role in the biogeochemical cycle of carbon and nutrients and thus the trophic state of a lake. The adsorption behavior of HAs to sediments in the absence and presence of phosphate was investigated in this study. Three types of HAs were used, AHA from algae-dominated lake sediments, MHA from macrophyte-dominated lake sediments, and a reference HA (RHA) with terrestrial sources. The adsorption capacity of lake sediments was highest for AHA, which can be explained by that AHA contained more carboxyl-containing molecules, proteinaceous compounds and polysaccharides that were preferentially adsorbed by minerals. Phosphate showed a stronger inhibitory effect on MHA adsorption than on AHA adsorption, suggesting that AHA can more effectively replace phosphate adsorbed to sediments. Our findings show that the functional groups of organic compounds control not only their fractionation and burial but also their ability to replace phosphate in sediments. We propose a novel mechanism to explain the legacy effect of lake eutrophication. That is, as lakes shift from a macrophyte-dominated state to more eutrophic, algae-dominated state, increasing algae-derived organic compounds can promote the release of phosphate from sediments, forming a positive feedback loop that sustains internal phosphorus loading and hence lake eutrophication.

Carbonate accelerated transformation of ferrihydrite in the presence of phosphate

In soils and rhizospheres, iron (oxyhydr)oxides and oxyanions like carbonate and phosphate occur ubiquitously and their interaction have important implications for nutrients and metals cycling. An elevated activity of carbonate in soils and sediments (e.g., pCO2, ∼2%) above current atmospheric CO2 (∼0.04%) is observed. The level of agronomic soil test phosphorus (P) in intensively managed agricultural soils can be up to several hundred mg kg−1. Although it is known that the transformation of iron (oxyhydr)oxides is suppressed by phosphate adsorption, it is unclear how increasing carbonate activities exert the reaction process. Here, the effects of carbonate on the transformation of ferrihydrite were evaluated at pH 7.5 in the presence of phosphate using experimental geochemistry, Fe K-edge X-ray absorption spectroscopy, transmission electron microscopy-energy dispersive X-ray spectroscopy, and Fourier transform infrared spectroscopy. Our results, for the first time, showed that the inhibitory effect of phosphate on ferrihydrite transformation was retarded by carbonate, and it transformed into hematite. Under pCO2 = 408 ppm (0.213 mM carbonate), an increase in [phosphate]o from 0 to 0.5 mM decreased the transformation rate of ferrihydrite from 0.009 to 0.003 d−1. However, the suppression was drastically perturbed when carbonate was added to the phosphate-ferrihydrite system. When 11.42 mM carbonate (pCO2 = 20000 ppmv) was present, the rate increased from 0.009 to 0.033 d−1 without phosphate and from 0.003 to 0.018 d−1 with 0.5 mM of phosphate. Moreover, carbonate promoted the formation of more crystalline rhombic hematite particles, while phosphate modified the morphology of hematite from rhombic to ellipsoidal-like with rough surfaces. The distinctive difference of the influence of carbonate and phosphate on the transformation kinetics, products distribution, and morphologies come from the interaction between carbonate/phosphate and ferrihydrite. Although phosphate formed strong inner-sphere complexes on the ferrihydrite surface, which inhibited the direct contact and dissolution of ferrihydrite, co-adsorbed carbonate still promoted the olation reaction, facilitating the formation of hematite. These results suggest that as an important geochemical process, the increasing activity of carbonate or pCO2 can override the retarding impact of phosphate on the transformation of ferrihydrite and control the occurrence/crystallization of hematite in the subsurface soils under the scenario of climate change.

Coprecipitation of humic acid and phosphate with Fe(III) enhances the sequestration of carbon and phosphorus in sediments

The coprecipitation of dissolved organic matter (DOM) and phosphate with Fe(III) at the sediment-water interface plays an important role in the biogeochemical cycle of organic carbon (OC) and phosphorus (P) in aquatic ecosystems. However, studies remain limited towards understanding the strength of ternary DOM-phosphate-Fe(III) coprecipitation in sequestering OC and P. Here, we investigated the behaviors of humic acid (HA) and phosphate during their coprecipitation with Fe(III) and compared the coprecipitation behaviors with those during the adsorption process. The results showed that the coprecipitation of HA and phosphate with Fe(III) enhanced as pH decreased or initial Fe(III) concentrations increased. The presence of phosphate had a moderate inhibition effect on HA coprecipitation, leading to 3%–37% of reduction in precipitated HA. Changes in optical properties of DOM (i.e., SUVA254, E2/E3, and fluorescent components) showed that macromolecular, aromatic molecules preferentially coprecipitated, and this selectivity pattern was not influenced by the presence of phosphate. Compared to the adsorption of HA and phosphate to goethite, coprecipitation with Fe(III) displayed stronger binding ability, sequestering more than10 times of HA and phosphate, and was more efficient in sequestering protein-like compounds. The inhibitory effect of phosphate on HA sequestration was weaker during coprecipitation than during adsorption. Moreover, coprecipitation led to a lower degree of compositional fractionation of HA than adsorption, which may be explained by that inclusion, an important pathway for coprecipitation, has low selectivity for chemical compositions. Collectively, our results demonstrate that coprecipitation plays a crucial role in sequestering both OC and P in aquatic systems, arguing that the formation of ternary OC-Fe-P coprecipitates may be an important pathway for sequestering OC and P over a long term and reducing the internal loading of P in eutrophic lakes.

Effect of phosphate on the adsorption of antibiotics onto iron oxide minerals: Comparison between tetracycline and ciprofloxacin

With the broadly application of antibiotics to treat infectious diseases in humans and animals, antibiotic contaminants such as tetracycline (TC) and ciprofloxacin (CIP) have been detected in soil environments, where iron oxide minerals and phosphate are ubiquitous. To date, the influence of phosphate on the adsorption behaviors of TC/CIP onto iron oxides is still poorly understood. In this study, the effects of phosphate on the adsorptions of TC and CIP onto iron oxide minerals were investigated. Adsorption isotherms showed that the adsorption affinities of TC and CIP onto the three iron oxide minerals were in the order of goethite > hematite > magnetite with or without phosphate, the trend was dominated by different surface area and amount of surface hydroxyl groups of iron oxide minerals. Meanwhile, TC contains more functional groups than CIP for bonding, which resulted in greater adsorption affinity of three iron oxides to TC than that to CIP. Interestingly, phosphate weakened TC adsorption, while enhanced CIP adsorption, on the three iron oxides. This observation was ascribed to that phosphate anion enhanced the surface negative charge of iron oxides, which reinforced the electrostatic repulsion between iron oxides and negatively charged TC, also reinforced the electrostatic attraction between iron oxides and positively charged CIP. Furthermore, the inhibitory effect of phosphate on TC adsorption was dramatically enhanced at high pH, while the promoting effect of phosphate on CIP adsorption was slightly changed with various pH. Our results highlight the importance of phosphate in exploring the environmental fate of antibiotics in natural environment.

Mobility of multiple heavy metalloids in contaminated soil under various redox conditions: Effects of iron sulfide presence and phosphate competition

The mobility of heavy metalloids including As, Sb, Mo, W, and Cr in soil was investigated under both reducing and oxidizing conditions. The effects of soil mineralogy and the presence of competitive anions were studied as important factors affecting the mobility of these contaminants. Batch experiments conducted with the addition of oxidized and fresh FeS exhibited enhanced sorption rates for As and W under oxidizing conditions, and for Mo under reducing conditions. The inhibitory effect of phosphate on the sorption rates was most apparent for As and Mo under both oxidizing and reducing conditions, while only a small phosphate effect was observed for Sb and W. For Sb and W mobility, pH was determined to be the most important controlling factor. The results of long-term batch experiments revealed that differences in the mobility of metalloids, particularly As, were also influenced by microbial activity in the oxidizing and reducing conditions.

Impact of phosphate and other medium components on physiological regulation of bacterial laccase production.

Laccases are multicopper oxidases known to catalyze the transformation of a wide range of phenolic and non-phenolic substrates using oxygen as electron acceptor and forming water as the only by product. Their potential relevance in several industries requires the constant search for novel laccases. Positive outcome of the isolation of laccase producing bacteria depends on the nature and concentration of media constituents. Several attempts to isolate laccase producing bacteria failed when the phosphate-containing M9 minimal medium was used. Shift to phosphate-less M162 medium led to successful isolations. Seven bacterial isolates belonging to genera Bacillus, Lysinibacillus, Bhargavaea and Rheinheimera were used to study the effect of medium constituents on laccase production. Inorganic phosphate (≥50 mM) was found to regulate laccase synthesis negatively though no inhibitory effect of phosphate (10-500 mM) was seen on laccase activity. All isolates ceased laccase synthesis when grown in the presence of tryptone (0.2-1%), with R. tangshanensis as an exception, or yeast extract (1.5-2%) as the only C/N source in M162 medium. Supplementation upto 0.1% of glucose in basal M162 medium increased laccase production in five isolates but decreased at higher concentrations. The influence of medium components on laccase synthesis was further affirmed by zymographic studies. These observations offer possibilities of isolating promising laccase producers from diverse environmental sources. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:541-548, 2017.

Effect of phosphate on heterogeneous Fenton oxidation of catechol by nano-Fe3O4: Inhibitor or stabilizer?

The effect of phosphate on adsorption and oxidation of catechol, 1,2-dihydroxybenzene, in a heterogeneous Fenton system was investigated. In situ attenuated total reflectance infrared spectroscopy (ATR-FTIR) was used to monitor the surface speciation at the nano-Fe3O4 catalyst surface. The presence of phosphate decreased the removal rate of catechol and the abatement of dissolved organic compounds, as well as the decomposition of H2O2. This effect of phosphate was mainly due to its strong reaction with surface sites on the iron oxide catalyst. At neutral and acid pH, phosphate could displace the adsorbed catechol from the surface of catalyst and also could compete for surface sites with H2O2. In situ IR spectra indicated the formation of iron phosphate precipitation at the catalyst surface. The iron phosphate surface species may affect the amount of iron atoms taking part in the catalytic decomposition of H2O2 and formation of hydroxyl radicals, and inhibit the catalytic ability of Fe3O4 catalyst. Therefore, phosphate ions worked as stabilizer and inhibitor in a heterogeneous Fenton reaction at the same time, in effect leading to an increase in oxidation efficiency in this study. However, before use of phosphate as pH buffer or H2O2 stabilizer in a heterogeneous Fenton system, the possible inhibitory effect of phosphate on the actual removal of organic pollutants should be fully considered.

Effects of Phosphate on Arsenate Uptake and Translocation in Nonmetallicolous and Metallicolous Populations of Pteris Vittata L. Under Solution Culture

An arsenic hyperaccumulator, Pteris vittata L., is common in nature and could occur either on As-contaminated soils or on uncontaminated soils. However, it is not clear whether phosphate transporter play similar roles in As uptake and translocation in nonmetallicolous and metallicolous populations of P. vittata. Five populations were used to investigate effects of phosphate on arsenate uptake and translocation in the plants growing in 1.2 L 20% modified Hoagland's nutrient solution containing either 100 μM phosphate or no phosphate and 10 μM arsenate for 1, 2, 6, 12, 24 h, respectively. The results showed that the nonmetallicolous populations accumulated apparently more As in their fronds and roots than the metallicolous populations at both P supply levels. Phosphate significantly (P < 0.01) decreased frond and root concentrations of As during short time solution culture. In addition, the effects of phosphate on As translocation in P. vittata varied among different time-points during time-course hydroponics (1–24 h). The present results indicated that the inhibitory effect of phosphate on arsenate uptake was larger in the three nonmetallicolous populations than those in the two metallicolous populations of P. vittata.

Inhibition effect of phosphate on the crystal grain growth of nanosized titania

The inhibitory effect of phosphate on the crystal grain growth of nanosized titania during high temperature calcination was investigated. Nanosized titanium dioxide powders prepared by hydrolysis of titanium tetrachloride were soaked in phosphate solutions with different concentrations. The obtained powders calcined at various temperatures were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectronic spectroscopy (XPS). The grain size of the samples without phosphate treatment increased quickly when calcined at high temperatures, while the grain size of the samples with phosphate modification increased slowly when calcined at the same temperature. This phenomenon implies that phosphate treatment plays an important role in inhibiting the crystal grain growth of titania. The possible mechanism of the inhibition effect of phosphate on titania is discussed.

Phosphate inhibition on thermophilic acetoclastic methanogens: a warning

The inhibitory effect of phosphate on acetoclastic-methanogens was investigated for three different thermophilic (55 degrees C) anaerobic consortia. When 70 mM of phosphate was tested, acetoclastic methanogens was completely inhibited in "Eerbeek" sludge which is dominated by Methanosaeta-like methanogens. For the "Hoogezand" sludge the specific methanogenic activity dropped by 79%, indicating that any of the acetate-consuming methanogens present in the sludge was more resistant to the phosphate applied. The results demonstrated that the inhibitory effect of phosphate may affect both methane production rate and final methane concentration and might also be time dependent. This study indicates that the degree of inhibition is species-dependent, and even more resistant species may be affected during long-term experiments. Such inhibition is a matter of concern for researchers since misleading conclusions might be taken from growth and specific methanogenic activity tests when considerable concentrations of phosphate buffer are used and no interference is expected.

New insights in the regulation of the afp gene encoding the antifungal protein of Aspergillus giganteus.

The antifungal protein (AFP) secreted by the mould Aspergillus giganteus is a small, highly basic polypeptide with antifungal activity. Previous work has shown that transcription of the corresponding afp gene is regulated by ambient pH, being suppressed under acidic and strongly induced under alkaline conditions. This observation suggested that the afp gene is regulated by the wide-domain transcriptional factor PacC. Here, we show that two putative PacC binding sites within the afp promoter, denoted afpP1 and afpP2, are efficiently recognised in vitro by a PacC fusion protein of A. nidulans. In addition, we found that phosphate, which was used as a buffering agent during cultivation, plays an important role in regulating afp expression. AFP production was nearly completely inhibited in the presence of external phosphate. The results of Northern analysis indicate that the inhibitory effect of phosphate is mediated at the transcriptional level.

Involvement of glycolytic metabolism in developmental inhibition of rat two-cell embryos by phosphate.

To elucidate the mechanism by which phosphate induces developmental inhibition of rat 2-cell embryos, we examined the mutual effects of glucose and other glycolytic and non-glycolytic sugars, the non-metabolizable glucose analogue, and glycolytic inhibitors on the inhibitory effect of phosphate. In the absence of glucose, 30-49% of embryos treated with 10-500 microM phosphate were able to develop to morula and blastocysts. On the other hand, in the presence of 5 mM glucose, 10 microM phosphate decreased the developmental rate of 2-cell embryos to the 4-cell stage and completely inhibited the development beyond the 4-cell stage. In contrast, glucose showed no influence on development in phosphate-free medium. Similarly to glucose, the other glycolytic sugars fructose (5 mM) and mannose (5 mM) enhanced the inhibitory effect of 10 microM phosphate but had no influence in the absence of phosphate. In contrast, the non-glycolytic sugar and non-metabolizable glucose analogue N-acetylglucosamine and 3-O-methylglucose (3-O-MGlc), respectively, did not enhance the effects of phosphate. 2-Deoxyglucose (2DGlc), another glucose analogue that is non-metabolizable but is converted by hexokinase to 2DGlc 6-phosphate, at concentrations as low as 0.1 mM completely inhibited cell cycle progression of 2-cell embryos cultured in glucose-free (Glc(-)) medium with 10 microM phosphate. In contrast, in the absence of phosphate, 2DGlc at the same concentration allowed 55% of 2-cell embryos to develop to morula and blastocyst stages. Addition of an inhibitor of enolase in glycolysis, sodium fluoride (NaF), at 1 mM to the Glc(-) medium also enhanced the inhibitory effects of 10 microM phosphate, whereas 1 mM NaF in the absence of phosphate showed no inhibitory effects on the development of 2-cell embryos to morula and blastocyst stages. From these results, disturbance of glycolysis is a critical reason for the developmental inhibition caused by phosphate in early rat embryos in culture.

Inhibitory effect of phosphate on in vitro development of 2-cell rat embryos is overcome by a factor(s) in oviductal extracts

It has been found that inorganic phosphate (P) at concentrations as low as 10 μM markedly inhibits in vitro development of early rat embryos at the 1-cell or 2-cell stage to the blastocyst stage, although P is present at concentrations of 0.37–1.19 mM in oviductal fluid, in which the development of early embryos occurs. We show here that fractions (PTF, 50–250 μg/ml) of rat oviductal extracts (OVEs) passed through a Blue CL6B affinity column have the ability to overcome the inhibitory effects of P on the development of 2-cell rat embryos in a dose-dependent manner, whereas 250 μg/ml OVE or 250 μg/ml of the bound fractions (BF) induced degenerative changes in the embryos at the 2-cell stage. Moreover, PTF at concentrations of ≥100 μg/ml stimulated the hatching of blastocysts both in medium with and without P, although none of the blastocysts in medium without PTF hatched from their zona pellucida.

Characterization of a chloride-selective channel from rough endoplasmic reticulum membranes of rat hepatocytes: evidence for a block by phosphate.

We have characterized the conduction and blocking properties of a chloride channel from rough endoplasmic reticulum membranes of rat hepatocytes after incorporation into a planar lipid bilayer. Our experiments revealed the existence of a channel with a mean conductance of 164 +/- 5 pS in symmetrical 200 mm KCl solutions. We determined that the channel was ten times more permeable for Cl- than for K+, calculated from the reversal potential using the Goldman-Hodgkin-Katz equation. The channel was voltage dependent, with an open probability value ranging from 0.9 at -20 mV to 0.4 at +60 mV. In addition to its fully open state, the channel could also enter a flickering state, which appeared to involve rapid transitions to zero current level. Our results showed a decrease of the channel mean open time combined with an increase of the channel mean closed time at positive potentials. An analysis of the dwell time distributions for the open and closed intervals led to the conclusion that the observed fluctuation pattern was compatible with a kinetic scheme containing a single open state and a minimum of three closed states. The permeability sequence for test halides determined from reversal potentials was Br- > Cl- > I- approximately F-. The voltage dependence of the open probability was modified by the presence of halides in trans with a sequence reflecting the permeability sequence, suggesting that permeant anions such as Br- and Cl- have access to an internal site capable of controlling channel gating. Adding NPPB to the cis chamber inhibited the channel activity by increasing fast flickering and generating long silent periods, whereas channel activity was not affected by 50 microM DNDS in trans. The channel was reversibly inhibited by adding phosphate to the trans chamber. The inhibitory effect of phosphate was voltage-dependent and could be reversed by addition of Cl-. Our results suggest that channel block involves the interaction of HPO2-4 with a site located at 70% of the membrane span.

Modulation of the electrophoretic ATP-induced K(+)-transport in yeast mitochondria by delta pH.

The modulation of the ATP-induced K(+)-transport pathway of the yeast inner mitochondrial membrane by delta pH was investigated in two ways. First, the inhibitory effect of phosphate was compared to the effect of other permeant acids, demonstrating that a part of the effect of phosphate was linked to its electroneutral transport down delta pH. However, an additional effect specific for phosphate also occurred inside the matrix. Second, the stimulation of the respiration by ATP in the presence of K+ was compared to the effects of the protonophore ClCCP2 and of the K(+)-ionophore valinomycin. Quite unexpectedly, the effect of ATP looked more like the effect of ClCCP than the effect of valinomycin. This and previous results (Manon et al., Biochimica et Biophysica Acta 1231, 282-288 (1995)) show that the rate of electrophoretic K(+)-entry is limited by the rate of electroneutral K(+)-exit via the K+/H+ exchange and is therefore delta pH-dependent.

On the protection by inorganic phosphate of calcium-induced membrane permeability transition.

The role of inorganic phosphate as inhibitor of mitochondrial membrane permeability transition was studied. It is shown that in mitochondria containing a high phosphate concentration, i.e., 68 nmo/mg, Ca2+ did not activate the pore opening. Conversely, at lower levels of matrix phosphate, i.e., 38 nmol/mg, Ca2+ was able to induce subsequent pore opening. The inhibitory effect of phosphate was apparent in sucrose-based media, but it was not achieved in KCI media. The matrix free Ca2+ concentration and matrix pH were lowered by phosphate, but they were always higher in K+-media. In the absence of ADP, phosphate strengthened the inhibitory effect of cyclosporin A on carboxyatractyloside-induced Ca2+ efflux. Acetate was unable to replace phosphate in the induction of the aforementioned effects. It is concluded that phosphate preserves selective membrane permeability by diminishing the matrix free Ca2+ concentration.

Purification and properties of an extracellular conidial trehalase from Humicola grisea var. thermoidea

An extracellular trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) was purified from conidia of Humicola grisea var. thermoidea. The purified enzyme is a glycoprotein and migrates as a single polypeptide band during polyacrylamide gel electrophoresis under non-denaturing conditions. The apparent molecular weight of the enzyme was estimated as 580 000 by gel filtration chromatography. The enzyme is separable into three polypeptide bands of 105 000, 98 000 and 84 000 daltons on SDS-PAGE. It is specific for trehalose and its activity is not inhibited by other dissaccharides. It has a K m of 2.3 mM, an optimum pH of 5.6 in sodium acetate buffer and a temperature optimum of 60°C. The enzyme is activated by Ca 2+, Co 2+ and Mn 2+ and inhibited by inorganic phosphate, AMP, ADP or ATP. The inhibitory effect of phosphate, AMP and ADP, but not that of ATP, was abolished in the presence of Ca 2+.

Phosphate is an inhibitor of copper, zinc superoxide dismutase

Bovine etyrhrocyte Cu, Zn superoxide dismutase (SOD) in its oxidized form has been shown by X-ray crystallography [1] to be a dimer of two equivalent subunits, with one Cu(II) and one Zn(II) ion per subunit. The Cu ion is bound to four histidyl imidazole ligands and a water molecule making the overall geometry five-coordinate. One of these imidazoles is deprotonated and acts as a bridging ligand between Cu and Zn. The remaining ligands to Zn are two additional histidyl imidazoles and aspartyl carboxylate, forming a distorted tetrahedral geometry. Cu, Zn SOD's are found to be extremely efficient catalysts of the disproportionation of superoxide (2O 2 + 2H + → O 2 + H 2O 2) has been proposed that this activity is their primary biological function in vivo [2]. A number of anions have been observed to bind to Cu, Zn SOD e.g. CN −, N in2 3 , SC in2N , OC N , and halides. Each of these anions binds to copper(II) in the enzyme, as indicated by the pronounced spectral shifts observed upon binding [3]. Inhibitory effects of these anions have been attributed to this binding [3]. There have been several observations reported in the literature that suggest that phosphate also interacts with Cu, Zn SOD. For example, it has been mentioned in passing that phosphate interferes with the binding of C N to the enzyme [4] and that it has inhibitory effect on the SOD activity [5]. In addition, it has been reported that phosphate influences the mode of binding of cobalt to the apoprotein [6] and that it interacts with the four-copper derivative (where Cu has been substituted for Zn in the native protein) [7]. We have no visible or ESR spectral changes in solutions of Cu, Zn SOD at high concentrations of phosphate, suggesting strongly that any interaction of phosphate with the enzyme does not occur by binding of that anion to Cu(II). Contrary to the reports of McAdam [8] and Cudd and Fridovich [9], who assumed that the inhibitory effect of phosphate on the SOD activity could be entirely attributed to ionic strength effects, we found that the SOD activity of native bovine Cu, Zn SOD measured at constant ionic strength decreased significantly in the presence of increasing concentrations of phosphate. In addition, we have titrated the native enzyme with NaN 3 in differing concentrations of phosphate at constant ionic strength and thereby demonstrated that the presence of phosphate decreased the affinity of the enzyme for the azide anion. This result is reminiscent of the decrease in the binding affinity of this protein when arginine-141 is chemically modified with phenylglyoxal [10]. Arginine-141 may well be the site phosphate binding to this protein, since the binding of phosphate to the arginine-modified protein is apparently not affected by the presence of phosphate. Spectroscopic evidence concerning the nature of the interaction between phosphate and Cu, Zn SOD has also been obtained.

Control by phosphate of candicidin production

Phosphate concentration was found to control the biosynthesis of the antibiotic candicidin by resting cells of Streptomyces griseus. Phosphate concentrations above 1 mM decreased the rate of incorporation of [ 14C]propionate and [ 14C] p-aminobenzoic acid into candicidin in relation to the concentration of phosphate. The inhibitory effect of phosphate on incorporation of labeled precursors into candicidin was not caused by inhibition of cellular uptake of precursors. Protein synthesis, sensitive to chloramphenicol, was not affected by phosphate levels that inhibit antibiotic synthesis. Similarly, phosphate concentrations inhibitory to antibiotic synthesis did not affect rifampinsensitive RNA synthesis.