Phosphate transport and arsenate resistance in the cyanobacterium Anabaena variabilis.

Cells of the cyanobacterium Anabaena variabilis starved for phosphate for 3 days took up phosphate at about 100 times the rate of unstarved cells. Kinetic data suggested that a new transport system had been induced by starvation for phosphate. The inducible phosphate transport system was quickly repressed by addition of Pi. Phosphate-starved cells were more sensitive to the toxic effects of arsenate than were unstarved cells, but phosphate could alleviate some of the toxicity. Arsenate was a noncompetitive inhibitor of phosphate transport; however, the apparent Ki values were high, particularly for phosphate-replete cells. Preincubation of phosphate-starved cells with arsenate caused subsequent inhibition of phosphate transport, suggesting that intracellular arsenate inhibited phosphate transport. This effect was not seen in phosphate-replete cells.     

Uptake kinetics of arsenic species in rice plants

Arsenic (As) finds its way into soils used for rice (Oryza sativa) cultivation through polluted irrigation water, and through historic contamination with As-based pesticides. As is known to be present as a number of chemical species in such soils, so we wished to investigate how these species were accumulated by rice. As species found in soil solution from a greenhouse experiment where rice was irrigated with arsenate contaminated water were arsenite, arsenate, dimethylarsinic acid, and monomethylarsonic acid. The short-term uptake kinetics for these four As species were determined in 7-d-old excised rice roots. High-affinity uptake (0-0.0532 mM) for arsenite and arsenate with eight rice varieties, covering two growing seasons, rice var. Boro (dry season) and rice var. Aman (wet season), showed that uptake of both arsenite and arsenate by Boro varieties was less than that of Aman varieties. Arsenite uptake was active, and was taken up at approximately the same rate as arsenate. Greater uptake of arsenite, compared with arsenate, was found at higher substrate concentration (low-affinity uptake system). Competitive inhibition of uptake with phosphate showed that arsenite and arsenate were taken up by different uptake systems because arsenate uptake was strongly suppressed in the presence of phosphate, whereas arsenite transport was not affected by phosphate. At a slow rate, there was a hyperbolic uptake of monomethylarsonic acid, and limited uptake of dimethylarsinic acid.     

Interaction of arsenate with phosphate-transport systems in wild- type and mutant Streptococcus faecalis

Harold, F. M. (National Jewish Hospital, Denver, Colo.), and J. R. Baarda. Interaction of arsenate with phosphate-transport systems in wild-type and mutant Streptococcus faecalis. J. Bacteriol. 91:2257-2262. 1966.-Arsenate competitively inhibits the growth of Streptococcus faecalis, primarily by competition with phosphate for a common transport system. Arsenate is itself accumulated by the cells; the uptake requires metabolic energy, and the intracellular arsenate level may reach 0.01 m. Cells loaded with arsenate have lost the capacity to take up radioactive glutamate, rubidium, phosphate, or arsenate itself, apparently by the uncoupling of adenosine triphosphate generation. The pH dependence of arsenate uptake is complex. At low concentrations of extracellular arsenate, uptake by the wild-type strain 9790 exhibits a single maximum about pH 8; mutant PT-1, previously shown to be defective in phosphate uptake, takes up essentially no arsenate. At high concentrations of arsenate, uptake by the wild type is bimodal with maxima at pH 5.5 and 9; the uptake curve for mutant PT-1 corresponds to the shoulder in the curve for the wild type. The apparent dissociation constant for arsenate uptake by the wild type is approximately 10(-5)m from pH 5 to 9, whereas that for mutant PT-1 is about 5 x 10(-5) M at pH 5 and rises rapidly with increasing pH. The results confirm the earlier conclusion that the lesion in mutant PT-1 resides in the transport of phosphate and arsenate. It is proposed that the wild type has two distinct transport systems, whereas the mutant has lost the one with alkaline pH optimum.     

Phosphate transport into brush-border membrane vesicles isolated from rat small intestine.

Uptake of Pi into brush-border membrane vesicles isolated from rat small intestine was investigated by a rapid filtration technique. The following results were obtained. 1. At pH 7.4 in the presence of a NaCl gradient across the membrane (sodium concentration in the medium higher than sodium concentration in the vesicles), phosphate was taken up by a saturable transport system, which was competitively inhibited by arsenate. Phosphate entered the same osmotically reactive space as D-glucose, which indicates that transport into the vesicles rather than binding to the membranes was determined. 2. The amount of phosphate taken up initially was increased about fourfold by lowering the pH from 7.4 to 6.0.3. When Na+ was replaced by K+, Rb+ or Cs+, the initial rate of uptake decreased at pH 7.4 but was not altered at pH 6.0.4. Experiments with different anions (SCN-,Cl-, SO42-) and with ionophores (valinomycin, monactin) showed that at pH 7.4 phosphate transport in the presence of a Na+ gradient is almost independent of the electrical potential across the vesicle membrane, whereas at pH 6.0 phosphate transport involves the transfer of negative charge. It is concluded that intestinal brush-border membranes contain a Na+/phosphate co-transport system, which catalyses under physiological conditions an electroneutral entry of Pi and Na+ into the intestinal epithelial cell. In contrast with the kidney, probably univalent phosphate and one Na+ ion instead of bivalent phosphate and two Na+ ions are transported together.     

Continuous culture of Rhodotorula rubra: kinetics of phosphate-arsenate uptake, inhibition, and phosphate-limited growth

The pink yeast Rhodotorula rubra of marine origin was found to be capable of extended growth at very low phosphate concentrations (K(0.5) = 10.8 nm). Average intracellular phosphate concentrations, based on isotope exchange techniques, were 15 to 200 nm, giving concentration gradients across the cell envelope of about 10(6). Sensitivity to metabolic inhibitors occurred at micromolar concentrations. Inability of the phosphate transport system, K(s) = 0.5 to 2.8 mum, V(max) = 55 mumoles per g of cells per min, to discriminate against arsenate transport led to arsenate toxicity at 1 to 10 nm, whereas environmental arsenate levels are reportedly much higher. Phosphate competitively prevented arsenate toxicity. The K(i) for phosphate inhibition of arsenate uptake was 0.7 to 1.2 mum. Phosphate uptake experiments showed that maximal growth rates could be achieved with approximately 4% of the total phosphate-arsenate transport system. Organisms adapted to a range both of concentration of NaCl and of pH. Maximal affinity for phosphate occurred at pH 4 and at low concentrations of NaCl; however, V(max) for phosphate transport was little affected. Maximal specific growth rates on minimal medium were consistent in batch culture but gradually increased to the much higher rates found with yeast extract media when the population was subjected to long-term continuous culture with gradually increasing dilution rates. Phosphate initial uptake rates that were in agreement with the steady-state flux in continuous culture were obtained by using organisms and medium directly from continuous culture. This procedure resulted in rates about 500 times greater than one in which harvested batch-grown cells were used. Discrepancies between values found and those reported in the literature for other organisms were even larger. Growth could not be sustained below a threshold phosphate concentration of 3.4 nm. Such thresholds are explained in terms of a system where growth rate is set by intracellular nutrient concentrations. Threshold concentrations occur in response to nutrient sinks not related to growth, such as efflux and endogenous metabolism. Equations are presented for evaluation of growth rate-limiting substrate concentrations in the presence of background substrate and for evaluating low inhibitor concentration inhibition mechanisms by substrate prevention of inhibitor flux.     

Arsenic-lipid complex formatinon during the active transport of arsenate in yeast

In studying formation of an arsenic-lipid complex during the active transport of (74)As-arsenate in yeast, it was found that adaptation of yeast to arsenate resulted in cell populations which showed a deficient inflow of arsenate as compared to the nonadapted yeast. Experiments with both types of cells showed a direct correlation between the arsenate taken up and the amount of As-lipid complex formed. (74)As-arsenate was bound exclusively to the phosphoinositide fraction of the cellular lipids. When arsenate transport was inhibited by dinitrophenol and sodium azide, the formation of the As-lipid complex was also inhibited. Phosphate did not interfere with the arsenate transport at a non-inhibitory concentration of external arsenate (10(-9)m). The As-adapted cells but not the unadapted cells were able to take up phosphate when growing in the presence of 10(-2)m arsenate.     

Mechanisms of arsenic hyperaccumulation in Pteris vittata. Uptake kinetics,interactions with phosphate,and arsenic speciation

The mechanisms of arsenic (As) hyperaccumulation in Pteris vittata, the first identified As hyperaccumulator, are unknown. We investigated the interactions of arsenate and phosphate on the uptake and distribution of As and phosphorus (P), and As speciation in P. vittata. In an 18-d hydroponic experiment with varying concentrations of arsenate and phosphate, P. vittata accumulated As in the fronds up to 27,000 mg As kg(-1) dry weight, and the frond As to root As concentration ratio varied between 1.3 and 6.7. Increasing phosphate supply decreased As uptake markedly, with the effect being greater on root As concentration than on shoot As concentration. Increasing arsenate supply decreased the P concentration in the roots, but not in the fronds. Presence of phosphate in the uptake solution decreased arsenate influx markedly, whereas P starvation for 8 d increased the maximum net influx by 2.5-fold. The rate of arsenite uptake was 10% of that for arsenate in the absence of phosphate. Neither P starvation nor the presence of phosphate affected arsenite uptake. Within 8 h, 50% to 78% of the As taken up was distributed to the fronds, with a higher translocation efficiency for arsenite than for arsenate. In fronds, 49% to 94% of the As was extracted with a phosphate buffer (pH 5.6). Speciation analysis using high-performance liquid chromatography-inductively coupled plasma mass spectroscopy showed that >85% of the extracted As was in the form of arsenite, and the remaining mostly as arsenate. We conclude that arsenate is taken up by P. vittata via the phosphate transporters, reduced to arsenite, and sequestered in the fronds primarily as As(III).     

Phosphate transport in arsenate-resistant mutants of Micrococcus lysodeikticus.

Two types of arsenate-resistant mutants of Micrococcus lysodeikticus were found: (i) mutants that grow in the presence of 10 mM but not 1 mM phosphate (Pi) with low uptake rate for Pi and arsenate, and (ii) mutants able to grow in the presence of 10 mM and 1 mM Pi, with a near-normal uptake rate for Pi but a low one for arsenate. The Km values for Pi transport and the Ki values for its competitive inhibition by arsenate were similar for the mutants and the wild type. Similar to the wild type, the mutants also accumulated Pi to high concentrations. In all strains, the transport of Pi was subject to repression by Pi. Mutant types showed lower Vmax but unaltered Km values for arsenate as compared to the wild type, and they accumulated arsenate to markedly lower levels. The results suggest a two-component transport system common to Pi and arsenate.     

Suppression of the High Affinity Phosphate Uptake System: A Mechanism of Arsenate Tolerance in Holcus lanatus L.

In Holcus lanatus L. phosphate and arsenate are taken up bythe same transport system. Short-term uptake kinetics of thehigh affinity arsenate transport system were determined in excisedroots of arsenate-tolerant and non-tolerant genotypes. In tolerantplants the V_max of ion uptake in plants grown in phosphate-freemedia was decreased compared to non-tolerant plants, and theaffinity of the uptake system was lower than in the non-tolerantplants. Both the reduction in V_max and the increase in K_m ledto reduced arsenate influx into tolerant roots. When the twogenotypes were grown in nutrient solution containing high levelsof phosphate, there was little change in the uptake kineticsin tolerant plants. In non-tolerant plants, however, there wasa marked decrease in the V_max to the level of the tolerant plantsbut with little change in the K_m. This suggests that the lowrate of arsenate uptake over a wide range of differing rootphosphate status is due to loss of induction of the synthesisof the arsenate (phosphate) carrier. Key words: Arsenate, Holcus lanatus L., phosphate uptake, tolerance mechanisms, uptake mechanisms     

Effect of arsenate on inorganic phosphate transport in Escherichia coli.

The effect of arsenate on strains dependent on the two major inorganic phosphate (Pi) transport systems in Escherichia coli was examined in cells grown in 1 mM phosphate medium. The development of arsenate-resistant Pi uptake in a strain dependent upon the Pst (phosphate specific transport) system was examined. The growth rate of Pst-dependent cells in arsenate-containing medium was a function of the arsenate-to-Pi ratio. Growth in arsenate-containing medium was not due to detoxification of the arsenate. Kinetic studies revealed that cells grown with a 10-fold excess of arsenate to Pi have almost a twofold increase in capacity (Vmax) for Pi, but maintained the same affinity (Km). Pi accumulation in the Pst-dependent strain was still sensitive to changes in the arsenate-to-Pi ratio, and a Ki (arsenate) for Pi transport of 39 microM arsenate was determined. The Pst-dependent strain did not accumulate radioactive arsenate, and showed only a transient decrease in intracellular adenosine triphosphate levels after arsenate was added to the medium. The Pi transport-dependent strain ceased growth in arsenate-containing media. This strain accumulated 74As-arsenate, and intracellular adenosine triphosphate pools were almost completely depleted after the addition of arsenate to the medium. Arsenate accumulation required a metabolizable energy source and was inhibited by N-ethylmaleimide. Previously accumulated arsenate could exchange with arsenate or Pi in the medium.     

Characterization of essential arginine residues implicated in the renal transport of phosphate and glucose.

We have characterized the reaction of arginine-specific reagents with the phosphate and glucose carriers of the kidney brush-border membrane. The inhibition of phosphate and glucose transport by phenylglyoxal follows pseudo-first-order kinetics. The rate of inactivation of phosphate transport by 50 mM phenylglyoxal was about 3-fold higher than that for glucose transport (kapp was 0.052 s-1 for the uptake of phosphate and 0.019 s-1 for the uptake of glucose). The order of the reaction, n, with respect to phenylglyoxal was 1.25 and 1.31 for the inactivation of phosphate and glucose transport, respectively. The inactivation of phosphate flux by p-hydroxyphenylglyoxal also follows pseudo-first-order kinetics, but the inhibition rate (kapp = 0.0012 s-1) was slower than with phenylglyoxal. The inactivation increased with the alkalinity of the preincubation medium for both phosphate and glucose fluxes and was maximal at pH 9.0. The inactivation of phosphate flux by phenylglyoxal depends upon the presence of an alkaline intravesicular pH. Extravesicular pH does not affect the reaction. Phenylglyoxal does not interfere with the recycling of the protonated carrier since phosphate uptake is inhibited independently of the pH used for transport measurements. Moreover, phenylglyoxal completely abolished trans stimulation by phosphate. Trans sodium inhibited phosphate uptake and abolished the pH profile of phosphate uptake.     

Succinate transport in Bacillus subtilis. Dependence on inorganic anions

Cations were generally ineffective in stimulating succinate transport in a succinate dehydrogenase mutant of Bacillus subtilis unless accompanied by polyvalent anions; phosphate and sulfate being particularly active. The K_m values for the phosphate or sulfate requirement were approx. 3 mM.Biphasic kinetics were characteristic of both the succinate (K_m values 0.1 and 1 mM), and inorganic phosphate (K_m values 0.1 and 3 mM) transport system(s). The phosphate transport system(s) was repressed by high inorganic phosphate and a coordinate increase in the transport of phosphate, arsenate, and phosphate-stimulated succinate transport accompanied growth in low phosphate media.A class of arsenate resistant mutants were simultaneously defective in the transport of arsenate, phosphate and succinate when cells were repressed for phosphate transport, however, the transport of these ions was regained in these mutants when grown in low phosphate media. Organic phosphate esters did not stimulate succinate transport in arsenate resistant mutants but were effective after growth in low phosphate media. Growth under phosphate limitation permitted the simultaneous regain of both phosphate and sulfate dependent succinate transport activities whereas sulfate limitation alone was ineffective.Succinate was not transported by an anion exchange diffusion mechanism since phosphate efflux was low or absent during succinate transport.The transport of C₄-dicarboxylates in B. subtilis is strongly stimulated by intracellular polyvalent anions. The absence of an anion permeability mechanism precludes succinate transport but partial escape from this restriction is mediated by the derepression of a phosphate transport system.     

Arsenate substitutes for phosphate in the human red cell sodium pump and anion exchanger

The sodium pump of human red blood cells mediates a Rb:Rb exchange that is dependent for maximal rates upon the simultaneous presence of intracellular ATP (or ADP) and phosphate. We have measured ouabain-sensitive 86Rb uptake into resealed ghosts of human red cells containing ADP and show that arsenate will substitute for phosphate in supporting the Rb:Rb exchange transport mode. The concentration dependence of arsenate-supported Rb:Rb exchange in ghosts containing 2 mM ADP shows both activating and inhibiting phases; the dependence upon phosphate shows similar characteristics. Elevation of the external [Rb] lowers the apparent affinity for arsenate since there is a shift to higher concentrations of arsenate in the activating and inhibiting phases of the arsenate concentration dependence curve. Similarly, elevation of [ADP] substantially reduces the inhibition of Rb:Rb exchange observed at higher [arsenate]. These effects are also observed in phosphate-supported Rb:Rb exchange. The phosphate requirement for Rb:Rb exchange involves phosphorylation of the sodium pump protein; the close agreement between the effects of arsenate and phosphate in supporting Rb:Rb exchange makes it likely that arsenylation of the sodium pump occurs during Rb:Rb exchange. Arsenate efflux from red blood cell ghosts into arsenate-free chloride medium is partially inhibited (77-80%) by DNDS (4,4'-dinitro-2,2'-stilbenedisulfonic acid), this compares with 82-87% inhibition by DNDS of phosphate efflux under the same conditions. It appears that Band III, the red cell anion transport system, accepts arsenate in a similar fashion to phosphate and that a fraction of the flux of both anions may occur through pathways other than Band III. Thus, in human red blood cells, both the sodium pump and the anion exchange transport system will accept arsenate as a phosphate congener and the protein-arsenate interactions are very similar to those with phosphate.     

Phosphate and glucose accumulation by Pseudomonas cultures in relation to their arsenic resistance

The effect of arsenite and arsenate on 14C-glucose and 32-P-phosphate transport was studied in the cells of Pseudomonas aeruginosa 561 sensitive to arsenite and in the cells of Pseudomonas putida 18 oxidizing arsenite and resistant to arsenic. Transport and accumulation of phosphate and glucose were inhibited in the presence of arsenite in the cells of P. aeruginosa 561 whereas arsenate inhibited only phosphate accumulation. Arsenite and arsenate had hardly any effect at the initial transport rate and on the overall accumulation of phosphate and glucose in the cells of P. putida 18. The resistance to arsenite is supposed to be caused by selective impermeability of the cellular membranes to arsenite and arsenate.     

Evidence for a functional change in the plasma membrane of yeasts associated with the mechanism of arsenate resistance.

In previous reports experimental evidence has been presented indicating a possible relationship between the formation of arseno-phosphoinositides and the active transport of arsenate-phosphate in yeast cells. There is an increment in the amount of inositides in yeast cells adapted to grow in the presence of toxic concentrations of arsenate. These cells exhibit a highly reduced arsenate uptake but maintain their capacity to transport phosphate. Since, in normal (nonadapted) yeast cells, both arsenate and phosphate anions share the same transport system, a study was conducted to obtain further information about the plausible role played by the phosphoinositides in the active transport system of arsenate and their inhibition that allows the cells to grow in the presence of the toxic. Studies on [³²P]orthophosphate and [⁷⁴As]arsenate incorporation into phospholipids in normal and arsenate-adapted yeast show that: The ³²P incorporation into phospholipids is two times larger in normal yeast as compared to arsenateadapted ones. The ³²P labeling was maximum for phosphatidylinositol in normal yeasts while in the arsenate-adapted cells it was maximum for phosphatidylcholine. This incorporation was largely inhibited by arsenate in normal yeasts and minimal in the arsenate-adapted ones. Cell fractionation shows that the maximum incorporation of [³²P]orthophosphate resides in the microsomal fraction, while the incorporation of [⁷⁴As]arsenate resides mainly in the cell envelope fraction which incorporates 86% of the ⁷⁴As label. Phosphate is capable of inhibiting the ⁷⁴As-inositide complex formation and destroying the previously formed one. Yeast cells prelabeled with [2C-³H]myoinositol showed a reduced turnover rate of phosphoinositides even when transporting nontoxic amounts of arsenate. The involvement of the inositides as a regulatory mechanism in the phosphate-arsenate active transport system in yeast cells is discussed.     

Uptake kinetics of different arsenic species by Brassica carinata

The time- and concentration-dependent uptake kinetics for arsenate and arsenite were determined in 15-day-old excised roots. In both cases, arsenite showed a mono-phasic influx with the isotherm data fitting a linear model better than a non-linear one. The time- and the concentration-dependent uptake of arsenate displayed a hyperbolic kinetic. Greater uptake of arsenate, compared with arsenite, was found especially at lower external substrate concentrations. Competitive inhibition of uptake with phosphate showed that arsenite and arsenate were taken up by different uptake systems because arsenate uptake was strongly inhibited in the presence of phosphate, whereas arsenite uptake was not affected.     

Arsenate transport and reduction in the hyper-tolerant fungus Aspergillus sp. P37

Aspergillus sp. P37 is able to grow at arsenate concentrations of 0.2 M--more than 20-fold higher than that withstood by reference microorganisms such Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans. This paper examines the transport of arsenate and phosphate and the reduction of arsenate in Aspergillus sp. P37. These properties were compared with the corresponding properties of the archetype strain Aspergillus nidulans TS1. Both uptake and efflux of arsenate were inhibited by carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, suggesting that the transport system(s) is(are) membrane-potential dependent. As uptake of arsenate and phosphate are higher in Aspergillus sp. P37 than in A. nidulans, the increase in arsenate resistance cannot be accounted for by a change in uptake. Cells of both strains loaded with arsenic slowly released the oxyanion. Speciation of the arsenic in the medium showed an enhanced level of arsenate reduction in Aspergillus sp. P37. These data suggest that increased arsenate reduction is at least in part responsible for the hyper-tolerant phenotype of this fungus.     

Relationship between phospholipid biosynthesis and the efficiency of the arsenate transport system in yeasts

In studying the possibility that phosphoinositides which formed complexes with arsenic were involved in the arsenate transport system of yeasts, a comparative study of the phospholipid composition and metabolism was carried out both in Saccharomyces carslbergensis and in its arsenate-adapted variant, which showed a deficient inflow of arsenate. It was found that the lipid composition of the two organisms was quite similar, the main classes of phospholipids being phosphatidylcholine, phosphatidylethanolamine, and phosphoinositides. The only difference was a 1.5- to 2-fold increase in the proportion of inositides in the arsenate-adapted cells. When the transport of arsenate became inactivated in the nonadapted yeasts after a 30- to 60-min exposure to 10(-2)m arsenate, an increment of inositides of 29 to 50% over the original level was also detected. A study of the incorporation of radioactivity from uniformly labeled (14)C-maltose and from (32)P-orthophosphate ((32)P(i)) demonstrated a decreased rate of lipid biosynthesis in the arsenate-adapted cells as compared to the normal nonadapted ones. The turnover of the phosphate in phospholipids demonstrated no turnover in phosphatidylcholine and phosphatidylethanolamine, and a slow turnover in phosphoinositides. It could be inferred that a normal rate of phospholipid (phosphoinositides) biosynthesis is necessary to have a normal arsenate uptake and that inositide accumulation impairs both the mechanism responsible for the uptake and accumulation of arsenate and the rate of lipid biosynthesis. No differences were found in the deoxyribonucleic acid or protein content of the two types of cells. Also, the arsenate-adapted cells, once freed of external arsenate, showed an increased uptake of (32)P(i) from low external concentrations of phosphate (10(-6) to 10(-8)m, 10-fold over that observed in AsS cells). These results are indicative of independent behavior in phosphate and arsenate transport systems.     

Phosphate transport in fertilized sea urchin eggs. I. Kinetic aspects

Properties of the fully developed phosphate transport system in the fertilized egg of the sea urchin, Strongylocentrotus purpuratus, were investigated. The rates of phosphate transport at concentrations of external phosphate of 1 to 44 μM, both in the absence and in the presence of 100 μM arsenate, exhibit typical saturation kinetics. At sea water concentrations of 2 μM phosphate, the rate of uptake is about 2 × 10^?9 μm/egg/minute at 15°C. Arsenate is a competitive inhibitor of phosphate transport, fully and immediately reversible in its effects, yielding K_i values ranging from 10.5 to 14.1 × 10^?6 M in comparison to the corresponding apparent K_M (Michaelis-Menten) constants for phosphate of 5.6 to 7.5 × 10^?6 M (pH 8.0, 15°C). The rate of arsenate uptake in a phosphate deficient medium amounts to 2.8 to 2.9 × 10^?10 μm arsenate/egg/minute at an arsenate concentration of 2.9 to 10.2 μM arsenate (HAsO₄^??), which is 9.5 and 5.6% of the rate of phosphate uptake at corresponding phosphate concentrations. Arsenate has essentially the same developmental effects at initial concentrations of 5–10 μM and 100 μM arsenate, namely no observable effects for exposure periods of 7.5 hours, although longer periods result in blockage of development at the early blastula stage. Outward flux of phosphate ions cannot be demonstrated by washing prelabelled eggs with sea water containing low or high concentrations of phosphate, even when phosphorylation has been blocked by exposing the eggs to a metabolic inhibitor. Phosphate uptake rates measured in the pH range from 5.0 to 10.0 reveal a sharp optimum at pH 8.8–8.9. Reference to the apparent pK' values of the phosphoric acid system indicate that the entering species is the HPO₄^?? ion. The effects on rates of phosphate uptake of exposure to sea water at pH values between 7 and 10 for 30 minute periods are fully reversible, but at lower pH values, reversal is delayed, and is only partial. Sodium molybdate (0.01 M), sodium pyrophosphate (1.5 × 10^?4 M), and adenosine triphosphate (1–5 × 10^?4 M) for exposure periods ranging from 40 to 180 minutes did not significantly affect phosphate uptake. Omission of Ca⁺⁺ ion from artificial sea water is without effect on phosphate uptake but the absence of both Ca⁺⁺ and Mg⁺⁺ results in profound and irreversible depression of both phosphate uptake and development. The data of this and the following paper are consistent with the conclusion that the transport of phosphate involves a surface located carrier. The apparent secondary and tertiary ionization constants of phosphoric acid in sea water (ionic strength = 0.6885) were measured, resulting in a value for pK′₂ = 6.14 and for pK′₃ = 10.99, at 15°C and phosphate at infinite dilution.