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

Abstract

AbstractProperties 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 Ki values ranging from 10.5 to 14.1 × 10−6 M in comparison to the corresponding apparent KM (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.