A STUDY OF THE MECHANISM OF PHOSPHATE TRANSPORT IN SEA URCHIN EGGS BY ION EXCHANGE ANALYSIS OF RAPIDLY LABELED COMPOUNDS

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Abstract
1. The mechanism involved in the transport of phosphate into fertilized sea urchin eggs has been studied by exposing eggs to pulses of P32 and examining the radioactive compounds formed. These have been identified in part by ion exchange and paper chromatography in control eggs and in the presence of metabolic inhibitors. 2. Control eggs of Strongylocentrotus purpuratus at the two-cell stage incorporated 6.4% to 39.1% of the P32 from sea water in 10 seconds to 10 minutes; most of this (98.4% for a one-minute pulse, and 94.5% for a 10-minute pulse) was in perchloric acid-soluble compounds. 3. The distribution of the label after one-minute and 10-minute pulses was similar. In a representative one-minute experiment 27.0% was found in arginine phosphate; 7.7% in inorganic phosphate; 0.8% in glucose-6-phosphate; 2.0% in ADP; 53.7% in ATP; and 5.7% was distributed among 5 unidentified fractions. Glucose-1-phosphate, while present in the extract, was without significant label. 4. In experiments in which one-minute and 10-minute pulses of p32 were given to eggs in the presence of 5 x 10-3 M sodium azide, or 10-4 M potassium cyanide, the incorporation of label was as much or more than in the controls, but its distribution was greatly altered, with similar changes for both azide and cyanide. In a one-minute pulse in the presence of azide, incorporation into arginine phosphate was reduced to 1.2% and into ATP to 14.3% while inorganic phosphate increased to 76.9%: a decrease of 65.2% in high energy phosphates and an increase of 69.2% in inorganic phosphate. Minor changes occurred in other fractions. Similar but much less marked changes were produced by 0.1 M monoiodoacetate. 5. A modified procedure was used with very short P32 pulses of 10 and 30 seconds, in an attempt to determine the initially labeled compound. The shorter the pulse, the greater was the percentage incorporation into inorganic phosphate, with parallel decreases in arginine-phosphate and ATP. 6. The results are considered to be incompatible with any mechanism for transport involving the initial formation of high energy phosphate; or with transport as the result of surface phosphorolysis of glycogen to form glucose-1-phosphate; or with transport resulting from glycolytic esterifications. The results are compatible with a carrier type of transport which would release inorganic phosphate unchanged into the metabolic pool in the eggs.