Metabolism of the frog outer segments: a kinetic study

1. The reaction of the high-energy phosphate esters of the frog isolated rods, with the firefly lantern extract, has been studied by recording the luminescence in a stopped-flow apparatus. The onset of the reaction was determined by the rapid mixing of the firefly lantern extract with the high-energy phosphates released from the rods fragmented during the mixing. 2. The time course of the reaction, i.e. of the luminescence, was not typical of adenosine-5′-triphosphate (ATP), indicating that the major part of the rod nucleotides is not ATP. 3. The isolated rods were fragmented 1 sec-2 min after a flash of light. As soon as 1 sec after illumination, a substantial decrease of the luminescent yield has been detected in a range of light bleaching from 0·007 to 20% of the rhodopsin, indicating an early reduction of the high-energy phosphate esters. 4. At longer times, flashes that bleached 7 or 20% of the rhodopsin induced a progressive decrease of the luminescent yield that was half completed in 6-9 sec, and was nearly complete in 20 sec, whereas a more or less complete recovery was observed after flashes bleaching from 0·7 to 0·007% of the rhodopsin, indicating the presence of buffering mechanisms. 5. Also, the time course of the reaction was modified in a complex way after stimulation of the rods, suggesting that other nucleotides beside ATP are hydrolysed. 6. The light-induced reduction of the high-energy phosphates has been observed also in broken rods, in the presence of the calcium chelating agent EGTA, suggesting that calcium is not needed for this effect. 7. Cyclic guanosine monophosphate (10-4 M) interfered with the photic effect. 8. The rapid effect of light on the content of high-energy phosphate esters suggests a physiological role in the mechanism of excitation.