Metabolism of the frog outer segments: a kinetic study

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Abstract
The reaction of the high-energy phosphate esters of the frog [Rana catesbiana] isolated rods, with the firefly lantern extract, was 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-enregy phosphates released from the rods fragmented during the mixing. The time course of the reaction, i.e., of the luminescence, was not typical of ATP, indicating that the major part of the rod nucleotides was not ATP. The isolated rods were fragmented 1 s 2-min after a flash of light. As soon as 1 s after illumination, a substantial decrease of the luminescent yield was detected in a range of light bleaching from 0.007-20% of the rhodopsin, indicating an early reduction of the high-energy phosphate esters. 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 s, and was nearly complete in 20 s, 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. The time course of the reaction was modified in a complex way after stimulation of the rods, suggesting that other nucleotides beside ATP were hydrolyzed. The light-induced reduction of the high-energy phosphates was observed in broken rods, in the presence of the calcium chelating agent EGTA [ethylene glycol bis(.beta.-aminoethyl ether)tetraacetate] suggesting that Ca was not needed for this effect. Cyclic GMP (10-4 M) interfered with the photic effect. The rapid effect of light on the content of high-energy phosphate esters suggested a physiological role in the mechanism of excitation.