Studies on the Bioluminescence of Renilla reniformis*

In earlier investigations on the bioluminescent system of the sea pansy, Renilla renifformis, we reported that adenine-containing nucleotides (either adenosine mono-, di-, or triphosphate), an oxidizable substrate (Renilla luciferin), molecular oxygen, and an enzyme (RenilZa luciferase) were required for luminescence in crude extracts of this organism (1, 2). In addition, several lines of evidence indicate that adenosine triphosphate functions indirectly, i.e. by first being converted to the functional nucleotide (2). These are: (a) kinetics of the reaction with ATP is much slower than that with AMP or ADP; (b) relatively crude luciferase preparations respond to AMP, ADP, and ATP, whereas more purified fractions will emit light in the presence of AMP or ADP but not ATP. Crude preparations contain a phosphatase that converts ATP to ADP and AMP and, during purification of the enzyme, the phosphatase activity is removed; and (c) ATP activity in purified extracts can be restored by the addition of commercial alkaline phosphatase plus magnesium. These data were interpreted to mean that ATP was being converted to AMP and ADP and that one of the latter two nucleotides was responsible for driving the light reaction. This seemed to be a reasonable conclusion in view of the fact that rechromatography of ADP on formate-charged Dowex 1 yielded ADP fractions that were all active, and that the nucleoside mono-, di-, and triphosphates of guanosine, cytidine, uridine, and inosine were found to be inactive (2). The data presented in this paper are intended to show, however, that the activity exhibited by AMP and ADP is due to contamination by trace amounts of an adenine-containing nucleotide that has been isolated from crystalline AMP preparations and found to be identical with 3’ ,5’-diphosphoadenosine. On the other hand, it will be shown that the ATP preparations that we are using are not contaminated by 3’) 5’-diphosphoadenosine and thus the activity of ATP in crude extracts can be explained in terms of synthesis of DPAl from ATP. It will be shown that the luminescent reaction represents the most sensitive assay known for DPA, since concentrations of 5 x 10mg M are easily detected. A discussion of the possible relationship between the involvement of DPA in the Renilh bioluminescent system and its role in the transfer of active sulfate between phenolic compounds, as shown by Gregory and Lipmann (3)) is presented.