BENTLEY GLASS 857 



quired. However, not only ATP, l)ui also ADl' and AMP will supj^ly 

 this requirement, although no other nucleotides will serve. The sys- 

 tem was found to contain a phosphatase which will convert ATP 

 to ADP, and ADP to AMP; but there is no phosphorylating mechanism 

 for doing the reverse. In short, ATP here serves as a generating sys- 

 tem for ADP and AMP, and the kinetics show that the response to 

 ATP is much slower than that to the diphosphate and monophosphate, 

 and has a lag period. ATP, but not ADP or AMP, requires an ac- 

 ti\ating metal ion (Mg or MN) in this system. With a purified en- 

 zyme preparation loss of phosphatase activity is accompanied by a loss 

 of ability to utilize ATP in the production of light. ADP and AMP 

 are not affected by this loss of phosphatase activity. 



The Renilla luminescent system differs from others in still another 

 way. Normal oxygen tension will not saturate the system; flushing 

 with oxygen causes an increase in the intensity of the light. Increas- 

 ing concentration of luciferin leads to saturation; increasing enzyme 

 concentration led to a directly proportional increase in light intensity. 

 The pW optimum is at 7.5; the temperature optimum is at 32°C. 

 Strong inhibitors of bacterial luminescence, such as cytochrome c, 

 do not inhibit Renilla luciferin. It is not oxidized like the flavin 

 substrate of the bacterial luminous system. It is not inhibited by 

 fluoride (except when ATP is utilized) , by cyanide, or by azide, by 

 arsenate, or by pyrophosphate. Unlike the firefly luminescence, that 

 of Renilla is not affected by pyrophosphate or by inorganic pyro- 

 phosphatase. This is further evidence of the subsidiary role of ATP 

 in the Renilla system. A requirement for sulfhydryl groups is indi- 

 cated by the inhibitory effect on luminescence of sulfhydryl inhibitors. 

 Metal ions, (except for zinc, which is a potent inhibitor) have no 

 effect, but a chelating agent such as Versene proves to be a strong 

 inhibitor of luminescence, and the inhibition cannot be reversed by 

 adding metal ions, such as Mg+ + . The Versene must have a different 

 type of action here. Unlike the Gonyaiilax system, there is no re- 

 quirement for salt in the luminescent system. On the contrary, sodium 

 and potassium chlorides inhibit luminescence linearly with increasing 

 salt concentration. 



Evidence for the formation of an intermediate is foimd in the 

 kinetics of a system preincubated wdth enzyme before oxygen is ad- 

 mitted. The steady-state value of the light intensity is doubled by 

 doubling of the anaerobic preincubation time. When such a preincu- 

 bated system is heated to 100°C for li/^ minutes, then cooled, and 

 fresh enzyme added, the presence of a heat-stable intermediate is 



