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THIRTEEN FIGURESThis study was undertaken in effort to analyze in part, some of the factors controlling bacterial growth rates, and the action of quinine as an inhibitor of the over-all process. P a rticular emphasis has been placed on temperature, because of the theory and relationships discussed in the preceding papers (Johnson and Lewin, '45; '46a, b) and the literature referred to therein. The effects of coenzyme have been included inasmuch as the addition of coenzyme may greatly influence not only the activity of certain dehydrogenase systems, particularly in glucose dehydrogenation (Yudkin, '33, '34) but also the inhibitory action of quinine on these systems (Johnson and Lewin, '46a). METHODS

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SEVEN FIGURESAccording to evidence recently obtained with luminous bacteria, a thermodynamic equilibrium between catalytically active and inactive forms of certain enzymes appears to exist normally within the living cell. This equilibrium has temperature and entropy characteristics resembling a protein denaturation. It is apparently a determining factor with regard to the optimum temperature for luminescence in a given species. I n addition, it is sensitive to hydrostatic pressure and apparently also to certain narcotics. The quantitative data and theoretical considerations which support this hypothesis have been discussed at sonie length in previous papers (Johnson, Brown and Marsland, '42, a, b ; Brown, Johnson and Marsland, '42; Eyring and Magee, '42; Johnson and Chase, '42).The purpose of the present study has been to extend the data and theory concerning the temperature relations of bacterial luminescence intensity in order to elucidate further the significance of the phenomena observed in both normal and inhibitor-containing cell suspensions. Particular reference has been made to the action of sulfanilamide, whose inhibition of luminescenee may be reversed to a large extent by increase in temperature. The action of urethane also has been investigated further, since its inhibition is partially or wholly reversible with increase in hydrostatic pressure. In this effect, the urethane inhibition of luminescence resembles that due to heat alone, which is also reversible, in part, by pressure. The action of all these inhibitory agents is related to the optimum temperature of luminescence in the specific organism concerned. The results of the present study make it possible to evaluate of it given process, and to designate more precisely the action of added more clearly biological differences in the velocity-temperature re1 a t' lolls 1 These studies have been aided, in part, by n graut to one of us (3'. H. J.) from the Penrose Fund of the American Philosophical Society.

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The bioluminescent reaction of Oplophorus takes place when the oxidation of coelenterazine (the luciferin) with molecular oxygen is catalyzed by Oplophorus luciferase, resulting in light of maximum intensity at 462 nm and the products CO2 and coelenteramide. Oplophorus luciferase has now been obtained in a highly purified state. Optimum luminescence occurs at pH 9 in the presence of 0.05--0.1 M NaCl at 40 degrees C, and, due to the unusual resistance of this enzyme to heat, visible luminescence occurs at temperatures above 70 degrees C when partially purified enzyme is used. The specific activity of purest preparations is 1.75 X 10(15) photons s-1 mg-1 at 23 degrees C. At pH 8.7, native luciferase has a molecular weight of approximately 130 000, apparently comprising 4 monomers of 31 000; at lower pHs, the native luciferase tends to polymerize. The quantum yield of coelenterazine is 0.34 at 22 degrees C with this enzyme. After the luminescent reaction, the spent solution is nonfluorescent, and likewise solutions of luciferase alone. When the bioluminescent reaction was carried out in the presence of 18O2, the product CO2 contained more than 50% C18O16O, supporting the dioxetane mechanism, but without ruling out the linear peroxide mechanism.