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In some luminous organisms (firefly, Cypridina, and bacteria) it is possible to obtain cell-free luminescence by the combination of a cold water extract (luciferase) with a hot water extract (luciferin). Failure in the past to observe such a reaction with extracts of luminous fungi can now be shown to be due to the following factors: (a) in crude aqueous extracts the luciferase is either too dilute or is inhibited. An active crude luciferase can be prepared from extracts by ammonium sulfate precipitation; (b) the substance in the hot water extract is very labile; (c) finally, the presence of reduced pyridine nucleotides is essential for light emission. The following experimental evidence is presented to support these conclusions. The mycelial mates from Collybia velutipes or Amillaria mellea were dried over P205 under reduced pressure. An acetone powder was made from the dried mats. Boiling 0.05 M potassium phosphate buffer (pH 7.0) containing 10-3 M ethylenediaminetetracetate (Versene) was used to extract the acetone powder. This mixture was heated at 100 C for 1 min and immediately cooled. The supernatant fluid obtained after centrifuging is designated according to Dubois' criteria as luciferin. The cold water fraction was prepared by grinding the acetone powder in a cold mortar with sand and buffer. The suspension was centrifuged and the crude luciferase was precipitated from the supernatant fluid by adding solid ammonium sulfate to 75 per cent saturation. The resulting precipitate was dissolved in a small volume of buffer. The results in figure 1 indicate that reduced diphosphopyridine nucleotide along with luciferase and luciferin is required for significant light emission. Reduced triphosphopyridine nucleotide will also stimulate light emission. Freshly

LIGHT EMISSION FROM EXTRACTS OF LUMINOUS FUNGI