| Summary: | There are many questions associated with the understanding of the origin and regulation of bioluminescence. A key question underlying this thesis was why coelenterazine evolved as the most common marine luciferin. Another problem addressed was the coelenterazine source of the bioluminescent hydroid Obelia, which is unknown. Coelenterazine is an integral part of Obelia’s photoprotein, triggered by Ca2+. The thesis also investigated the uncertainty over how Ca2+ enters Obelia’s photocytes. Obelia is considered to have four species, but they have been misidentified. Green fluorescent protein (GFP) acts as a fluor in Obelia producing green light. The selective advantage of this green bioluminescence requires confirmation. Results showed that Obelia geniculata cultures rapidly lost their bioluminescence, indicating Obelia requires a dietary supply of coelenterazine. Adding coelenterazine to Obelia briefly restored its bioluminescence. Levels of obelin dropped in Obelia geniculata cultures. Obelia longissima had a lower level of coelenterazine and bioluminescence than Obelia geniculata. Coelenterazine was detected in species living on Obelia and, three non luminous species of copepod. A range of species were identified in zooplankton, as possible coelenterazine sources. Ratios of obelin to apoobelin changed in older colonies. The problem of misidentification of Obelia species was solved by utilising the fluorescent patterns of Obelia’s photocytes. GFP was recorded for the first time in the hydrotheca and tentacles of Obelia dichotoma. Fluorescence maxima for Obelia geniculata and Obelia dichotoma iv were different. GFP in Obelia was found to photobleach far slower than GFP in EGFP. This supports the hypothesis that Obelia has a molecular mechanism which protects its GFP from photobleaching. Light emission from Obelia geniculata was different from Obelia longissima. This suggests that the mode of entry of Ca2+ into the photocytes of these two species is different. To investigate the exact pathway by which initial stimulation of Obelia causes Ca2+ to enter the photocytes, experiments were conducted using K+ channel blockers. The potassium ion channel blockers tetraethyl ammonium chloride and 4-aminopyridine both produced a bioluminescent response in Obelia geniculata. This suggested that the pathway included K+channels. In a polar solvent coelenterazine produced low chemiluminescence, which increased with increasing luciferin concentration. Human albumin and BSA increased this effect. This supported the solvent cage hypothesis that bioluminescent proteins originally evolved as primitive oxygenase enzymes.
|
|---|
