I. The function of aldehyde in the bacterial luminescent reaction was investigated using {1-('14)C} decanol in a coupled enzyme system. The substrates for the bioluminescent reaction, {1-('14)C} decanal and FMNH(,2), were generated in the reaction mixture by horse liver dehydrogenase and bacterial FMN reductase. Analysis of the reaction products indicated that aldehyde is exclusively convertted to the corresponding acid by bacterial luciferase. A linear relationship between light emission and acid production was obtained with a resulting quantum yield of 0.10 quanta per molecule of acid produced. / II. In order to determine if the lag period in luminescence observed during early exponential growth of Beneckea harveyi reflects the repression of synthesis of one or both subunits of luciferase at the level of gene transcription/translation or the synthesis of one or both polypeptide(s) in the form of an inactive precursor, a double labelling experiment was performed such that, the proteins synthesized during the luminescence lag and inductions periods were labelled with ('3)H-leucine and ('14)C-leucine respectively. Analysis of the radioactivity incorporated into the (alpha) and (beta) subunits of luciferase indicates that both subunits are coordinately controlled and that the induction of luminescence is reflected by preferential de-novo synthesis of luciferase during the induction period. Analysis of the soluble protein indicates that five additional polypeptides are co-induced with luciferase and that one polypeptide appears to be repressed during the induction period. Analysis of the radioactivity incorporated into NAD(P)H:flavin oxidoreductase (the major NADH dependent FMN reductase and a supplier of FMNH(,2) for the luminescent reaction) in Beneckea harveyi indicates that its synthesis is constitutive. / III. NAD(P)H:flavin oxidoreductase has been purified with the aid of affinity chromatography of (epsilon)-amino hexanoyl-FMN-Sepharose. The enzyme is composed of a single polypeptide chain of 24,000 daltons. Kinetic studies indicate that the higher specificity of the enzyme for NADH, riboflavin and FMN as opposed to NADPH and FAD is due primarily to variations in the Michaelis constants for the different substrates. Initial velocity studies with all pairs of substrates gave intersecting patterns supporting a sequential mechanism for the NAD(P)H:flavin oxidoreductase.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.77110 |
| Date | January 1981 |
| Creators | Michaliszyn, George A. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biochemistry) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 000137439, proquestno: AAINK54862, Theses scanned by UMI/ProQuest. |
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