An investigation of the reaction kinetics of luciferase and the effect of ionizing radiation on the reaction rate.

The bioluminescence produced by luciferase, a firefly enzyme, requires three substrates: luciferin, ATP and oxygen. We find that ionizing radiation, in the form of a proton beam from a cyclotron, will eliminate dissolved oxygen prior to any damage to other substrates or to the protein. The dose constant for removal of oxygen is 70 +/- 20 Gy, a much smaller dose than required to cause damage to protein. Removal of oxygen, which is initially in excess, leads to a sigmoidal response of bioluminescence to radiation dose, consistent with a Michaelis-Menten relationship to substrate concentration. When excess oxygen is exhausted, the response becomes exponential. Following the irradiation, bioluminescence recovers due to a slow leak of oxygen into the solution. This may also explain previous observations on the response of bioluminescent bacteria to radiation. We have studied the dependence of the reaction rate on enzyme and substrate concentration and propose a model for the reaction pathway consistent with this data. The light output from unirradiated samples decreases significantly with time due to product inhibition. We observe that this inhibition rate changes dramatically immediately after a sample is exposed to the beam. This sudden change of the inhibition rate is unexplained but shows that enzyme regulatory function responds to ionizing radiation at a dose level less than 0.6 Gy.

Choanoflagellate lorica construction and assembly: the nudiform condition. II. Acanthoeca spectabilis Ellis.

Acanthoeca spectabilis is one of the most common loricate choanoflagellates found in marine biofilms everywhere. However, it is special for two reasons; firstly, it is probably the most distinctive member of the small nudiform clade of loricate choanoflagellates. Secondly, the lorica chamber of Acanthoeca comprises a closely wound left-handed coil of costae that is unique amongst choanoflagellates. Mathematical analysis of the lorica chamber shows that the helical costae undergo two turns. This species, more than any, demonstrates that the helical coiling of costae can only be achieved by a rotational movement generated by the cell during lorica assembly. Comparison of the lorica morphology of Acanthoeca with that of the closely related genus Polyoeca indicates that the helical costae of Acanthoeca are probably homologous with the outer longitudinal costae of Polyoeca. This is unusual because helical costae are usually the innermost layer of costae. However, since there is no 'true' inner layer in the chamber of Acanthoeca the outer layer of costae are adjacent to the cell surface and therefore available for coiling. In contrast to tectiform choanoflagellates, which number more than one hundred species and inhabit a wide variety of microniches, the six known extant nudiform species must either represent a minor evolutionary development or be the remnants of a previously more extensive radiation.

The kinetics of radiation damage to the protein luciferase and recovery of enzyme activity after irradiation.

Experimental observations are reported which follow the bioluminescence intensity of luciferase during irradiation by a 5 MeV proton beam. Bioluminescence is a measure of the protein enzyme activity and provides an assay of the enzyme rate of reaction in real time. Transient responses after a pulse of protons show recovery of the reaction rate with two time constants of 0.3 s(-1) and 0.01 s(-1). Changes in the reaction rate are due to radiation damage to the active form of the protein luciferase. Quantitative analysis of the radiation damage and recovery of the protein shows that products of the radiolysis of water play major part in the process of enzyme damage at room temperature. A few minutes after the pulse of protons, most of the enzyme activity has recovered. We attribute the fast recovery to the removal of charged ions, while the slow recovery involves refolding of denatured protein.