1993). The luminescence intensity is a relative measure, calibrated by determining the absolute amount of enzyme present, for example, the active site concentration, which yields the specific luminescence intensity. This parameter is a function of two processes, the rate of catalysis and the luminescence yield (as V. mol product formed s?1). The latter appears to correlate with CO2/O2 specificity of different Rubiscos (Tcherkez et?al. 2006). Determination of the rate Oxygenase
of catalysis by dinoflagellate Rubisco is likely to require at least partial purification of the enzyme: Cox, Lilley & Andrews (1999) used highly purified enzyme to determine these parameters for R. rubrum Rubisco. The time course of luminescence intensity in assay mixtures containing crude Symbiodinium extracts resembles that of Form learn more
II Rubisco purified from R. rubrum (Lilley et?al. 1993) in that there is an initial fast peak of intensity followed by a decline to a lower stable level. However, for Symbiodinium extracts, the peak intensity is relatively lower and the time to reach it (about 10?s, Fig.?1 and 2a) is much slower than for R. rubrum (<0.25?s). These differences require further examination, but are likely to provide clues about the nature of the dinoflagellate enzyme. The later, more stable emission by Symbiodinium extracts declined in a linear fashion during the 80�C200?s time frame of the assay at rates between 7 and 14% per min (Figs?1, 3 & 5a). Temperature control in assays is important since the luminescence intensity is strongly affected <a href="http://www.selleckchem.com/products/BKM-120.html
">BKM120 by assay temperature (data not shown). The luminescence yield of R. rubrum Rubisco Mn2+-chemiluminescence varies with temperature within the range of 20�C40?��C (Cox et?al. 1999), in contrast to that from Form I (L8S8) enzymes. The catalytic rate and luminescence yield remain to be determined for Symbiodinium, but there is likely to also be a large effect of temperature on the yield. Of the injected reagents, RuBP was pre-equilibrated to room temperature (20�C25?��C), but the added cell extract was at 2?��C. However, its volume (1.5% of the final assay volume) was sufficiently small that any disturbance made to the temperature on addition to the assay vial was negated by the 80?s time before the start of the intensity measurement period. The design of this luminometer, with all sample vials held within a thermostatted aluminium carousel, minimizes temperature fluctuations within the sample vials. The addition of cell extract in assay mixtures sometimes resulted in a short-lived burst of chemiluminescence (Fig.?5a, extract from cells pre-treated at 37?��C). This chemiluminescence was not RuBP dependent and occurred with some cell extracts but not others. It usually decayed in the 20?s before RuBP addition, but occasionally persisted for longer. However, it always decayed entirely within 50?s after RuBP addition, thus having no effect on the subsequent activity measurement, which commenced at 80?s.