. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. PER1PHYLLA LUCIFERASE STORED IN OVARY 343 100 '<:• 80 70 60 * 50- o. 24 26 28 30 Elution volume (ml) Figure 2. Molecular weight estimation of luciferases A. B, C, and L. The gel filtration was carried out on a column of Superdex 200 Prep (1 X cm), in 20 mM acetate buffer (pH ), containing 1 M KCI and lauroylcholine chloride. Calibration standards: aldolase (1). BSA (2), ovalbumin (3). carbonic anhydrase (4). myoglobin (5). ribonuclease A (6). SDS-PAGE (polyaerylamide gel electrophoresis) analy- sis of lucife
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. PER1PHYLLA LUCIFERASE STORED IN OVARY 343 100 '<:• 80 70 60 * 50- o. 24 26 28 30 Elution volume (ml) Figure 2. Molecular weight estimation of luciferases A. B, C, and L. The gel filtration was carried out on a column of Superdex 200 Prep (1 X cm), in 20 mM acetate buffer (pH ), containing 1 M KCI and lauroylcholine chloride. Calibration standards: aldolase (1). BSA (2), ovalbumin (3). carbonic anhydrase (4). myoglobin (5). ribonuclease A (6). SDS-PAGE (polyaerylamide gel electrophoresis) analy- sis of luciferases A, B, and C under reducing condition (with 2-mercaptoethanol) showed only one major band cor- responding to a molecular mass of 24 kDa (Fig. 3); thus, the purity of these proteins and the oligomeric nature of lucif- erases B and C are verified. Although the molecular mass of the luciferase monomer obtained by gel filtration (19 kDa) does not match well with that obtained by SDS-PAGE (24 kDa), we chose to use the value of 20 kDa for luciferase A (and 40 kDa and 80 kDa for luciferases B and C) as a reasonable approximation, pending the determination of its precise value in the future. We also note here that luciferase L (32 kDa) also yielded luciferase A upon treatment with 2-mercaptoethanol (Fig. 3; see Discussion). The spectral properties of luciferase A, B, and C were practically identical with those of luciferase L; their absorp- tion and fluorescence spectra indicate that the luciferases are simple proteins, without any chromophore that absorbs or fluoresces in the visible region. En-\iuutic properties The enzymatic properties of luciferases A. B, and C are generally similar to those of luciferase L previously reported (Shimomura and Flood. 1998), but with some notable differ- ences. Thus, the luminescence intensity of luciferase A was highest at 27 °C, and those of luciferases B and C at 30 °C, whereas the luminescence intensity of luciferase L showed no maximum,
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