Characterization and properties of Pholas luciferase as a metalloglycoprotein.
The luciferase of the bioluminescent boring mollusc, Pholas dactylus, has been purified by a new method which includes centrifugation in cesium chloride gradients. Homogeneous preparations have been obtained and molecular weight determinations and subunit analysis support the idea that this preparation is an oxyluciferin-luciferase complex. The preparation catalyzes oxidation of ascorbic acid in presence of H2O2, and this peroxidase activitity has been used for characterization (thermal and pH stabilities, activity as a function of pH, isoelectric point, turnover number). The existence of two atoms of copper has been established and their involvement in the peroxidase activity indicated. Chemical analyses have shown that Pholad luciferase is a glycoprotein and the existence of glucosamine, fucose, mannose, and galactose residues has been demonstrated. The apparent buoyant dentisty (1.340), the sedimentation coefficient (10.7 S), the Stoke's radius (83 A), the partial specific volum (0.707), and the molecular weight (350,000) have been determined. The frictional ratio (flf0 = 1.8) derived from the Stoke's radius indicates that the molecule is asymmetric. The quaternary structure has been examined. Subunits of molecular weight 150,000 and 46,000 have been observed. The latter has electrophoretic properties identical with luciferin or oxyluciferin.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002614:Chelating Agents / Q000494:pharmacology; D003300:Copper / Q000032:analysis; D004050:Ditiocarb / Q000494:pharmacology; D004355:Drug Stability; D004578:Electron Spin Resonance Spectroscopy; D005643:Fucose / Q000032:analysis; D005690:Galactose / Q000032:analysis; D005944:Glucosamine / Q000032:analysis; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008156:Luciferases / Q000302:isolation & purification / Q000378:metabolism; D008358:Mannose / Q000032:analysis; D008667:Metalloproteins / Q000302:isolation & purification / Q000378:metabolism; D008974:Mollusca / Q000201:enzymology*; D011487:Protein Conformation
D000596:Amino Acids; D002614:Chelating Agents; D006023:Glycoproteins; D008667:Metalloproteins; D005643:Fucose; D003300:Copper; D004050:Ditiocarb; D008156:Luciferases; D005944:Glucosamine; D008358:Mannose; D005690:Galactose