| Abstract: | A particle bound tryptophan-5-monooxygenase in bovine pineal glands was solubilized by a combination of high pH (PH 8.5), high salt concentration (0.5 M-KCl or NaCl), and sonication (20 kHz, 200 W, 30min), We recovered 75% or more of the monooxygenase activity of the tissue homogenate in the 105,000 g supernatant thus confirming our previous study (Hori et al., 1976). The solubilized enzyme preparation contained an activating substance which was also particle bound and which activated the monooxygenase when preincubated together with the enzyme and dithiothreitol. The solubilized tryptophan-5-monooxygenase was purified about 6-fold over the tissue homogenate using ammonium sulphate fractionation and column chromatography on hydroxylapatite and Sephacryl S-200. The activating substance was separated from the monooxygenase during hydroxylapatite column chromatography. The activating substance was shown to be different from phospholipids, such as L-α-lysophosphatidyl choline and L-α-phosphatidyl-L-serine, and from heparin or bovine serum albumin, although bovine serum albumin significantly activated the monooxygenase. Further experiments have suggested that both the monooxygenase and the activating substance are modified by dithiothreitol and that these modified materials interact to give the active form of the monooxygenase. |
