Abstract
Purification of mammalian ATase is essential for the understanding of the regulation of de novo purine synthesis in mammals. The basic properties of rat liver ATase after step 4 of purification methods described below includes a) feedback inhibition by adenosine or guanosine mono-, di- and tri-phosphates, with the decreasing order of magnitude, b) Inhibition by 1, 10-orthophenanthroline as a Fe++ chelator, but not by 1, 7-metaphenanthroline, and c) Inactivation by exposure to oxygen with a half life of 15 min at 37°C with the stable activity under argon. In this study ogygen inactivation of ATase was overcome by the use of oxygen-free buffer and HPLC methods. The purification procedures include the following 8 steps. 1. 55,000xg supernatant of rat liver homogenate, 2. Heat treatment to 56°C, 3. Acid precipitation to pH 5.1, 4. Ammonium sulfate fractionation between 30 and 55%, 5. Gel filtration by Toyopeari HW55F, 6. DEAE-5PW HPLC column, 7. Hydroxyapatite HPLC column, 8. SDS polyacrylamide gel and electroelution. After step 7, ATase was purified to 958-fold with 4.1% recovery. After step 8, ATase was finally purified to homogeneity proved by a single protein band with silver stain. The monomer size of ATase was 62K on SDS polyacrylamide gel. The amount of ATase in rat liver is estimated as about 10 μg/g liver. The methods of purification of mammalian ATase open the research direction to molecular cloning and monoclonal antibody production.
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Yamaoka, T., Kagita, F., Yoshikawa, H. et al. 182 PURIFICATION OF RAT LIVER AMIDOPHOSPHORIBOSYLTRANS FERASE (ATase). Pediatr Res 24, 141 (1988). https://doi.org/10.1203/00006450-198807000-00206
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DOI: https://doi.org/10.1203/00006450-198807000-00206