Characterization of an adenosine 3':5'-cyclic monophosphate phosphodiesterase from baker's yeast. Its binding to subcellular particles, catalytic properties and gel-filtration behaviour.
1. The 3':5'-cyclic AMP phosphodiesterase in the microsomal fraction of baker's yeast is highly specific for cyclic AMP, and not inhibited by cyclic GMP, cyclic IMP or cyclic UMP. Catalytic activity is abolished by 30 micrometer-EDTA. At 30 degrees C and pH8.1, the Km is 0.17 micrometer, and theophylline is a simple competitive inhibitor with Ki 0.7 micrometer. The pH optimum is about 7.8 at 0.25 micrometer-cyclic AMP, so that over the physiological range of pH in yeast the activity changes in the opposite direction to that of adenylate cyclase [PH optimum about 6.2; Londesborough & Nurminen (1972) Acta Chem. Scand. 26, 3396-3398].2. At pH 7.2, dissociation of the enzyme from dilute microsomal suspensions increased with ionic strength and was almost complete at 0.3 M-KCl. MgCl2 caused more dissociation than did KCl or NaCl at the same ionic strength, but at low KCl concentrations binding required small amounts of free bivalent metal ions. In 0.1 M-KCl the binding decreased between pH 4.7 and 9.3. At pH 7.2 the binding was independent of temperature between 5 and 20 degrees C. These observations suggest that the binding is electrostatic rather than hydrophobic. 3. The proportion of bound activity increased with the concentration of the microsomal fraction, and at 22 mg of protein/ml and pH 7.2 was 70% at I0.18, and 35% at I0.26. Presumably a substantial amount of the enzyme is particle-bound in vivo. 4. At 5 degrees C in 10 mM-potassium phosphate, pH 7.2, the apparent molecular weight of KCl-solubilized enzyme decreased with enzyme concentration from about 200 000 to 40 000. In the presence of 0.5M-KCl, a constant mol.wt. of about 55 000 was observed over a 20-fold range of enzyme concentrations.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000418:Albumins / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012965:Sodium Chloride / Q000494:pharmacology; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D013806:Theophylline / Q000494:pharmacology
D000418:Albumins; D002712:Chlorides; D012965:Sodium Chloride; D011189:Potassium Chloride; D013806:Theophylline; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D008274:Magnesium