A novel phosphodiesterase from cultured tobacco cells

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Abstract
A novel phosphodiesterase was purified from cultured tobacco [Nicotiana tabacum] cells to a state which appeared homogeneous on polyacrylamide gel electrophoresis. The enzyme hydrolyzed various phosphodiester and pyrophosphate bonds, including p-nitrophenyl thymidine 5''-phosphate, p-nitrophenyl thymidine 3''-phosphate, cyclic nucleotides, ATP, NAD+, inorganic pyrophosphate, dinucleotides and poly(ADP ribose), which is a polymer synthesized from NAD+. It did not hydrolyze highly polymerized polynucleotides. The MW of the native enzyme was estimated as 270,000-280,000 by gel filtration on Sephadex G-200 and Bio-Gel A-5m. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the enzyme was composed of subunits with MW calculated to be 75,000. The enzyme did not require divalent cations for activity being fully active in the presence of EDTA. The pH optimum for the enzyme was approximately 6 with p-nitrophenyl thymidine 5''-phosphate or cyclic AMP and 5.3 with NAD+. Double reciprocal plots of the initial velocity against the concentration of p-nitrophenyl thymidine 5''-phosphate gave 2 apparent Km values of 0.17 and 1.3 mM, suggesting the presence of at least 2 active sites.