Partial purification and properties of a multifunctional 3′,5′-cyclic nucleotide phosphodiesterase from Lactuca cotyledons

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Abstract The extraction, partial purification and properties of a 3′,5′-cyclic nucleotide phosphodiesterase from lettuce cotyledons is described. Purification involved fractional precipation with (NH 4 ) 2 SO 4 , chromatography on Sephadex G-200, affinity chromatography on Affi-Gel Blue and non-denaturing polyacrylamide gel electrophoresis. The behaviour of the final enzyme preparation on SDS-polyacrylamide gel electrophoresis was examined and inidcated an M , of ca 62 000. The enzyme from 3′,5′-cyclic nucleotide phosphodiesterases previously isolated from plant tissues in that it exhibits activity towards pyrimidine as well as purine cyclic nucleotides. Furthermore, it hydrolyses cyclic CMP at a comparable rate to that with which it hydrolyses cyclic AMP and cyclic GMP. Both 3′- and 5′-AMP were released, with the 5′-nucleotide being the major product. Whereas the K m with all three substrates remained constant during the purification procedure, V max with cyclic AMP was lower than that for cyclic CMP but increased as purification proceeded. The effects were examined of a range of di- and trivalent metal ions on the enzyme activity. Fe 3+ significantly stimulated the activity, more so when cyclic GMP was the substrate. Cu 2+ inhibited the activity.