The cyclic nucleotide phosphodiesterases of spinach chloroplasts and microsomes

Abstract

Cyclic nucleotide phosphodiesterase was extracted from intact chloroplasts and partially purified. Peak 1 c activity from Sephadex G-200 was resolved by electrophoresis into two major bands (MWs 1.87 × 10 5 and 3.7 × 10 5). Both also possessed acid phosphatase, ribonuclease, nucleotidase and ATPase. The chloroplast peak 1 c cyclic nueleotide phosphodiesterase was located in the envelope. Peak 1 m cyclic nucleotide phosphodiesterase obtained from the microsomal fraction had a MW of 2.63 × 10 5. Electrophoresis separated 1 m into two bands of cyclic nucleotide phosphodiesterase activity (MWs 2.63 × 10 5 and 1.28 × 10 5). Both contain ATPase, ribonuclease, nucleotidase, but not acid phosphatase. Peak 1 c has high activity towards 3′:5′-cyclic AMP and 3′:5′-cyclic GMP but little towards 2′:3′-cyclic nucleotides. Peak 1 m showed most activity towards 2′:3′-cyclic AMP, 2′:3′-cyclic GMP and 2′:3′-cyclic CMP with little activity towards 3′:5′-cyclic nucleotides. With 1 c, 3′:5′-cyclic AMP and 3′:5′-cyclic GMP exhibit mixed-type inhibition towards one another. The 2′:3′-cyclic AMP phosphodiesterase 1 m was competitively inhibited by 2′:3′-cyclic GMP. p-Chloromercuribenzoate inhibits 1 c but not 1 m. Electrophoresis after dissociation indicates that 1 c and 1 m are both enzyme complexes. After dissociation, the 1 c complex but not that of 1 m could be reassociated. The ribonuclease of the 1 m complex hydrolyses RNA to yield 2′:3′-cyclic nucleotides as the main products. These results are compatible with the 1 c cyclic nucleotide phosphodiesterase complex being involved in the metabolism of 3′:5′-cyclic AMP, and the 1 m complex being concerned with RNA catabolism.