SINGLE-STEP AFFINITY PURIFICATION, PARTIAL STRUCTURE AND PROPERTIES OF HUMAN PLATELET CGMP INHIBITED CAMP-PHOSPHODIESTERASE

The human platelet cilostamide-and cGMP-inhibited cAMP phosphodiestera se (cGI-PDE) was rapidly purified approximate to 19000-fold to apparen t homogeneity using single step affinity chromatography on the isothio cyanate derivative of cilostamide coupled to aminoethyl agarose. Withi n 24 h, 30 mu g of enzyme protein was obtained from 20 ml of packed pl atelets. V-max for cAMP and cGMP was 6.1 and 0.9 mu mol/min per mg pro tein, respectively. Several polypeptides (110/105, 79, 62, 55/53 kDa) were identified after SDS-PAGE, all of which were immunologically rela ted to cGI-PDE and represented approx. 5, 20, 50 and 20% of the total protein, respectively. Limited proteolysis of the cGI-PDE with chymotr ypsin produced a major fragment of approximate to 47 kDa (and at least two smaller peptides) with catalytic activity and sensitivity to cGMP and OPC 3911 similar to controls. Phosphorylation of the cGI-PDE by c AMP-dependent protein kinase (A-kinase) resulted in maximal incorporat ion of 0.6-1.8 mol of P-32/mol 110/105 and 79 kDa polypeptides; much l ower and variable amounts of phosphate were incorporated into the 62 a nd 55/53 kDa polypeptides. After digestion of cGI-PDE with several pro teinases a number of peptides were isolated and sequenced. Most of the peptide sequences obtained could be aligned within the carboxy termin al domain of the deduced sequence of the human cardiac cGI-PDE. These and other results suggest that the subunit size of the intact platelet cGI-PDE is 110 kDa and that proteolytic fragments of 79, 62 and 55/53 kDa are produced during purification. The smaller fragments (62 and 5 5/53 kDa) contain the catalytic domain; the larger fragments (110 and 79 kDa) also contain the regulatory domain with phosphorylation sites for A-kinase