Single-step affinity purification, partial structure and properties of human platelet cGMP inhibited cAMP phosphodiesterase.
The human platelet cilostamide- and cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) was rapidly purified approximately 19,000-fold to apparent homogeneity using single step affinity chromatography on the isothiocyanate derivative of cilostamide coupled to aminoethyl agarose. Within 24 h, 30 micrograms of enzyme protein was obtained from 20 ml of packed platelets. Vmax for cAMP and cGMP was 6.1 and 0.9 mumol/min per mg protein, respectively. Several polypeptides (110/105, 79, 62, 55/53 kDa) were identified after SDS-PAGE, all of which were immunologically related to cGI-PDE and represented approx. 5, 20, 50 and 20% of the total protein, respectively. Limited proteolysis of the cGI-PDE with chymotrypsin produced a major fragment of approximately 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 cAMP-dependent protein kinase (A-kinase) resulted in maximal incorporation of 0.6-1.8 mol of 32P/mol 110/105 and 79 kDa polypeptides; much lower and variable amounts of phosphate were incorporated into the 62 and 55/53 kDa polypeptides. After digestion of cGI-PDE with several proteinases a number of peptides were isolated and sequenced. Most of the peptide sequences obtained could be aligned within the carboxy terminal 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 55/53 kDa) contain the catalytic domain; the larger fragments (110 and 79 kDa) also contain the regulatory domain with phosphorylation sites for A-kinase.
Degerman
E
E
Department of Medical and Physiological Chemistry, University of Lund, Sweden.
Moos
M
M
Rascón
A
A
Vasta
V
V
Meacci
E
E
Smith
C J
CJ
Lindgren
S
S
Andersson
K E
KE
Belfrage
P
P
Manganiello
V
V
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D001792:Blood Platelets / Q000201:enzymology*; D002846:Chromatography, Affinity; D006152:Cyclic GMP / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D010446:Peptide Fragments / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D011233:Precipitin Tests; D017421:Sequence Analysis
D010446:Peptide Fragments; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP