Purification and characterization of high-affinity cyclic adenosine 5'-monophosphate phosphodiesterases from human acute myelogenous leukemic cells.
Soluble, high-affinity cyclic adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterases extracted from blast cells of patients with acute myelogenous leukemia have been characterized by physical, kinetic, and immunological criteria and fractionated to a high degree of purity. Procedures used in this study were similar to those used to purify the high-affinity enzyme from dog kidney. Two forms of high-affinity enzyme were found in blast cells. Form A was similar to the known type IV phosphodiesterases, including those of normal lymphocytes and monocytes. It showed a molecular weight near 60,000, a rate of hydrolysis of cyclic AMP 7 to 10 times that of cyclic guanosine 3':5'-monophosphate (cyclic GMP), competitive inhibition by cyclic GMP for cyclic AMP hydrolysis, and identical immunoreactivity by Western transfer analysis. This enzyme form was purified to apparent homogeneity by physical criteria but showed a low maximum velocity relative to other phosphodiesterase forms. A second, different form of high-affinity phosphodiesterase (Form B) was also resolved and partially purified. By comparison with Form A, this enzyme eluted from diethylaminoethyl cellulose at slightly lower ionic strength, had a lower molecular weight, appeared specific for cyclic AMP as substrate, showed no inhibition of cyclic AMP hydrolysis by cyclic GMP, and displayed no immunological cross-reactivity to the Mr 60,000 enzyme. Neither enzyme form was activated by calmodulin or proteolysis, whereas both showed comparable inhibition by 6,7-dimethoxy-1-veratrylisoquinoline, 1-methyl-3-isobutylxanthine, and 1,3-dimethylxanthine.
Onali
P
P
Strada
S J
SJ
Chang
L
L
Epstein
P M
PM
Hersh
E M
EM
Thompson
W J
WJ
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000276:immunology / Q000302:isolation & purification*; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D006801:Humans; D006882:Hydroxyapatites; D007700:Kinetics; D015470:Leukemia, Myeloid, Acute / Q000201:enzymology*; D008970:Molecular Weight
D006882:Hydroxyapatites; D015105:3',5'-Cyclic-AMP Phosphodiesterases