Partial purification and characterization of adenosine- and guanosine-3',5'-monophosphate phosphodiesterases from human lung tissue.
Crude extracts of human lung tissue were examined for cyclic adenosine- and guanosine-3',5'-monophosphate (cAMP and cGMP) phosphodiesterase activities. Nonlinear reciprocal plots were observed for each substrate. DEAE-Sephadex chromatography of the extracts revealed four main fractions of activity, which were further purified by Sephadex gel filtration. The phosphodiesterase activity of the resulting individual fractions was partially characterized with respect to substrate specificity, kinetic parameters, apparent molecular weight (gel filtration), thermal stability at 30 and 37 degrees C, effect of the cyclic nucleotide not utilized as substrate, and the possible influence of Ca2+-dependent protein activator. The results indicate that the tissue contains phosphodiesterases with strict specificity and a high apparent affinity for each of the two cyclic nucleotides (the Km values determined were approximately 0.3-0.4 muM). The high affinity cAMP phosphodiesterase activity was enriched in two of the purified fractions; both activities probably represent fragments of the native high affinity cAMP specific enzyme. A third purified phosphodiesterase showed mixed substrate specificity. The Km value recorded for hydrolysis of either substrate with this enzyme was approximately 25 muM. A fourth, irregularly occurring, phosphodiesterase activity also showed mixed substrate specificity. The Km value registered for hydrolysis of either substrate with this fraction was approximately 0.4 muM. There was no evidence for a Ca2+-dependent specific activation by a boiled lung tissue supernatant of any of the purified enzymes.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000302:isolation & purification / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D011489:Protein Denaturation
D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP; D002118:Calcium