Apparent multiple forms of cyclic AMP phosphodiesterase from rat erythrocytes.
Bio-Gel A-5m chromatography has been used to separate apparent multiple forms of cyclic nucleotide phosphodiesterase from rat erythrocytes. Cyclic AMP phosphodiesterase was resolved by gel filtration into three peaks of activity with apparent molecular weights of about 300,000, 225,000 and 100,000, while cyclic GMP phosphodiesterase activity in gel column fractions was too low to permit meaningful estimates of its molecular weight. All three of the separated peaks of cyclic AMP phosphodiesterase activity displayed anomalous kinetic behaviour suggestive of negative cooperativity. The possibility that multiple phosphodiesterase activities could arise from in vitro alterations of a single enzyme was investigated. Similar changes in gel filtration profiles resulted when erythrocyte extracts were treated with trypsin or ammonium sulfate or were incubated at 37 degrees C. After these treatments, a large proportion of the enzyme activity occurred in low (ca. 100,000) molecular weight regions. The low molecular weight phosphodiesterase activities from untreated, incubated, and trypsin-treated extracts possessed similar properties. All were inhibited by methylxanthines, had pH optima of approximately 8.0, and similar kinetic properties and requirements for divalent cations. These observations raise the possibility that preparative procedures or limited proteolysis occurring during preparation and handling of extracts can contribute to the apparent multiplicity of enzyme forms seen after gel filtration of phosphodiesterase from rat erythrocytes and perhaps other cell types.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000097:blood* / Q000302:isolation & purification; D000818:Animals; D002850:Chromatography, Gel; D004912:Erythrocytes / Q000201:enzymology*; D005260:Female; D007527:Isoenzymes / Q000097:blood* / Q000302:isolation & purification; D007700:Kinetics; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000097:blood*; D051381:Rats
D007527:Isoenzymes; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases