Purification and characterization of calmodulin-dependent functional protein, phosphodiesterase, in the lens.
Calmodulin and calmodulin-dependent functional protein play an important role in the maintenance of lens transparency and homeostasis. In the present study, phosphodiesterase, one of the typical calmodulin-dependent functional proteins, was purified from bovine lens by DEAE-cellulose chromatography, calmodulin-Sepharose 4B chromatography and Superose 12 chromatography. Moreover, calmodulin-dependent phosphodiesterase, and independent phosphodiesterase were separated from crude lens extract using DEAE-cellulose column. The calmodulin-dependent phosphodiesterase was purified 4500-fold with a 0.7% yield; it was a dimer formed with two single polypeptides of 59K as the molecular weight. The enzyme had a higher affinity for cyclic GMP than for cyclic AMP, and functioned at calcium ion concentration above 10(-6) M in the incubation mixture. W-7 as calmodulin antagonist indirectly inhibited the enzyme activity and nifedipine as calmodulin-dependent phosphodiesterase antagonist directly inhibited the enzyme activity. These results suggest that an appearance of calmodulin-dependent phosphodiesterase activity depends on the interrelation between the calcium ion and calmodulin in the lens.
Yamamoto
T
T
Department of Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Meijo University, Nagoya, Japan.
Iwata
S
S
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D007908:Lens, Crystalline / Q000201:enzymology*; D009543:Nifedipine / Q000494:pharmacology; D009994:Osmolar Concentration; D013449:Sulfonamides / Q000494:pharmacology
D013449:Sulfonamides; C017967:W 7; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D009543:Nifedipine; D002118:Calcium