Involvement of arginine residues in the activation of calmodulin-dependent 3',5'-cyclic-nucleotide phosphodiesterase.
Pretreatment of an affinity-purified, brain calmodulin (CaM)-dependent phosphodiesterase (EC 3.1.4.17) with p-hydroxyphenylglyoxal (pHPG), a specific arginine-modifying reagent, resulted in a time-dependent loss in CaM-stimulated hydrolysis of cyclic AMP and cyclic GMP with no change in basal, CaM-independent activity. The loss in CaM-stimulated activity was preceded by a transient increase in CaM-dependent activity. Phenylglyoxal was 10-fold more effective than pHPG in promoting the loss of CaM-stimulated activity with a second-order rate constant of 13.3 M-1 min-1. Other arginine-modifying reagents, 1,2-cyclohexanedione and 2,3-butanedione, were not effective. The pHPG-modified enzyme was activated by 100 microM lysophosphatidylcholine to levels comparable to CaM-stimulated activity. The arginyl-modified enzyme was also activated by chymotrypsin and trypsin but not to the extent of the untreated enzyme stimulated with CaM. The presence of CaM during chemical modification with pHPG protected the enzyme from inactivation. Both the extent of activation and the amount of CaM necessary for 50% maximal activation were affected by pHPG treatment of the enzyme. The approximate number of modified arginines estimated by [7-14C]phenylglyoxal incorporation and amino acid analysis after complete inactivation of CaM stimulation was seven residues per catalytic subunit assuming enzyme homogeneity. The Stokes radius and sedimentation coefficient of the enzyme were unchanged by the modification. These results suggest that arginine residues are critical for functional interaction between phosphodiesterase and CaM and that controlled modification can selectively alter CaM-stimulated enzyme activity.
Nibhanupudy
N
N
Department of Biochemistry, Howard University College of Medicine, Washington, D.C. 20059.
Jones
F
F
Rhoads
A R
AR
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000447:Aldehydes / Q000494:pharmacology*; D000818:Animals; D001120:Arginine; D001921:Brain / Q000201:enzymology*; D002147:Calmodulin / Q000494:pharmacology; D002417:Cattle; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D004789:Enzyme Activation; D007700:Kinetics; D010447:Peptide Hydrolases; D010658:Phenylglyoxal / Q000031:analogs & derivatives / Q000494:pharmacology*
D000447:Aldehydes; D002147:Calmodulin; C028465:4-hydroxyphenylglyoxal; D001120:Arginine; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D010447:Peptide Hydrolases; D010658:Phenylglyoxal