Inhibition of calmodulin-dependent cyclic AMP phosphodiesterase by phenoxazines.
Phenoxazine derivatives were examined for their ability to inhibit the calmodulin-mediated activation of phosphodiesterase, which is based on the hydrolysis of cAMP to AMP by phosphodiesterase in the presence or absence of inhibitor, followed by quantitative analysis by HPLC method. Anticalmodulin activity of phenoxazines with respect to substitution at C-2 position follows the order: 2-trifluoromethyl>2-chloro>unsubstituted phenoxazines. The interaction of phenoxazines with calmodulin using fluorescence spectroscopy has been performed. Binding study showed that calmodulin has two types of binding sites for phenoxazines. One is high affinity binding site (Kd value 0.07-0.46 microM) and the other, a low affinity binding site (Kd value 0.7-34.5 microM). The change in secondary structure of calmodulin upon binding to phenoxazines was studied by circular dichroism (CD) method, which showed that the percentage of helicity decreased with an extensive change in tertiary structure of calmodulin. Kinetic analysis of the phenoxazine-calmodulin interaction showed that phenoxazines competitively inhibited the activation of phosphodiesterase without affecting Vmax. Thus, these studies showed a good correlation between the ability of phenoxazines to block the activation of phosphodiesterase and their ability to bind to the activator.
Jagadeesh
Shankar
S
Cell Biology Department, Georgetown University Medical Center, Washington, DC 20057, USA. sj36@georgetown.edu
Padma
Thimmaiah
T
Parimala
Hanumesh
H
Chandramouli
K H
KH
D'Souza
Cletus J M
CJ
Thimmaiah
Kuntebommanahalli N
KN
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002942:Circular Dichroism; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D004305:Dose-Response Relationship, Drug; D008024:Ligands; D010078:Oxazines / Q000737:chemistry / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D002147:Calmodulin; D008024:Ligands; D010078:Oxazines; C039203:phenoxazine; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1