Conformational transition accompanying the binding of Ca2+ to the protein activator of 3',5'-cyclic adenosine monophosphate phosphodiesterase.
The Ca2+-dependent protein activator of 3':5'-cyclic adenosine monophosphate phosphodiesterase is shown to undergo a conformational transition upon binding of 2 mol of Ca2+/mol of activator. Circular dichroic studies indicate that Ca2+ induces an increase of 5-8% in alpha-helix content with a concomitant decrease in the amount of random coil. In the absence of Ca2+ and in the presence of [ethylenebis(oxoethylenenitrilo)]tetraacetic acid (EGTA), the protein contains 30-35% alpha helix, 50% random coil, and 15-20% beta-pleated sheat. Spectrophotometric titration indicates that the two tyrosyl residues have pK's of 10.4 and 11.9 and are therefore in different environments. The Ca2+-induced conformational change is accompanied by an increased exposure to protons of the partially exposed tyrosine, as shown by a shift in its pK from 10.4 to 10.). Increased solvation is also consistent with a negative difference spectrum at 287 and 279 nm as seen upon Ca2+ binding. Modification in the environment of all or some of the phenylalanine residues also is part of the conformational change accompanying Ca2+ binding. A new and rapid purification procedure which yields large amounts (25-30% yields) of homogenous protein activator and a direct and sensitive assay procedure for cAMP phosphodiesterase and its activator are also described.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000302:isolation & purification / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology*; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000502:physiology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
D000596:Amino Acids; D009419:Nerve Tissue Proteins; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D002118:Calcium