PROGRESSIVE DECLINE IN THE ABILITY OF CALMODULIN ISOLATED FROM AGED BRAIN TO ACTIVATE THE PLASMA-MEMBRANE CA-ATPASE

TO identify possible relationships between the loss of calcium homeost asis in brain associated with aging and alterations in the function of key calcium regulatory proteins, we have purified calmodulin (CaM) fr om the brains of Fischer 344 rats of different ages and have assessed age-related alterations in (i) the secondary and tertiary structure of CaM and (ii) the ability of CaM to activate one of its target protein s, the plasma membrane (PM) Ca-ATPase. There is a progressive, age-dep endent reduction in the ability of CaM to activate the PM-Ca-ATPase, w hich correlates with the oxidative modification of multiple methionine s to their corresponding methionine sulfoxides. No other detectable ag e-related posttranslational modifications occur in the primary sequenc e of CaM, suggesting that the reduced ability of CaM to activate the P M-Ca-ATPase is the result of methionine oxidation. Corresponding age-r elated changes in the secondary and tertiary structure of CaM occur, r esulting in alterations in the relative mobility of CaM on polyacrylam ide gels, differences in the intrinsic fluorescence intensity and solv ent accessibility of Tyr(99) and Tyr(138), and a reduction in the aver age a-helical content of CaM at 20 degrees C. Shifts in the calcium- a nd CaM-dependent activation of the PM-Ca-ATPase are observed for CaM i solated from senescent brain, which respectively requires larger conce ntrations of either calcium or CaM to activate the PM-Ca-ATPase. The o bservation that the oxidative modification of CaM during normal biolog ical aging results in a reduced calcium sensitivity of the PM-Ca-ATPas e, a lower affinity between CaM and the Phl-Ca-ATPase, and the reducti on in the maximal velocity of the PM-Ca-ATPase is consistent with earl ier results that indicate the calcium handling capacity of a range of tissues including brain, heart, and erythrocytes isolated from aged an imals declines, resulting in both longer calcium transients and elevat ed basal levels of intracellular calcium. Thus, the oxidative modifica tion of selected methionines in CaM may explain aspects of the loss of calcium homeostasis associated with the aging process.