Effects of fluorescent pseudo-ATP and ATP-metal analogs on secondary structure of Na+/K+-ATPase

The secondary structure of Na+/K+-ATPase after modification of the ATP-bind ing sites was analyzed. Consistently with recent reports, we found in tryps in-treated Na+/K+-ATPase additionally to alpha-helix also beta-sheet struct ures in the transmembrane segments. However, binding of fluorescein 5'-isot hiocyanate (FITC), the pseudo-ATP analog, to the ATP-binding site did not a ffect the secondary structure of undigested Na+/K+-ATPase. Consequently, fl uorescence intensity changes of FITC-labeled Na+/K+-ATPase commonly used to observe conformational transitions of the enzyme reflect physiological cha nges of the native structure. The metal complex analogues of ATP, Cr(H2O)(4 )ATP and Co(NH3)(4)ATP, on the other hand, affected the secondary structure of Na+/K+-ATPase. We propose that these changes in the secondary structure are responsible for inhibition of backdoor phosphorylation, (C) 2000 Elsev ier Science B.V. All rights reserved.