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The amino acid sequence of a fluorescein labeled peptide from the active site of sodium potassium atpase ec 3.6.1.3






Journal of Biological Chemistry 259(15): 9532-9535

The amino acid sequence of a fluorescein labeled peptide from the active site of sodium potassium atpase ec 3.6.1.3

(Na,K)-ATPase is an active-transport protein that couples the energy obtained from the hydrolysis of ATP to the transport of Na+ and K+ across animal cell membranes. In order to investigate the enzymatic mechanism of this activity, a peptide derived from the ATP-binding site of [canine kidney] (Na,K)-ATPase was purified and its amino acid sequence was determined. The peptide was identified by the covalent incorporation of a fluorescent probe, fluorescein 5'-isothiocyanate, into the active site before tryspin digestion of the protein. The labeling of (Na,K)-ATPase by fluorescein 5'-isothiocyanate was associated with the irreversible inhibition of enzymatic activity, and both the labeling of the tryptic peptide and inhibition of activity were prevented when the reaction was performed in the presence of ATP. An apparent Kd of 5.7 .mu.M was calculated when the reaction between (Na,k)-ATPase and fluorescein 5'-isothiocyanate was performed under pseudo 1st-order conditions. The amino acid sequence of the active-site peptide, His-Leu-Leu-Val-Met-Lys-Gly-Ala-Pro-Glu-Arg, is similar to the sequence of a fluorescein-labeled peptide derived from the active site of the sarcoplasmic reticulum Ca2+-transport ATPase.


Accession: 006599176



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