The amino acid sequence of a fluorescein-labeled peptide from the active site of (Na,K)-ATPase.
(Na,K)-ATPase in 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 (Na,K)-ATPase has been purified and its amino acid sequence has been determined. The peptide was identified by the covalent incorporation of a fluorescent probe, fluorescein 5'-isothiocyanate, into the active site before trypsin 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 microM was calculated when the reaction between (Na,K)-ATPase and fluorescein 5'-isothiocyanate was performed under pseudo first-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 (Mitchinson, C., Wilderspin, A. F., Trinaman, B. J., and Green, N. M. (1982) FEBS Lett. 146, 87-92).
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis*; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis*; D013861:Thiocyanates
D000596:Amino Acids; D005452:Fluoresceins; D010446:Peptide Fragments; D013861:Thiocyanates; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate