The fluorescein isothiocyanate-binding site of the plasma-membrane H+-ATPase of Neurospora crassa.
The mammalian (Na+,K+), Ca2+-, and (H+,K+)-ATPases contain a well-characterized lysine residue that reacts with fluorescein 5'-isothiocyanate (FITC); enzymatic activity is protected by ATP, suggesting that the residue is located in or near the nucleotide-binding domain. In this study, the plasma-membrane H+-ATPase of Neurospora crassa is also shown to be sensitive to FITC. The reaction occurs with pseudo first-order kinetics, has a pKa of 8.0, and is stimulated by Mg2+. Enzymatic activity is protected by MgADP with a Kd of 0.2-0.3 mM, close to the Ki with which MgADP serves as a competitive inhibitor of ATP hydrolysis. A tryptic peptide labeled with FITC in the absence, but not in the presence, of MgADP has been isolated and sequenced. The FITC-sensitive residue is Lys474, located in a region that exhibits significant homology with the mammalian cation-transporting ATPases.
Pardo
J P
JP
Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510.
Slayman
C W
CW
D000595:Amino Acid Sequence; D001665:Binding Sites; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013861:Thiocyanates
D005452:Fluoresceins; D013861:Thiocyanates; D006180:Proton-Translocating ATPases; D016650:Fluorescein-5-isothiocyanate; D008274:Magnesium