Fluorescence quenching by nucleotides of the plasma membrane H+-ATPase from Kluyveromyces lactis.
The yeast plasma membrane H+-ATPase isolation procedure was improved; a highly pure enzyme (90-95%) was obtained after centrifugation on a trehalose concentration gradient. H+-ATPase kinetics was slightly cooperative: Hill number = 1.5, S0.5 = 800 microM ATP, and turnover number = 36 s-1. In contrast to those of other P-type ATPases, H+-ATPase fluorescence was highly sensitive to nucleotide binding; the fluorescence decreased 60% in the presence of both 5 mM ADP and AMP-PNP. Fluorescence titration with nucleotides allowed calculation of dissociation constants (Kd) from the binding site; Kd values for ATP and ADP were 700 and 800 microM, respectively. On the basis of amino acid sequence and homology model analysis, we propose that binding of the nucleotide to the N-domain is coupled to the movement of a loop beta structure and to the exposure of the Trp505 residue located in the loop. The recombinant N-domain also displayed a large hyperbolic fluorescence quenching when ATP binds; however, it displayed a higher affinity for ATP (Kd = 100 microM). We propose for P-type ATPases that structural movements during nucleotide binding could be followed if a Trp residue is properly located in the N-domain. Further, we propose the use of trehalose in enzyme purification protocols to increase the purity and quality of the isolated protein and to perform structural studies.
Sampedro
José G
JG
Area Académica de Nutrición and Area Académica de Farmacia, ICSA Universidad Autónoma del Estado de Hidalgo, Abasolo 600 Colonia Centro, CP 42000, Pachuca, México. jg.sampedro@gmail.com
Ruiz-Granados
Yadira G
YG
Nájera
Hugo
H
Téllez-Valencia
Alfredo
A
Uribe
Salvador
S
D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D006868:Hydrolysis; D007716:Kluyveromyces / Q000201:enzymology*; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases