Human plasma gelsolin reversibly binds Mg-ATP in Ca(2+)-sensitive manner.
Gelsolin is a Ca(2+)-regulated actin-modulating protein found in a variety of cellular cytoplasm and also in blood plasma. Affinity separation of human plasma gelsolin was successfully accomplished by eluting the protein with a low concentration of nucleoside polyphosphate from immobilized Cibacron Blue F3GA (1, 2). This finding was followed by the demonstration that the protein had one class of ATP binding site with Kd = 2.8 x 10(-7) M, which saturated at an ATP/gelsolin ratio of 0.6 in the absence of Ca2+ (3). To obtain further information on the nucleotide binding properties of gelsolin, binding studies were done in the presence of EGTA with GTP, ADP, and GDP by equilibrium dialysis. Incubation of plasma gelsolin with GTP resulted in binding of 0.6 mol of GTP per mol of protein with a dissociation constant of 1.8 x 10(-6) M, indicating that ATP binds to gelsolin with higher affinity than GTP. Neither ADP nor GDP at up to 100 microM appreciably bound to gelsolin at a physiological salt concentration. Then, the effects of divalent metal ions on the ATP binding to plasma gelsolin were examined. Gelsolin bound to ATP with Kd = 2.4 x 10(-6) M in a solution containing 2 mM MgCl2, whereas micromolar free Ca2+ concentrations inhibited ATP binding. Furthermore, addition of Ca2+ rapidly reversed the preformed nucleotide binding to gelsolin, suggesting that Ca2+ binding to gelsolin leads to a conformational change which disrupts a nucleotide binding fold in the protein molecule.
Kambe
H
H
Third Department of Internal Medicine, Osaka University School of Medicine.
Ito
H
H
Kimura
Y
Y
Okochi
T
T
Yamamoto
H
H
Hashimoto
T
T
Tagawa
K
K
D000227:Adenine Nucleotides / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000097:blood* / Q000737:chemistry; D018260:Gelsolin; D006150:Guanine Nucleotides / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008840:Microfilament Proteins / Q000097:blood* / Q000737:chemistry; D008969:Molecular Sequence Data
D000227:Adenine Nucleotides; D002135:Calcium-Binding Proteins; D018260:Gelsolin; D006150:Guanine Nucleotides; D008840:Microfilament Proteins; D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium