Interdependence of profilin, cation, and nucleotide binding to vertebrate non-muscle actin.
The interaction of profilin and non-muscle beta,gamma-actin prepared from bovine spleen has been investigated under physiologic ionic conditions. Profilin binding to actin decreases the affinity of actin for MgADP and MgATP by about 65- and 13-fold, respectively. Kinetic measurements indicate that profilin binding to actin weakens the affinity of actin for nucleotides primarily due to an increased nucleotide dissociation rate constant, but the nucleotide association rate constant is also increased about 2-fold. Removal of the actin-bound nucleotide and divalent cation produces the labile intermediate species in the nucleotide exchange reaction, nucleotide free actin (NF-actin), and increases the affinity of actin for profilin about 10-fold. Profilin binds NF-actin with high affinity, K(D) = 0.013 microM, and slows the observed denaturation rate of NF-actin. Addition of ATP to NF-actin weakens the affinity for profilin and addition of Mg(2+) to ATP-actin further weakens the affinity for profilin. The high-affinity Mg(2+) of actin regulates binding of both nucleotide and profilin to actin and is important for actin interdomain coupling. The data suggest that profilin binding to actin weakens nucleotide binding to actin by disrupting Mg(2+) coordination in the actin central cleft.
Kinosian
H J
HJ
Center for Molecular Biology and Cancer Research and Department of Medicine, Albany Medical College, Albany, New York 12208, USA. henry.kinosian@med.va.gov
Selden
L A
LA
Gershman
L C
LC
Estes
J E
JE
D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D002417:Cattle; D003285:Contractile Proteins; D004985:Ethenoadenosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D008956:Models, Chemical; D051304:Profilins; D011489:Protein Denaturation; D020033:Protein Isoforms / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D000199:Actins; D002413:Cations, Divalent; D003285:Contractile Proteins; D004985:Ethenoadenosine Triphosphate; D008840:Microfilament Proteins; D051304:Profilins; D020033:Protein Isoforms; C042937:aza-epsilon-ADP; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D008274:Magnesium