Non-muscle actin filament elongation from complexes of profilin with nucleotide-free actin and divalent cation-free ATP-actin.
Using vertebrate cytoplasmic actin consisting of a mixture of beta and gamma isoforms, we previously characterized profilin and nucleotide binding to monomeric actin (Kinosian, H. J., et al. (2000) Biochemistry 39, 13176-13188) and F-actin barbed end elongation from profilin-actin (PA) (Kinosian, H. J., et al. (2002) Biochemistry 41, 6734-6743). Our initial calculations indicated that elongation of F-actin from PA was more energetically favorable than elongation of F-actin from monomeric actin; therefore, the overall actin elongation reaction scheme described by these two linked reactions appeared to be thermodynamically unbalanced. However, we hypothesized that the profilin-induced weakening of MgATP binding by actin reduces the negative free energy change for the formation of profilin-MgATP-actin from MgATP-actin. When this was taken into account, the overall reaction scheme was calculated to be thermodynamically balanced. In our present work, we test this hypothesis by measuring actin filament barbed end elongation of nucleotide-free actin (NF-A) and nucleotide-free profilin-actin (NF-PA). We find that the free energy change for elongation of F-actin by NF-PA is equal to that for elongation of F-actin from NF-A, indicating energetic balance of the linked reactions. In the absence of actin-bound divalent cation, profilin has very little effect on ATP binding to actin; analysis of elongation experiments with divalent cation-free ATP-actin and profilin yielded an approximately energetically balanced reaction scheme. Thus, the data in this present report support our earlier hypothesis.
Kinosian
Henry J
HJ
Center for Cell Biology and Cancer Research, Albany Medical College, Albany, New York 12208, USA. hkinosi1@nycap.rr.com
Selden
Lynn A
LA
Gershman
Lewis C
LC
Estes
James E
JE
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D003285:Contractile Proteins / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D051304:Profilins; D020033:Protein Isoforms / Q000378:metabolism; D013816:Thermodynamics
C022920:ATP-G-actin; D000199:Actins; D003285:Contractile Proteins; D008840:Microfilament Proteins; D051304:Profilins; D020033:Protein Isoforms; D000255:Adenosine Triphosphate