Non-Muscle Actin Filament Elongation from Complexes of Profilin with Nucleotide-Free Actin and Divalent Cation-Free ATP-Actin
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- 作者：Henry J. Kinosian ; Lynn A. Selden ; Lewis C. Gershman ; and James E. Estes
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：May 25, 2004
- 年：2004
- 卷：43
- 期：20
- 页码：6253 - 6260
- 全文大小：123K
- 年卷期：v.43,no.20(May 25, 2004)
- ISSN：1520-4995
文摘
Using vertebrate cytoplasmic actin consisting of a mixture of 
chars/beta2.gif" BORDER=0 ALIGN="middle"> and chars/gamma.gif" BORDER=0 > isoforms, we previouslycharacterized profilin and nucleotide binding to monomeric actin (Kinosian, H. J., et al. (2000) Biochemistry39, 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 fromPA was more energetically favorable than elongation of F-actin from monomeric actin; therefore, theoverall actin elongation reaction scheme described by these two linked reactions appeared to bethermodynamically unbalanced. However, we hypothesized that the profilin-induced weakening of MgATPbinding by actin reduces the negative free energy change for the formation of profilin-MgATP-actinfrom MgATP-actin. When this was taken into account, the overall reaction scheme was calculated to bethermodynamically balanced. In our present work, we test this hypothesis by measuring actin filamentbarbed end elongation of nucleotide-free actin (NF-A) and nucleotide-free profilin-actin (NF-PA). Wefind that the free energy change for elongation of F-actin by NF-PA is equal to that for elongation ofF-actin from NF-A, indicating energetic balance of the linked reactions. In the absence of actin-bounddivalent cation, profilin has very little effect on ATP binding to actin; analysis of elongation experimentswith divalent cation-free ATP-actin and profilin yielded an approximately energetically balanced reactionscheme. Thus, the data in this present report support our earlier hypothesis.
chars/beta2.gif" BORDER=0 ALIGN="middle"> and chars/gamma.gif" BORDER=0 > isoforms, we previouslycharacterized profilin and nucleotide binding to monomeric actin (Kinosian, H. J., et al. (2000) Biochemistry39, 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 fromPA was more energetically favorable than elongation of F-actin from monomeric actin; therefore, theoverall actin elongation reaction scheme described by these two linked reactions appeared to bethermodynamically unbalanced. However, we hypothesized that the profilin-induced weakening of MgATPbinding by actin reduces the negative free energy change for the formation of profilin-MgATP-actinfrom MgATP-actin. When this was taken into account, the overall reaction scheme was calculated to bethermodynamically balanced. In our present work, we test this hypothesis by measuring actin filamentbarbed end elongation of nucleotide-free actin (NF-A) and nucleotide-free profilin-actin (NF-PA). Wefind that the free energy change for elongation of F-actin by NF-PA is equal to that for elongation ofF-actin from NF-A, indicating energetic balance of the linked reactions. In the absence of actin-bounddivalent cation, profilin has very little effect on ATP binding to actin; analysis of elongation experimentswith divalent cation-free ATP-actin and profilin yielded an approximately energetically balanced reactionscheme. Thus, the data in this present report support our earlier hypothesis.