Interactions of Acanthamoeba Profilin With Actin and Nucleotides Bound to Actin

Biochemistry. 1998 Aug 4;37(31):10871-80. doi: 10.1021/bi980093l.

Abstract

Three methods, fluorescence anisotropy of rhodamine-labeled profilin, intrinsic fluorescence and nucleotide exchange, give the same affinity, Kd = 0.1 microM, for Acanthamoeba profilins binding amoeba actin monomers with bound Mg-ATP. Replacement of serine 38 with cysteine created a unique site where labeling with rhodamine did not alter the affinity of profilin for actin. The affinity for rabbit skeletal muscle actin is about 4-fold lower. The affinity for both actins is 5-8-fold lower with ADP bound to actin rather than ATP. Pyrenyliodoacetamide labeling of cysteine 374 of muscle actin reduces the affinity for profilin 10-fold. The affinity of profilin for nucleotide-free actin is approximately 3-fold higher than for Mg-ATP-actin and approximately 24-fold higher than for Mg-ADP-actin. As a result, profilin binding reduces the affinity of actin 3-fold for Mg-ATP and 24-fold for Mg-ADP. Mg-ATP dissociates 8 times faster from actin-profilin than from actin and binds actin-profilin 3 times faster than actin. Mg-ADP dissociates 14 times faster from actin-profilin than from actin and binds actin-profilin half as fast as actin. Thus, profilin promotes the exchange of ADP for ATP. These properties allow profilin to bind a high proportion of unpolymerized ATP-actin in the cell, suppressing spontaneous nucleation but allowing free barbed ends to elongate at more than 500 subunits/second.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acanthamoeba / metabolism*
  • Actins / metabolism*
  • Adenosine Diphosphate / metabolism*
  • Adenosine Triphosphate / metabolism*
  • Animals
  • Binding, Competitive
  • Contractile Proteins*
  • Fluorescence Polarization
  • Fluorescent Dyes / metabolism
  • Indoles / metabolism
  • Microfilament Proteins / chemistry
  • Microfilament Proteins / metabolism*
  • Profilins
  • Protein Binding
  • Protozoan Proteins / chemistry
  • Protozoan Proteins / metabolism*
  • Rhodamines / metabolism
  • Spectrometry, Fluorescence

Substances

  • Actins
  • Contractile Proteins
  • Fluorescent Dyes
  • Indoles
  • Microfilament Proteins
  • Profilins
  • Protozoan Proteins
  • Rhodamines
  • rim 1
  • Adenosine Diphosphate
  • Adenosine Triphosphate