Mechanism for nucleotide exchange in monomeric actin.
Rabbit skeletal muscle G-actin has been treated to obtain ADP, 1,N6-ethenoadenosine diphosphate (epsilon-ADP), or 1,N6-ethenoadenosine triphosphate (epsilon-ATP) at the nucleotide binding site and either Mg2+ or Ca2+ at high- and moderate-affinity metal binding sites. Apparent rates or rate constants for the displacement of the actin-bound nucleotides by epsilon-ATP or ATP have been obtained by stopped-flow measurements at pH 8 and 20 degrees C of the fluorescence difference between bound and free epsilon-ATP or epsilon-ADP. In the presence of Ca2+, displacement of ADP by epsilon-ATP or epsilon-ADP by ATP is a biphasic process, but in the presence of low (less than 10 microM) Mg2+ concentrations, it is a slow first-order process. At high levels of Mg2+ (greater than 50 microM), low ADP concentrations displace epsilon-ATP from G-actin as a consequence of Mg2+ binding to moderate-affinity sites on the actin. Displacement of epsilon-ATP by ATP in the presence of either Ca2+ or Mg2+ is slow at low ATP concentrations, but the rate is increased by high ATP concentrations. Using ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, we find that nucleotide exchange is affected differently by the removal of Ca2+ from the high-affinity site compared to Ca2+ removal from moderate-affinity sites. A mechanism for the displacement reaction is proposed in which there are two forms of an actin-ADP complex and metal binding influences the ratio of these forms as well as the binding of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)
Frieden
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C
Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110.
Patane
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K
D000199:Actins / Q000302:isolation & purification / Q000378:metabolism*; D000227:Adenine Nucleotides / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D004985:Ethenoadenosine Triphosphate / Q000378:metabolism; D005456:Fluorescent Dyes; D007700:Kinetics; D009132:Muscles / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D000227:Adenine Nucleotides; D004985:Ethenoadenosine Triphosphate; D005456:Fluorescent Dyes; C015195:1,N(6)-ethenoadenosine diphosphate; D000244:Adenosine Diphosphate