Abstract
Actobindin, a 9.8-kDa protein purified from Acanthamoeba castellanii, contains two actin-binding sites that can simultaneously bind two actin monomers. However, actobindin inhibits actin polymerization to a greater extent than can be explained by its affinity for actin monomers (site-specific KD = 3.3 microM). This paradox would be resolved if actobindin could interfere with the nucleation phase of polymerization by using both binding sites to bind simultaneously to an actin oligomer because the interaction with oligomer would be thermodynamically favored over that with actin monomer. We now show that a covalently cross-linked actin dimer prepared from cross-linked F-actin binds to actobindin with high affinity (apparent KD = 11 nM) in accordance with theoretical predictions for simultaneous binding of two actin subunits per single actobindin and consistent with the hypothesis that actobindin might bind to native actin oligomers and prevent them from nucleating polymerization. Furthermore, the interaction with cross-linked dimer exhibits specificity in that an isomeric cross-linked actin dimer with more rapid electrophoretic mobility binds weakly to actobindin. However, only this isomeric dimer is produced when cross-linking reagents are added to actin undergoing polymerization in the presence of actobindin. Therefore, if actobindin inhibits polymerization by interacting with a native dimer whose conformation is similar to that of the cross-linked dimer with slower electrophoretic mobility, then actobindin must either block the cross-linking sites or convert the dimer to a different conformation.
Highlights
From the #Instituteof Physiology, Eberhard-Karls-UniuersitaTtiibingen, Gmelinstrasse 5, D-72076 Tiibingen, Federal Republic of Germany, the lllnstitute of Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland, and the Departmentof Biochemistry and Physiology, University of Barcelona, 08028 Barcelona, Spain
Currents induced by L-arginineand L-leucine were voltage-dependent. pH changes from6.25 to 8.75 did not rBAT-related protein is not the only transport system involved in dibasic amino acid transpoTrht.e ecotropic murine leukemia virus receptor was recently identifieads the dibasic amino acid transport system y’ [14, 15]
The present observations reveal that expression of the rBAT membrane protein mediates electrogenic transport of neutral
Summary
L-arginine-inducedcurrent was reduced after prior superfusion with L-lysine, and L-leucine-induced current was reduced after extended L-alanine transport (Fig. 9; n = 4) It appeared that each neutral amino acid couldstimulate the transport of dibasic amino acids and vice versa. We determined the reversal potential for transport of L-arginine (0.1 mM) and L-leucine (1 mM), respectively, as soon as themaximal current was induced (20 s after start of amino acid superfusion) and again after 10 min transport of the respective amino acid. The reversal potentials determined at thebeginning of amino acid superfusion were shifted after 10 min of L-leucine and L-arginine superfusion from -106 f 1.7 mV and 38.3 +: mV to -96.7 2 1.8mV and & 10.6mV, respectively ( n = 5) Both changes in extracellular aswell as intracellular amino acid concentrations alter the driving force for the individual amino acids
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