Evidence that the presumptive second nucleotide interacting site on actin is of low specificity and affinity.
The contractile protein actin contains one mole of firmly bound nucleotide and a number of divalent cations bound with different affinities. During recent years evidence for a second nucleotide interacting site on actin has been reported. Therefore, a specific search for the presence of a second nucleotide-interacting site on actin was undertaken. For this purpose G- and F-actin or actin in complex with deoxyribonuclease I (DNase I) was passed over ADP-agarose which was found to retain all three forms of actin. Nucleotide bound to the high affinity site of actin did not exchange during passage and retention to agarose-immobilized ADP, thus indicating the presence of a second nucleotide interacting site. This site was found to be equally accessible in G- and F-actin and in the actin-DNase I complex, whereas DNase I alone passed unretained through this column. A number of nucleotides and phosphorylated compounds were tested for their ability to compete with immobilized ADP for actin interaction. It was found that all forms of actin are liberated only by high concentrations (5mM) of ADP, ATP and NADH, by 1mM CTP and ITP, and by high salt concentrations (150mM NaCl). Since it was found that EDTA- and heat-treated actin were also retained on ADP-agarose, we conclude that this second nucleotide interacting site is of limited specificity, low affinity, and not dependent on the native configuration of actin. It exhibits characteristics of an unspecific, polyanionic site, but may represent the low affinity phosphate binding site.
Kiessling
P
P
Institute of Cytobiology and Cytopathology, Philipps-University, Marburg, Germany.
Polzar
B
B
Mannherz
H G
HG
D000199:Actins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000327:Adsorption; D000818:Animals; D002846:Chromatography, Affinity; D003570:Cytidine Triphosphate / Q000737:chemistry; D003850:Deoxyribonuclease I / Q000037:antagonists & inhibitors; D004591:Electrophoresis, Polyacrylamide Gel; D007293:Inosine Triphosphate / Q000737:chemistry; D008854:Microscopy, Electron; D009132:Muscles / Q000737:chemistry; D009711:Nucleotides / Q000378:metabolism*; D011817:Rabbits
D000199:Actins; D009711:Nucleotides; D007293:Inosine Triphosphate; D000244:Adenosine Diphosphate; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D003850:Deoxyribonuclease I