Interactions of actin and tubulin with human deoxyhemoglobin. Their possible occurrence within erythrocytes.
Short actin filaments are an essential component of the red-cell membrane skeleton, and microtubules are also present in nucleated erythrocytes as a marginal band. Actin and tubulin share the property of possessing a very anionic terminal peptide. Since deoxyhemoglobin (Hb) is known to be a strong polyanion-binding protein, we have considered how it may interact with actin and tubulin within the intact cell. Here we demonstrate that actin and tubulin form in vitro a high-affinity complex with Hb. This is shown by measuring, by stopped-flow experiments, the decrease of the binding rate constant of CO to Hb in the presence of increasing amounts of actin and tubulin. One tetramer of Hb is bound by an actin monomer, and about two tetramers by an alpha, beta-tubulin heterodimer. Binding assays in batch experiments with immobilized tubulin give the same stoichiometry. Formation of the complexes involves the 2,3-bisphosphoglycerate-binding site of Hb and a negatively charged domain, most likely the highly acidic N and C-terminal peptides of actin and tubulin. In addition to providing new opportunities to study the structural and functional properties of actin and tubulin, these results support the idea that in the case of partial metabolic depletion of bisphosphoglycerate and ATP in erythrocytes, Hb may interact with oligomeric actin and tubulin present in the cytoskeleton.
Laboratoire de Biophysique, Unité associée 1089 du Centre National de la Recherche Scientifique, Paris, France.
I
I
Lebbar
F
F
Stetzkowski-Marden
O
O
Mauffret
R
R
Cassoly
D000199:Actins / Q000097:blood / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism; D002263:Carboxyhemoglobin / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009132:Muscles / Q000378:metabolism; D011817:Rabbits; D013552:Swine; D014404:Tubulin / Q000097:blood / Q000378:metabolism*
D000199:Actins; D006454:Hemoglobins; D014404:Tubulin; D002263:Carboxyhemoglobin