Purification and characterization of erythrocyte caldesmon. Hypothesis for an actin-linked regulation of a contractile activity in the red blood cell membrane.
We have previously shown that in human or pig whole erythrocytes, only a single 71-kDa polypeptide cross-reacts with the affinity-purified antibody to pig platelet caldesmon (der Terrossian et al., 1989). In the present paper, we demonstrate that this polypeptide represents a genuine caldesmon which remains attached to the membrane prepared in the presence of an excess of free Mg2+ but not in its absence. Immunoreactivity of this peptide is specific towards the antibody to pig platelet caldesmon since it is not labelled with antibodies to other components of the red cell membrane. Erythrocyte caldesmon was purified to 95% homogeneity and displays well known characteristics of caldesmons from other sources. Together with tropomyosin, it has the ability to regulate platelet actin-activated rabbit skeletal muscle myosin ATPase activity. The stoichiometry of 1 caldesmon/1 tropomyosin/7-9 actin molecules indicates that the amount of caldesmon, in the red cell membrane, corresponds precisely to the amount of tropomyosin. Immunofluorescent labelling of whole erythrocytes gave similar punctate patterns with purified antibodies to myosin, to caldesmon, to tropomyosin and to actin (but not to spectrin), suggesting colocalization of these proteins. Together, and for the first time, our results give strong evidence that caldesmon, bound on the actin protofilament, might represent the inhibitory component, so far uncharacterized, of a thin-filament-like system in erythrocyte.
der Terrossian
E
E
INSERM U29. Laboratoire de Port Royal, Paris, France.
Deprette
C
C
Lebbar
I
I
Cassoly
R
R
D000199:Actins / Q000097:blood*; D000251:Adenosine Triphosphatases / Q000097:blood; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000097:blood* / Q000302:isolation & purification; D003285:Contractile Proteins / Q000097:blood* / Q000302:isolation & purification; D003599:Cytoskeleton / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism / Q000502:physiology*; D004912:Erythrocytes / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D011817:Rabbits; D013552:Swine
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D003285:Contractile Proteins; D000251:Adenosine Triphosphatases; D009218:Myosins; D008274:Magnesium