Kouichi Morimoto

Actin polymerization induced by calspectin, a calmodulin-binding spectrinlike protein

We have purified from a membrane fraction of bovine brain a calmodulin‐binding protein (calspectin) that shares a number of properties with erythrocyte spectrin: It has a heterodimeric structure with M r 240 000 and 235 000 and binds to (dimeric form) or crosslinks (tetrameric form) F‐actin. We show that calspectin (tetramer) is capable of inducing the polymerization of G‐actin to actin filaments by increasing nucleation under conditions where actin alone polymerizes at a much slower rate. Thus, brain calspectin behaves in the same manner as erythrocyte spectrin, supporting the idea that, in conjunction with actin oligomers it comprises the cytoskeletal meshwork underlying the cytoplasmic surface of the nerve cell.

Caldesmon, a calmodulin-binding, F actin-interacting protein, is present in aorta, uterus and platelets

Caldesmon, a protein originally found in chicken gizzard, was concluded also to be present in bovine aorta, uterus, and human platelets by demonstration of a protein with the following properties: (a) Ca2+‐dependent calmodulin‐binding; (b) binding to F actin in such way that the binding was broken on Ca2+‐dependent binding of calmodulin; (c) cross‐reactivity in immune blotting procedures with affinity‐purified antibody against gizzard caldesmon; (d) similar subunit M r‐values on SDS‐gel to those of gizzard caldesmon. Like gizzard caldesmon, platelet caldesmon was composed of two polypeptide bands of M r 150000 and 147000, but caldesmon in aorta and uterus gave a single band of M r 150000. A polypeptide of M r 165000 that was immunologically distinct from caldesmon but, like caldesmon, bound to calmodulin and F actin in a flip‐flop fashion, was also demonstrated in aorta and uterus.

Reconstitution of Ca2+-sensitive gelation of actin filaments with filamin, caldesmon and calmodulin

The discovery of Ca2+-activatable cyclic nucleotide phosphodiesterase [1 ] and the subsequent demonstration of a protein factor which confers Ca2÷-sensitivity upon this enzyme [2,3] coincided with the discovery of a protein activator of brain phosphodiesterase [4]. The identity of the 2 proteins as a Ca2÷-binding protein (calmodulin) was established subsequently [5]. The structural similarity of calmodulin and troponin Cs [6,7] suggests that both proteins may have stemmed from a common ancestral protein and may perform analogous functions as 4-domain calcium receptive proteins. However, surprisingly few studies have been done with regard to the interaction of calmodulin with components of the contractile or cytoskeletal system except for myosin light chain kinase. Calmodulin associates with skeletal muscle troponin components to form a soluble hybrid complex and neutralizes the inhibitory action of troponin I on the actomyosin ATPase activity [8]. A similar observation was made in [9]. We have purified, from erythrocytes [10,11 ], brain [12] and chicken gizzard smooth muscle [13,14], actin-related proteins that bind to calmodulin in the presence of Ca 2÷. The calmodulinbinding protein from chicken gizzard, named caldesmon, interacted with calmodulin and F actin in the presence or absence, respectively, of Ca 2÷ and forma. tions of the 2 species of protein complexes is regulated by [Ca 2+] in a flip-flop fashion [14]. Here, this mechanism is extended to the control of the filamininduced gelation of actin filaments: calmodulincaldesmon system conferred Ca2+-sensitivity upon the

Assignment of amides in the amino acid sequence of mammalian calmodulin by mass spectrometry

The primary structure of mammalian calmodulin from various sources was analyzed by secondary ion mass spectrometry. The assignments of amide groups at residues 24, 60, 129 and 135, which had been controversial in the previous reports, were in perfect agreement with the amino acid sequence deduced from the cDNA sequence of calmodulin of other vertebrates. The result is against the post-translational amidation or deamidation process of this protein.