Using desensitized actomyosin from chicken gizzard smooth muscle, we found that the activity of actin-myosin interaction, as determined by superprecipitation, is controled by both myosin-and thin filament-linked dual mechanism. While Ca"-and calmodulin-dependent phosphorylation of myosin light chain by myosin light chain kinase was prerequisite for the actin-myosin interaction, calmodulin-and caldesmonlinked flip-flop mechanism served as an on-off switch for the actin-myosin interaction. Presence of tropomyosin in the system was essential for the proper interaction of actin and myosin. The present results in combination with the previous work (29) provide evidence that this flip-flop switch is controled by the concentration of Ca" in such a way that, at decreased Ca" levels, association of caldesmon with actin filament eliminates the actin-myosin interaction and, at increased Ca" levels, Ca"dependent association of calmodulin with caldesmon dissociates actin filament from caldesmon thus making actin filament available for the interaction with myosin. Thus, Ca" controls the actin-myosin interaction of gizzard muscle through both myosin-linked and thin filament-linked regulatory systems and the effects of Ca" on both regulatory systems are mediated by calmodulin.

Previously, we have purified a spectrin-like calmodulin-binding protein termed calspectin from a membrane fraction of brain (14, 15). Calspectin, composed of oc (240,000-Mr) and ,6' (235,000-M,) subunits, is located predominantly in membranes and can be released from them with media used for the extraction of spectrin from erythrocyte ghosts but not with Triton X-100. By correlative biochemical and electronmicroscopic studies, we now provide evidence that the calspectin molecule undergoes dimer-tetramer interconversion in solution (existing as a tetramer in 0.1 M .I{Cl, and as a dimer in 0.6 M KCI). Although the dimer and tetramer are both bound to actin filaments, only the tetramer can cross-link actin to form a viscous gel. Electron microscopic images of calspectin molecules obtained by a low angle rotaryshadowing technique show extended, flexible rod-like shapes of 220 nm (tetramer) in length, which is very similar to the reported images of erythrocyte spectrin. The heterodimetric form appears to consist of on and ,8 monomers lying side by side.The tetrameric form appears to consist of two heterodimers joined head-to-head, with the actin binding sites appearing at the tail ends of the dimers. Calspectin and fodrin (24), which is recently purified and characterized as a spectrin-like calmodulinbinding protein (10), are probably one and the same.

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.

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, 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.

We used rotary-shadowing electron microscopy to map the calmodulin-and actin-binding sites on the brain spectrin, calspectin (or fodrin). Calspectin dimers appeared as rods 110 nm long and joined in a head-to-head manner to form tetramers 220 nm long. We determined calmodulinbinding sites by a ferritin-labeling method combined with biotin-avidin complex formation. Ferritin particles were found to attach to the head parts of calspectin dimers at a position 10-20 nm from the top of the head. The number of the calmodulin-binding sites seemed to be only one for each dimer and two for each tetramer. In contrast, the actin-binding sites were localized at the tail ends of the calspectin molecules. The tetramers attached to muscle F-actin with their tail ends and often cross-linked adjacent filaments. The results are discussed in view of the analogy to the erythrocyte spectrin.

The seasonal variation in protein expression in the sporophyte of Saccharina japonica (Areschoug) Lane, Mays, Druehl and Saunders was investigated. High-quality proteins that are available for protein profiling were extracted by the ethanol/phenol extraction method, and 564 protein spots in total were detected. Proteins were identified through database search by combining Mascot and MS BLAST for 100 spots, and significant difference of expression level between the samples collected in winter and in summer was observed in the case of 95 spots. Within 67 spots upregulated in the samples collected in summer, vanadium-dependent bromoperoxidase (vBPO) were identified for 21spots. It is thought that the elevation of expression level of vBPO in summer depend on the activation of the functions: (1) elimination of active oxygen species and protection of the algal body from oxygen injury, (2) prevention of the growth inhibition due to the adherence of attached organisms, in the season.

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