| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1993: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1992: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
Talin purified by the new method was found to bind to both F-and G-actin : Talin cosedimented with F-actin. On gel filtration of a mixture of talin and G-actin, a comprex of talin and actin was obtained. Talin stimulated the polymerization rate of G-actin. Interactions among the three major constituents of focal adhensions, talin, actin, and alpha-actinin, were studied. Both talin and alpha-actinin increased the rate and extent of polymerization of actin, and their effects were additive. Whereas talin alone exhibited very little actin-gelating activity, it potentiated markedly the gelation in the presence of alpha-actinin and lowered the concentration of alpha-actinin necessary for the gel formation. Its gelation-potentiating activity on prepoplymerized actin was much smaller than observed on G-actin. Treatment of talin with a cross-linking reagent, EDC or dimethyl suberimidate, resulted in the formation of its oligomeric polypeptides. The comprexes of talin and G-actin were also demon
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strated with the cross-linking reagents and fluorescence-labeled actin. On limited digestion of talin with caplain, it was cleaved into an N-terminal 47-kD fragment and a C-terminal 190-kD fragment. The 190-kD fragment showed comparable or higher levels of activities to bind to actin and to stimulate nucleation and gelation of actin asobserved for the intact talin, while the 47-kD fragment did not exhibit any significant levels of these activities. Anti-chicken gizzard talin antibodies raised against rabbits wre used for detecting talin and its proteolytic fragments in various chicken tissues including the brain, gizzard, skeletal muscle, cardiac muscle and oviducts. Most of these tissue extracts were found to contain high levels of immuno-reactive 190-kD polypeptide(s)in addition to the intact 230-kD polypeptide, indicating that the talin was very suspectible to proteolytic degradation during preparation of tissue extracts or possibly even in living cells.
These results indicate that the C-terminal domein plays crutial roles in organizing the cytoskeletal elements. On the other hand, N-terminal domein is likely to play some role in the interaction with membrane elements. Less
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