The effects of a 45 000 molecular weight protein from unfertilized sea urchin eggs and its 1:1 actin complex on actin filaments.
A 45 kDa actin-binding protein (SU45) has been isolated previously from egg extracts of the Hawaiian sea urchin Tripneustes gratilla by DEAE Sephacel, Sephadex G-75 and hydroxyapatite chromatography. Using pyrene-labelled rabbit skeletal muscle actin, we have found that when SU45 is added to actin in the presence of calcium and the salt concentration is increased, the initial rate of actin assembly is accelerated. Moreover, the final polymer concentration is reduced indicating that SU45 caps the preferred end of actin filaments shifting the critical concentration (Cc) to that of the nonpreferred end. Determination of the Cc as a function of the concentration of SU45 gave an apparent KD of 1 nM. Dilution of F-actin to below its Cc, into buffers containing SU45 and Ca2+ resulted in a sharp increase in the rate of depolymerization; reducing the Ca2+ concentration attenuated this effect. Incubation of SU45 with rabbit skeletal muscle G-actin yielded a 1:1 complex which held 45Ca2+ tightly with a dissociation half-time of 10.8 days. By kinetic analyses of assembly in the presence of the SU45-actin complex and dilution-induced disassembly of filaments precapped with complex, we have estimated both the association rate constant (4.0 X 10(4)M-1s-1) and the dissociation rate constant (0.05s-1) for the nonpreferred ends of actin filaments. Finally, dilution of F-actin to below its Cc, into complex in either Ca2+ or EGTA resulted in a much slower depolymerization consistent with a rapid capping of the preferred end by the SU45-actin complex.
D000199:Actins / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D005453:Fluorescence; D018260:Gelsolin; D008840:Microfilament Proteins; D008970:Molecular Weight; D010063:Ovum / Q000032:analysis; D011108:Polymers; D011817:Rabbits; D012617:Sea Urchins
D000199:Actins; D002352:Carrier Proteins; D018260:Gelsolin; D008840:Microfilament Proteins; D011108:Polymers; C016953:brevin; D004533:Egtazic Acid; D002118:Calcium