Abstract
Physarum contains at least two distinct DNase I-binding proteins, i.e. actin and Cap 42 (a + b). The latter, a tight (1:1) complex of Cap 42 (a) and Cap 42 (b) (Maruta, H., Isenberg. G., Schreckenbach, T., Hallmann, R., Risse, G., Schibayama, T., and Hesse, J. (1983) J. Biol. Chem. 258, 10144-10150), is a Ca2+-dependent F-actin capping protein. DNase I binds to Cap 42 (b) but not to Cap 42 (a). Consequently, DNase I-agarose was used for an affinity-purification of Cap 42 (a + b), after its separation from actin by DEAE-cellulose chromatography. Cap 42 (a + b) was dissociated into its subunits when released from DNase I-agarose by 8.8 M formamide. The two subunits were subsequently separated from each other on hydroxylapatite. Both Cap 42 (a) and Cap 42 (b) were Ca2+-dependent F-actin capping proteins that cap the fast growing end of actin filaments and block actin polymerization at this end. Like Cap 42 (a + b), Cap 42 (b) required Ca2+ for its capping activity only when phosphorylated. The phosphorylation of Cap 42 (b) was completely blocked by DNase I or a tertiary complex of Cap 42 (a), actin, and Ca2+. Cap 42 (b) is not identical with native (= polymerizable) actin because (i) Cap 42 (b) was unable to form filaments, (ii) the Cap 42 (b) kinase did not phosphorylate native actin, and (iii) fragmin formed a tight (1:1) complex with native actin but not with Cap 42 (b). Although it is unlikely that Cap 42 (b) is simply a denatured form of actin that has lost its polymerizability during the preparation, it still remains to be clarified whether Cap 42 (b) is a nonpolmerizable actin variant derived from a distinct actin gene or a post-translationally modified form of polymerizable actin.
Highlights
Physarurn contains at least two distinct DNase I- blocks actin polymerization at this end, and inducesrapid
Cap 42 was dissociated into its subunits when released from DNase Iagarose by 8.8 M formamide
Cap 42 (a) alone requires Ca2+ for its capping activity( 5 ) .unlike fragmin, Cap 42 (a) has no F-actinsevering activity( 5 ) .Cap 42 (b) isa phosphoprotein whose phosphorylation is completely inhibited by an equimolar complex of Cap 42 (a) and actin only in the presence of Ca2+(4)
Summary
CL were used as the source for Cap 42 (a b), Cap 42 (b) kinase, Physarum contains at leastfour proteins of 42,000 daltons which are functionally distinguishable from each other [1,2,3,4,5]. MM NaCl eluate fromDEAE-cellulose, containing Cap (a b) and Cap 42 (b) kinase,was further fractionated on 25 ml of DNase I-. + DNase I-Agarose Affinity Chromatography of Cap 42 (a the 75 mM phosphate eluatew, hereas mostof Cap 42 (a)was recovered in the 150 mM phosphate eluate Both fractions were subsequently dialyzed against TDSA buffer and kept onice. Further Purification of Cap 42 (b) Kinase-The 1.0 M NaCl eluate from DNase I-agarose, containing the Cap (b) kinase,was further fractionated on Sephadex G-150 (superfine, 1.6 X 60 cm, Pharmacia) equilibrated with TDA buffer containing 0.1 M NaC1, 1 mM EGTA, b)-We used DNase I-agaroseaffinity chromatographyto. Short fragments of S - 1 decorated and fixed actin filaments were used as peared in the flow through fractions, all the F-actin capping activity andmost of the Cap (b) kinase activity were bound to DNase I-agarose
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