Purification Actin

Key words Actin purification and assembly, Actin-binding proteins, Dictyostdium, NEM-myosin II, TIRF microscopy... 

We again consider a network crosslinked by actin binding protein. The number of crosslinks that are formed. A, is given by Equation 10, and a plot of G vs (the total amount of added ABP) is shown, schematically, in Figure 3. Both the slope and the intercept provide information about the kinetic parameters of network formation. Elasticity determinations clearly can be used to assess the amount of crosslinking protein in an assembly if all other conditions are kept constant, G varies linearly with consequently, after appropriate calibration (in principle with only two data points), elasticity measurements could be used for quantitative assessment of the efficacy of biochemical purification procedures. Parenthetically, we note that if conditions can be arranged such that K S 1, gives a direct measure of the number of nuclei iIq. 

Actin-binding proteins ABP-50, purification, 196, 78 ABP-120, purification, 196, 79 ABP-240 purification, 196, 76 effect on actin depolymerization, 215, 74 extraction, 196, 311 isolation from Dictyostelium discoideum, 196, 70 platelet-derived actin binding proteins [characterization, 215, 58 purification, 215, 58, 64 recombination with actin, 215, 73] 30-kDa Dictyostelium discoideum actin-crosslinking protein [assays, 196, 91 preparation, 196, 84] actin-depolymerizing factor [assay, 196, 132[ DNase assay, 196, 136 platelet-derived a-actinin [characterization, 215, 58 purification, 215, 58, 70 recombination with actin, 215, 73]. 

Pollard, T. D. (1984). Purification of a high molecular weight actin filament gelation protein from Acanthamoeba that shares antigenic determinants with vertebrate spectrins. /. Cell Biol. 99, 1970-1980. 

Zhang,J. Q., Weisberg, A., and Horowits, R. (1998). Expression and purification of large nebulin fragments and their interaction with actin. Biophys. J. 74, 349-359. 

Li, Y., Hua, F., Carraway, K.L., and Carothers Carraway, C.A. 1999. The pl85" -contain-ing glycoprotein complex of a microfilament-associated signal transduction particle. Purification, reconstitution, and molecular associations with p58 and actin. J Biol Chem 274(36) 25651-25658. 

The exact function of the annexin fold family is at present unclear. All of these proteins appear to show calcium-dependent binding to phosphatidylethanolamine or phosphatidylinositol liposomes. In addition, they can promote fusion of liposomes, and because of this property, it has been suggested that these proteins might mediate calcium dependent exocytosis. P36 and p35 have also been shown to bind to F-actin and spectrin [65,66]. Recently, Khanna et al. [70] have reported a procedure for the simultaneous purification of p35, p36 oligomer and p36 monomer from bovine lung, and identified all three proteins as substrates of protein kinase C. Furthermore, the work of Huang et al. [86] and Khanna et al. [69] has suggested that all three proteins are inhibitors of phospholipase A2. Further experiments will be required to clarify the function of these proteins. 

Shapland, C., Hsuan, ).)., Totty, N. F., Lawson, D. ). (1993). Purification and properties of transgelin a transformation and shape change sensitive actin-gelling protein. Cell Biol. 121,1065-1073. 

Machesky LM, Atkinson SJ, Ampc C et al. Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose. J Cell Biol 1994 127 107-15. 

The cross-axis CPC (coil planet centrifuge), with column holders at the off-center position on the rotary shaft, enables retention of the stationary phase of aqueous-aqueous polymer-phase systems such as PEG 1000-potassium phosphate and PEG 8000-dex-tran T500 [3,4], Since the last decade, various types of cross-axis CPC (types XL, XLL, XLLL, and L) have been developed for performing CCC with highly viscous aqueous polymer-phase systems. The separation and purification of protein samples, including lactic acid dehydrogenase [5], recombinant enzymes, profiUn-actin complex, and so on, were achieved using these cross-axis CPCs [6]. 

Sobue, K., Muramoto, K., Fujita, M. and Kakiuchi, S. (1981). Purification of a calmodulin-binding protein from chicken gizzard that interacts with F-actin. Proc. Natl. Acad. Sci. USA 78, 5652-5655. 

Pardee JD, Spudich JA (1982) Purification of muscle actin. In Methods Enzymology 85 164-81... 

Ohishi I, Hama Y (1992) Purification and characterization of heterologous component lls of botulinum C2 toxin. In Microbiol Immunol. 36 221 -9 Ohishi I, Iwasaki M, Sdkaguchi G. (1980) Purification and characterization of two components of botulinum 02 toxin. In Infect Immun. 30 668-73 Ohishi I, Miyake M (1985) Binding of the two components of 02 toxin to epithelial cells and brush borders of mouse intestine. In Infect Immun. 48 769-75 Ohishi I, Tsuyama S (1986) ADP-ribosylation of nonmuscle actin with component I of 02 toxin. In Biochem Biophys Res Comm. 136 802-6 Ohishi I, Yanagimoto A (1992) Visualizations of binding and internalization of two nonlinked protein components of botulinum 02 toxin in tissue culture cells. In Infect Immun. 60 4648-55... 

Derivation Chlorination of benzene in actinic light. Method of purification Fractional crystallization. The technical grade may run 10-15% y isomer but can be brought up to 99% (lindane). 

Fractionation and Purification. The differences in extractability make it possible to begin purification at the earliest stage by fractional extraction. This process has been exploited most in the purification of actin by Straub (1942-3). Most of the L-myosin and the globular proteins are removed from fresh brei by initial extraction with salt solutions (I = 0.6), and the residual sacroplasmic proteins are removed by repeated washing with water at alkaline pH the unextracted L-myosin is then denatured with acetone. When the dried residue is extracted... 

Tropomyosin extract prepared according to Bailey (1948) contains proteins of the myogen group and denatured G-actin (Dubuisson, 1950e). For complete purification a series of precipitations and crystallization are necessary (Bailey, 1948). 

The purification of GST-tagged DdVASP is described here exemplarily. The purification of other actin-binding proteins fused to GFP can be performed accordingly. 

Lu PJ, Shieh WR, Rhee SG, Yin HL, Chen CS (1996) Lipid products of phosphoinositide 3-kinase bind human profilin with high affinity. Biochemistry 35 14027—14034 Machesky LM, Atkinson SJ, Ampe C, Vandekerckhove J, Pollard TD (1994a) Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose. J Cell Biol 127 107-115... 

Maun, NA., Speicher, D.W., EMNubile, M.J. and Southwick, F.S. (1996). Purification and properties of a Ca -independent barbed-end actin filament capping protein, CapZ, from human polymorphonuclear leukocytes. Biochem. 35, 3518-3524. 

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