45] Macrophage actin-binding protein

Abstract

This chapter presents procedure for the purification of actin-binding protein from rabbit lung macrophages. The purification procedure involves following steps: homogenization of the cells; extract gelation; chromatography on 4% agarose; and DEAE-sepharose chromatography. actin-binding protein derived from the columns can be stored in 50% glycerol at –20 ° without the loss of actin sedimentation activity. sedimentation to be a useful assay for the quantitation of the crosslinking. The sedimentation assay measures actin filament aggregation. Filaments polymerized from purified muscle actin are extremely long but sufficiently dispersed under physiological conditions of ionic strength and pH that they sediment slowly at low g forces. Therefore, the aggregation of these filaments by the addition of small quantities of a crosslinking agent can bring about the more rapid sedimentation of a relatively large quantity of actin protein at relatively low g forces. The assay accurately reflects the capacity of proteins to crosslink actin filaments into gel networks, and the results concur with direct measurements of the critical gel point for solutions of actin, a The aggregation of the filaments is directly proportional to the crosslinker density. Therefore, sedimentation can be measured at concentrations of actin-binding protein both below and above the gel point, and the sedimentation rate is directly proportional to the amount of actin-binding protein in the assay.