Isolation and characterization of a 30,000-dalton calcium-sensitive actin cross-linking protein from Dictyostelium discoideum.

M Fechheimer,D L Taylor

doi:10.1016/s0021-9258(17)43076-6

Abstract

A calcium-sensitive actin-binding protein having a subunit molecular mass of 30,000 daltons (30K protein) has been isolated from Dictyostelium discoideum. Structural, immunological, and functional analyses demonstrated that the 30K protein was distinct from other actin-binding proteins of D. discoideum. A native molecular mass of 31,700 daltons was determined by equilibrium sedimentation, indicating that the protein is monomeric. The Stokes radius was 30 A. The frictional coefficient calculated from these measurements was 1.44, indicating an asymmetric shape. The 30K protein induced an increase in the viscosity of a solution of F-actin. Bundles of actin filaments were observed in negatively stained mixtures of actin and the 30K protein. Both the formation of filament bundles and the increases in viscosity of actin induced by the 30K protein were observed in the presence of 1 X 10(-8) M but not 2 X 10(-6) M calcium. Variation of the pH from 6.6 to 7.8 had no effect on the activity of the 30K protein. Calcium induced neither a large change in quaternary structure of the 30K protein nor a restriction of the lengths of actin filaments by the 30K protein. The apparent affinity of the 30K protein for actin was decreased in the presence of calcium. Reversible cross-linking of actin filaments by the 30K protein may contribute to regulation of the consistency and contractility of cytoplasm in D. discoideum.

Highlights

From the Department of Biological Sciences and Center for Fluorescence Research in Biomedical Sciences, Carnegie-Mellon University, Pittsburgh, Pennsylvania 15213
A calcium-sensitive actin-binding protein having a ture may influence contractility, since conditions which insubunit molecular mass of 3 0, 0 0 0 daltons (30K pro- duce solation of the myosin-depleted fractions initiate contein) has been isolated from Dictyostelium discoideum. traction in myosin-containing fractions
The fricand contractilityof cytoplasm in D. discoideumamoebae have beenidentified.Actin and myosin have been purifiedfrom tional coefficient calculated from these measurements was 1.44, indicating an asymmetric shape

Summary

Molecular weightwas determined by sedimentation in a Beckman

Model E analytical centrifugeusing the highspeed meniscus depletion sedimentationequilibrium method (Yphantis,1964).The 30K protein (0.4 mg/ml) was dialyzed versus 20mM Pipes, 50 mM KCI, 1 mM. EGTA, 1 mM EDTA, 1 mM dithiothreitol, 0.02% NaN3, pH 7.0. The dialysate was used asthe reference solution. Molecular weight was calculated from the slope of a plot of log protein concentration uersus the square of the distancefrom the axis of rotation in ninedifferent intervals within the ultracentrifuge cell. The results are expressed as the average molecular weight & S.E. of the mean. Present insolutions containing 5 mM EGTA, calcium, 1.5 mM MgCI2, and 1mM ATP at pH7.0 were kindly provided by J. The frictional coefficient and axial ratio of the 30,000-dalton protein were estimated from its Stokes radius and native molecular weight (Scheraga, 1961). After centrifugation at 200,000 X g for 90 min, the G-actin was adjusted to 3.2 mg/ml and polymerized by addition of 50 mM KC1

RESULTS

Protein a bcdefg

The native molecular weight was directly determined by

DISCUSSION