Phalloidin shift on velocity sedimentation sucrose gradient centrifugation for identification of...

Velocity sedimentation by sucrose density gradient centrifugation has been used to characterize ascites microvillar microfilament cores and to identify microfilament-associated proteins. Fluoride, calcium, phalloidin and chemical cross-linking treatments of microvilli during Triton X-100 extractions increase the sedimentation rate of the microfilament core, compared with untreated control samples. Electrophoretic analyses of the distributions of actin, alpha-actinin and other microfilament-associated proteins across the gradients indicate that the primary mechanism for stabilization of the microfilament core is the reduction of fragmentation of the microfilaments. Significantly, alpha-actinin could be completely removed from the microfilaments by calcium treatment without causing a decrease in the size of the microfilament core. Because of the specificity of phalloidin in the stabilization of microfilaments, the shift on the gradients of microfilaments and their associated proteins in the presence of phalloidin provides a diagnostic tool for the identification of microfilament-associated proteins. This phalloidin shift technique should have widespread utility in the analysis of actin forms and microfilament-associated proteins from complex cell fractions.