phalloidine and Ascites

The nuclear fraction isolated from Krebs II ascites cells following cell disruption by nitrogen cavitation was separated into four fractions by salt/detergent extraction: NP-40 soluble fraction, 130 mM KCl extract, DOC/Triton x 100 soluble fraction and salt/detergent treated nuclei. The protein composition of the individual fractions was studied by SDS-PAGE and the relative amounts of actin and a 35 kDa protein (p35) were measured from gel scans. There was a time-dependent shift of actin from the 130 mM KCl extract to the NP-40 soluble fraction upon storage of the nuclear fraction on ice, indicating a progressive depolymerization of microfilaments. Compared with actin there was a slower release of p35 into the NP-40 soluble fraction. The results suggest that p35 is not integrated in the microfilament network. Phalloidin, which stabilizes the microfilaments, enriched the amount of both proteins in the 130 mM KCl extracts, together with a series of other proteins in the range 50-205 kDa. The presence of phalloidin also resulted in a large increase in the actin content in both the DOC/Triton x 100 extract and the fraction containing salt/detergent treated nuclei. Incubation of cells with insulin and/or cycloheximide enriched the amount of actin in the 130 mM KCl fraction. The results show that short term incubation of cells with phalloidin, insulin or cycloheximide increases the actin content of the nuclear fraction and also affects the presence of several other proteins.

Topics: Actins; Animals; Ascites; Cell Nucleus; Cycloheximide; Insulin; Mice; Mice, Inbred BALB C; Microfilament Proteins; Molecular Weight; Neoplasm Proteins; Phalloidine

[14C]Glucosamine metabolic labeling and concanavalin A blots were used to identify four major glycoprotein species associated with ascites tumor cell microvillar microfilament cores and with a transmembrane complex containing actin. Phalloidin shift analysis of glucosamine-labeled microvilli showed that glycoproteins of 110-120, 80, 65, and 55 kDa are stably associated with the microfilament cores. Analysis of large (greater than 10(6) kDa) transmembrane complexes from microvillar membranes made under microfilament-depolymerizing conditions (Carraway, C. A. C., Jung, G., and Carraway, K. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 430-434) revealed glycoproteins of the same Mr values, showing the same relative staining or labeling patterns as those observed with the microfilament cores. Gel filtration of high salt, high pH extracts of intact microvilli, microfilament cores, or transmembrane complexes showed that in all of these fractions the glycoproteins are associated in a very large, stable complex. The glycoprotein multimer was isolated essentially free of actin and other components by Sephacryl S-1000 chromatography of microvilli, microvillar membranes prepared at pH 11, microfilament cores, or transmembrane complex fractions in Triton X-100, 1 M KCl, glycine, pH 9.5. Purified glycoprotein complex bound actin when incubated under polymerizing conditions. The presence of the glycoprotein heteromultimer in both microfilament cores and transmembrane complex from isolated membranes and the association of the purified glycoprotein complex with actin are consistent with our hypothesis that the glycoprotein-containing transmembrane complex is an association site for microfilaments at the plasma membrane.

Topics: Actins; Adenocarcinoma; Animals; Ascites; Blotting, Western; Chromatography, Gel; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Glucosamine; Membrane Glycoproteins; Microfilament Proteins; Microvilli; Phalloidine; Rats; Tumor Cells, Cultured

The association of microvillar microfilaments with the microvillar membrane actin-containing transmembrane complex of MAT-C1 13762 ascites tumor cell microvilli has been investigated by differential centrifugation, gel electrophoresis and electron microscopy of detergent extracts of the isolated microvilli. Several methods have been used to reduce breakdown and solubilization of the microfilament core actin during the detergent extractions for preparation of microvillar core microfilaments. Gel electrophoresis of differential centrifugation fractions demonstrated that over 70% of the total microvillus actin could be pelleted with microfilament cores at 10 000 g under extraction conditions which reduce filament breakdown. Transmission electron microscopy (TEM) of all of the core preparations showed arrays of microfilaments and small microfilament bundles. The major protein components of the microfilament cores, observed by sodium dodecyl sulfate (SDS) electrophoresis, were actin and alpha-actinin. Among the less prominent polypeptide components was a 58 000 Dalton polypeptide (58 K), previously identified as a member of the MAT-Cl transmembrane complex. This three-component complex contains, in addition to 58 K, actin associated directly and stably with a cell surface glycoprotein (Carraway, CAC, Jung, G & Carraway, K L, Proc. natl acad. sci. US 80 (1983) 430). Evidence that the apparent association of complex with the microfilament core was not due simply to co-sedimentation was provided by myosin affinity precipitation. These results provide further evidence that the transmembrane complex is a site for the interaction of microfilaments with the microvillar plasma membrane.

Topics: Actins; Adenocarcinoma; Animals; Ascites; Buffers; Cell Fractionation; Cell Line; Centrifugation; Cross-Linking Reagents; Cytoskeleton; Electrophoresis, Polyacrylamide Gel; Isoelectric Focusing; Mammary Neoplasms, Experimental; Microfilament Proteins; Microscopy, Electron; Microvilli; Phalloidine; Rats