Isolation and characterization of a 58-kDa membrane- and microfilament-associated protein from ascites tumor cell microvilli.
A 58-kDa protein is found in microvilli and in actin-containing transmembrane complexes of 13762 ascites tumor cells with immobile surface receptors; it is absent from sublines with mobile receptors. 58-kDa protein has been proposed to stabilize microvilli and restrict receptor mobility by stabilizing membrane-microfilament interactions. Antibodies against 58-kDa protein were blot-purified from antisera of rabbits injected with crude transmembrane complex and were used to monitor purification of the protein. 58-kDa protein was extracted from EDTA/EGTA-stripped microvillar microfilament cores with 1 M NaCl. A single depolymerization-polymerization cycle of the microfilaments, followed by solubilization of 58-kDa protein in 1 M NaCl and chromatography on hydroxyapatite-Sephadex G-150, purified the protein to greater than 95% homogeneity. The native molecular weight and frictional coefficient indicated a monomeric, asymmetric structure. 58-kDa protein bound F-actin in pelleting assays and inhibited polymerization of pyrenyl-actin. It also bound phosphatidylserine, phosphatidylinositol, and phosphatidylcholine vesicles in pelleting studies. Immunoblot analyses of endogenously and exogenously proteolyzed microvilli and their membranes and microfilament cores showed specific membrane and microfilament binding fragments of 28-30 kDa. The microfilament- and phospholipid-binding properties of 58-kDa protein and the localization of its proteolysis products are consistent with its proposed role in stabilizing membrane-microfilament interactions in the ascites cell microvilli.
Liu
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Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101.
Carraway
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KL
Carraway
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CA
D000199:Actins / Q000378:metabolism; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000032:analysis*; D002845:Chromatography; D002850:Chromatography, Gel; D017886:Durapatite; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D006882:Hydroxyapatites; D015151:Immunoblotting; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008840:Microfilament Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008871:Microvilli / Q000648:ultrastructure*; D008970:Molecular Weight; D011485:Protein Binding
D000199:Actins; D006882:Hydroxyapatites; D008565:Membrane Proteins; D008840:Microfilament Proteins; D017886:Durapatite; D004492:Edetic Acid