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Isolation and characterization of a 58-kDa membrane- and microfilament-associated protein from ascites tumor cell
Y Liu1, K L Carraway, C A Carraway
1Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101.
Abstract:
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.
Insights
A 58-kDa protein stabilizes microvilli by anchoring membrane receptors to the cytoskeleton in ascites tumor cells. This protein
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Microvilli are crucial for nutrient absorption and cell adhesion.
- Surface receptor mobility is linked to cell function and signaling.
- The interaction between membrane proteins and the actin cytoskeleton is vital for cellular structure.
Purpose of the Study:
- To identify and characterize a protein involved in stabilizing microvilli and restricting receptor mobility.
- To elucidate the molecular mechanisms underlying membrane-microfilament interactions in ascites tumor cells.
Main Methods:
- Antibody-based purification of the 58-kDa protein.
- Biochemical extraction and chromatographic purification.
- F-actin binding assays and vesicle binding studies.
- Proteolysis and immunoblot analysis.
Main Results:
- The 58-kDa protein was purified to >95% homogeneity and found to have an asymmetric structure.
- The protein binds F-actin, inhibits actin polymerization, and interacts with phospholipid vesicles.
- Specific fragments of the protein bind to both membranes and microfilaments.
Conclusions:
- The 58-kDa protein plays a key role in stabilizing microvilli by linking membrane receptors to the actin cytoskeleton.
- Its interaction with both actin filaments and phospholipids contributes to its function.
- This protein is absent in tumor cell sublines with mobile receptors, suggesting its role in restricting mobility.