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Nonmuscle tropomyosin from ascites tumor cell microvilli
Abstract:
Tropomyosin has been isolated from microvilli preparations from 13762 rat mammary adenocarcinoma ascites tumor cells by Triton extraction and pelleting of the microvillar microfilament core, extraction of the microfilament core with 1 M KCl, heat treatment, and hydroxyapatite chromatography. Three major isoforms, designated 31K-a (acidic), 31K-b (basic), and 29K, were identified as tropomyosins by two-dimensional isoelectric focusing-dodecyl sulfate electrophoresis, a urea shift on dodecyl sulfate electrophoresis, chemical cross-linking, amino acid analysis, and molecular weight determinations. The native (60,000) and subunit (31,000 and 29,000) molecular weights, the amino acid composition, and the stoichiometry for binding to F-actin (actin/tropomyosin, 6:1) were typical of nonmuscle tropomyosins. The amount of tropomyosin present in the microvilli preparations is sufficient to saturate about half of the microvillar F-actin. By two-dimensional isoelectric focusing-dodecyl sulfate electrophoresis, the 31K isoforms appeared similar to isoforms of normal rat kidney cells but the 29K isoform was apparently smaller than any normal rat kidney isoforms. All three isoforms bound to F-actin, but the 29K form bound most strongly. Its behavior was similar to that of muscle tropomyosin, exhibiting saturable binding as a function of both ionic strength and Mg2+ concentration. In contrast, the 31K isoforms bound more weakly and required higher concentrations of Mg2+ for binding than that required for saturation with 29K (4 mM). These results clearly indicate that nonmuscle tropomyosin isoforms from a single source and location (subplasmalemmal) in the cell can exhibit different properties.
Insights
Researchers identified three distinct tropomyosin isoforms in rat tumor cell microvilli. These nonmuscle tropomyosins exhibit varied F-actin binding properties, with one isoform resembling muscle tropomyosin.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Tropomyosin is a key actin-binding protein involved in muscle contraction and nonmuscle cellular functions.
- Understanding tropomyosin isoforms in cancer cells is crucial for elucidating their roles in cell structure and motility.
Purpose of the Study:
- To isolate and characterize tropomyosin isoforms from microvilli of 13762 rat mammary adenocarcinoma ascites tumor cells.
- To investigate the biochemical and functional properties of these nonmuscle tropomyosin isoforms, particularly their interaction with F-actin.
Main Methods:
- Isolation of microvillar core filaments using Triton extraction and high salt concentrations.
- Purification of tropomyosin isoforms via heat treatment and hydroxyapatite chromatography.
- Characterization using 2D electrophoresis, urea shift electrophoresis, cross-linking, amino acid analysis, and F-actin binding assays.
Main Results:
- Three tropomyosin isoforms (31K-a, 31K-b, and 29K) were identified, with molecular weights and amino acid compositions typical of nonmuscle tropomyosins.
- The 29K isoform exhibited strong, saturable F-actin binding similar to muscle tropomyosin, influenced by ionic strength and Mg2+.
- The 31K isoforms bound more weakly to F-actin and required higher Mg2+ concentrations for saturation compared to the 29K isoform.
Conclusions:
- Nonmuscle cells contain diverse tropomyosin isoforms with distinct F-actin binding characteristics.
- The 29K isoform's unique binding properties suggest specialized roles in microvillar structure or function within the tumor cell.
- These findings highlight the functional heterogeneity of tropomyosin isoforms even within a single cellular location.