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PMID: 6538573 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Studies on the spectrin-like protein from the intestinal brush border, TW 260/240, and characterization of its interaction with the cytoskeleton and actin.

The Journal of cell biology ·Vol. 98 ·No. 1 ·1984-01-00 ·Pages 66-78

Pearl M, Fishkind D, Mooseker M, Keene D, Keller T

Abstract

The terminal web of the intestinal brush border contains a spectrin-like protein, TW 260/240 (Glenney, J. R., Jr., P. Glenney, M. Osborne, and K. Weber, 1982, Cell, 28:843-854.) that interconnects the "rootlet" ends of microvillar filament bundles in the terminal web (Hirokawa, N., R. E. Cheng, and M. Willard, 1983, Cell, 32:953-965; Glenney J. R., P. Glenney, and K. Weber, 1983, J. Cell Biol., 96:1491-1496). We have investigated further the structural properties of TW 260/240 and the interaction of this protein with actin. Salt extraction of TW 260/240 from isolated brush borders results in a loss of terminal web cross-linkers primarily from the apical zone directly beneath the plasma membrane. Morphological studies on purified TW 260/240 using the rotary shadowing technique confirm earlier results that this protein is spectrin-like and is in the tetrameric state in buffers of low ionic strength. However, examination of TW 260/240 tetramers by negative staining revealed a molecule much straighter and more uniform in diameter than rotary-shadowed molecules. At salt concentrations at (150 mM KCl) and above (300 mM KCl) the physiological range, we observed a partial dissociation of tetramers into dimers that occurred at both 0 degree and 37 degrees C. We also observed (in the presence of 75 mM KCl) a concentration-dependent self-association of TW 260/240 into sedimentable aggregates. We have studied the interaction of TW 260/240 with actin using techniques of co-sedimentation, viscometry, and both light and electron microscopy. We observed that TW 260/240 can bind and cross-link actin filaments and that this interaction is salt- and pH-dependent. Under optimum conditions (25-75 mM KCl, at pH 7.0) TW 260/240 cross-linked F-actin into long, large-diameter bundles. The filaments within these bundles were tightly packed but loosely ordered. At higher pH (7.5) such bundles were not observed, although binding and cross-linking were detectable by co-sedimentation and viscometry. At higher salt (greater than 150 mM KCl), the binding of TW 260/240 to actin was inhibited. The presence of skeletal muscle tropomyosin had no significant effect on the salt-dependent binding of TW 260/240 to F-actin.

MeSH Terms
Actins/metabolism Animals Carrier Proteins/metabolism Chickens Cytoskeleton/metabolism Hydrogen-Ion Concentration Microfilament Proteins Microvilli/ultrastructure Protein Binding Protein Conformation Spectrin/metabolism Temperature Tropomyosin/metabolism
Chemicals
Actins Carrier Proteins F-actin-binding proteins Microfilament Proteins Tropomyosin Spectrin
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Pearl M
Fishkind D
Mooseker M
Keene D
Keller T
References (51)
51 references, click to expand
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.98.1.66
Published
1984-01-00
Pages
66-78
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2112984
Subset
IM
Grants
NIADDK NIH HHS · AM25387 · United States
External Links
PubMed source DOI: 10.1083/jcb.98.1.66 PMC Full Text View journal details
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