Abstract
Intestinal brush-border-derived membrane vesicles contain, after demembranation in the presence of Ca2+, a subset of polypeptides that are specifically solubilized by the addition of Ca2+ chelators. As described previously, this fractionation scheme leads to the enrichment of two major proteins (I and II), one of which has been shown to be identical to the cellular p36K target of Rous sarcoma virus-encoded tyrosine-specific protein kinase (Gerke, V., and Weber, K., (1984) EMBO J. 3, 227-233). We have applied a similar protocol to membrane vesicles from porcine liver and purified a third Ca2+-binding protein (III). All three proteins had wide tissue distributions, and were absent from brain, red blood cells, and cardiac and skeletal muscle. Relative amounts varied between tissues, with protein I low in liver and protein III very low in intestine. Despite their similar extractability the three proteins (I, II, and III) are clearly distinct as far as immunological, biochemical, and physicochemical properties are concerned. They also show characteristic differences in their affinities for Ca2+ ions. The association constants of Ca2+ binding for proteins I and III have been estimated by means of indirect methods to be 10(4) M-1 (protein I) and 10(6) M-1 (protein III), while the direct Hummel-Dreyer method reveals Ca2+ binding to protein II, characterized by an association constant of 0.4 X 10(5) M-1 in the absence and 0.2 X 10(5) M-1 in the presence of 2 mM MgCl2. Conformational changes upon binding Ca2+ are described for protein II using circular dichroism, fluorescence emission, and UV difference spectra. These alterations could be attributed to an increased exposure of tyrosine and tryptophan residues to a more aqueous environment, and led to increased hydrophobicity of protein II that would explain the observed Ca2+-dependent interaction with hydrophobic matrices like phenyl-Sepharose.
Highlights
Intestinal brush-border-derived membrane vesicles that are believed to regulate the organization of the tightly contain, after demembranation in thepresence of Ca2+, packed actin bundles in microvilli and theunderlying terminal a subsetof polypeptides that arespecifically solubilized web
The pellets were washed in buffer for the isolation of proteins I and I1 from porcine brushborder-derived membrane vesicles (l)p,roteins I1 and I11 were purified from liver tissue (Fig. 1,A and B )
In a previous study [1]we describedthe isolation and partial characterization of two Ca2+-bindingproteins, proteins I and 11, from intestinal epithelial cells. Both proteins are retained in the Triton-insoluble fraction of brush-border membrane vesicles in the presence of Ca2+,but are released
Summary
Intestinal brush-border-derived membrane vesicles that are believed to regulate the organization of the tightly contain, after demembranation in thepresence of Ca2+, packed actin bundles in microvilli and theunderlying terminal a subsetof polypeptides that arespecifically solubilized web (for review see Refs. 4 and 5). Despite monomer of M , 32,000 whosefunction has yet to be defined Their similar extractability the three proteins (I, 11, Using a similar fractionation scheme, i.e. preparation of and 111)are clearly distinct as far as immunological, cytoskeletal fractions in thepresence of Ca2+and extraction biochemical, and physicochemical properties are con- with EGTA-containing buffers, polypeptides migrating at cerned. They show characteristic differences in similar apparent molecular masses (36 kDa, 32 kDa) in SDS their affinities for Ca” ions. A similar 68-kDa protein has been purified from EGTA extracts of human lymphocytes [18,19,20]
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