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Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells. 1988

J M Anderson, and B R Stevenson, and L A Jesaitis, and D A Goodenough, and M S Mooseker
Department of Medicine and Liver Center, Yale School of Medicine, New Haven, Connecticut.

ZO-1, originally identified by mAb techniques, is the first protein shown to be specifically associated with the tight junction. Here we describe and compare the physical characteristics of ZO-1 from mouse liver and the Madin-Darby canine kidney (MDCK) epithelial cell line. The ZO-1 polypeptide has an apparent size of 225 kD in mouse tissues and 210 kD in canine-derived MDCK cells as determined by SDS-PAGE/immunoblot analysis. ZO-1 from both sources is optimally solubilized from isolated plasma membranes by either 6 M urea or high pH conditions; partial solubilization occurs with 0.3 M KCl. The nonionic detergents, Triton X-100 and octyl-beta-D-glucopyranoside, do not solubilize ZO-1. These solubility properties indicate that ZO-1 is a peripherally associated membrane protein. ZO-1 was purified to electrophoretic homogeneity from [35S]methionine metabolically labeled MDCK cells by a combination of gel filtration and immunoaffinity chromatography. Purified ZO-1 has an s20,w of 5.3 and Stokes radius of 8.6 nm. These values suggest that purified ZO-1 is an asymmetric monomeric molecule. Corresponding values for mouse liver ZO-1, characterized in impure protein extracts, were 6 s20,w and 9 nm. ZO-1 was shown to be a phosphoprotein in MDCK cells metabolically labeled with [32P]orthophosphate; analysis of phosphoamino acids from purified ZO-1 revealed only phosphoserine. ZO-1 epitope number was determined by Scatchard analysis of competitive and saturable binding of two different 125I-mAbs to SDS-solubilized proteins from liver and MDCK cells immobilized on nitrocellulose. Saturation binding occurs at 26 ng mAb/mg liver and 63 ng/mg of MDCK cell protein. This is equivalent to 30,000 ZO-1 molecules per MDCK cell assuming a single epitope/ZO-1 molecule.

UI MeSH Term Description Entries
D007118 Immunoassay A technique using antibodies for identifying or quantifying a substance. Usually the substance being studied serves as antigen both in antibody production and in measurement of antibody by the test substance. Immunochromatographic Assay,Assay, Immunochromatographic,Assays, Immunochromatographic,Immunoassays,Immunochromatographic Assays
D007365 Intercellular Junctions Direct contact of a cell with a neighboring cell. Most such junctions are too small to be resolved by light microscopy, but they can be visualized by conventional or freeze-fracture electron microscopy, both of which show that the interacting CELL MEMBRANE and often the underlying CYTOPLASM and the intervening EXTRACELLULAR SPACE are highly specialized in these regions. (From Alberts et al., Molecular Biology of the Cell, 2d ed, p792) Cell Junctions,Cell Junction,Intercellular Junction,Junction, Cell,Junction, Intercellular,Junctions, Cell,Junctions, Intercellular
D007668 Kidney Body organ that filters blood for the secretion of URINE and that regulates ion concentrations. Kidneys
D008099 Liver A large lobed glandular organ in the abdomen of vertebrates that is responsible for detoxification, metabolism, synthesis and storage of various substances. Livers
D008565 Membrane Proteins Proteins which are found in membranes including cellular and intracellular membranes. They consist of two types, peripheral and integral proteins. They include most membrane-associated enzymes, antigenic proteins, transport proteins, and drug, hormone, and lectin receptors. Cell Membrane Protein,Cell Membrane Proteins,Cell Surface Protein,Cell Surface Proteins,Integral Membrane Proteins,Membrane-Associated Protein,Surface Protein,Surface Proteins,Integral Membrane Protein,Membrane Protein,Membrane-Associated Proteins,Membrane Associated Protein,Membrane Associated Proteins,Membrane Protein, Cell,Membrane Protein, Integral,Membrane Proteins, Integral,Protein, Cell Membrane,Protein, Cell Surface,Protein, Integral Membrane,Protein, Membrane,Protein, Membrane-Associated,Protein, Surface,Proteins, Cell Membrane,Proteins, Cell Surface,Proteins, Integral Membrane,Proteins, Membrane,Proteins, Membrane-Associated,Proteins, Surface,Surface Protein, Cell
D008970 Molecular Weight The sum of the weight of all the atoms in a molecule. Molecular Weights,Weight, Molecular,Weights, Molecular
D010750 Phosphoproteins Phosphoprotein
D002460 Cell Line Established cell cultures that have the potential to propagate indefinitely. Cell Lines,Line, Cell,Lines, Cell
D002499 Centrifugation, Density Gradient Separation of particles according to density by employing a gradient of varying densities. At equilibrium each particle settles in the gradient at a point equal to its density. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Centrifugations, Density Gradient,Density Gradient Centrifugation,Density Gradient Centrifugations,Gradient Centrifugation, Density,Gradient Centrifugations, Density
D002846 Chromatography, Affinity A chromatographic technique that utilizes the ability of biological molecules, often ANTIBODIES, to bind to certain ligands specifically and reversibly. It is used in protein biochemistry. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Chromatography, Bioaffinity,Immunochromatography,Affinity Chromatography,Bioaffinity Chromatography

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