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Proteolysis of cardiac gap junctions during their isolation from rat hearts

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Summary

Gap junctions (GJ) isolated from rat hearts in presence of the protease inhibitor phenylmethylsulfonylfuoride (PMSF) contain a Mr 44,000 to 47.000 major polypeptide and have a urea-resistant layer of fuzz on their cytoplasmic surfaces, whereas junctions isolated without PMSF are proteolyzed to a Mr 29.500 polypeptide by a serine protease and have smooth cytoplasmic surfaces (C.K. Manjunath, G.E. Goings & E. PageAm. J. Physiol. 246:H865–H875, 1984). Rat liver GJ isolated with or without PMSF contain a Mr 28,000 polypeptide and have smooth cytoplasmic surfaces. Here we examine the origin, type and inhibitor sensitivity of the heart protease; why similar proteolysis is absent during isolation of rat liver gap junctions; and whether the Mr 44.000 to 47,000 cardiac GJ polypeptide is the precursor of the Mr 29,500 subunit. We show that the Mr 44,000 to 47,000 polypeptide corresponds to the unproteolyzed connexon subunit; that proteolysis of this polypeptide occurs predominantly during exposure to high ionic strength solution (0.6m KI) which releases serine protease from mast cell granules; that this protease is inhibitable with PMSF and (less completely) soybean trypsin inhibitor and chymostatin; and thatin vivo degranulation of mast cells by injecting rats with compound 48/80 fails to prevent breakdown of cardiac GJ during isolation. The results support the concept that GJ from rat heart and liver differ in protein composition.

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References

  • Alcala, J., Bradley, R., Huszak, J., Waggoner, P., Maisel, H. 1978. Biochemical and structural features of chick lens gap junctions.J. Cell Biol. 19:219a

    Google Scholar 

  • Bird, J.W.C., Carter, J.H. 1980. Proteolytic enzymes in striated and non-striated muscle.In: Degradative Processes in Heart and Skeletal Muscle. K. Wildenthal, editor. pp. 51–85. North Holland Biomedical, Amsterdam

    Google Scholar 

  • Brookhuyse, R.M., Kuhlmann, E.D., Stols, A.L.H. 1976. Lens membranes. II. Isolation and characterization of the main intrinsic polypeptide (MIP) of bovine lens fiber membranes.Exp. Eye Res 23:365–371

    PubMed  Google Scholar 

  • Busch, W.A., Stromer, M.H., Goll, D.E., Suzuki, A. 1972. Ca2+-specific removal of Z lines from rabbit skeletal muscle.J. Cell Biol. 52:367–381

    PubMed  Google Scholar 

  • Croall, D.E., De Martino, G.N. 1983. Purification and characterization of Ca2+-dependent proteases from rat heart.J. Biol. Chem. 258:5660–5665

    Google Scholar 

  • Dayton, W.R., Goli, D.E., Zeece, M.G., Robson, R.M., Reville, W.J.A. 1976. Ca2+-activated protease possibly involved in myofibrillar protein turnover. Purification from porcine muscle.Biochemistry 15:2150–2158

    PubMed  Google Scholar 

  • Drabikowski, W., Gorecka, A., Jakubiec-Puka, A. 1977. Endogenous proteinases in vertebrate skeletal muscle.Int. J. Biochem. 8:61–71

    Google Scholar 

  • Duckworth, W.C., Heinemann, M.A., Kitabachi, A.E. 1972. Purification of insulin-specific protease by affinity chromatography.Proc. Natl. Acad. Sci. USA 69:3698–3702

    PubMed  Google Scholar 

  • Everitt, M.T., Neurath, H. 1979. Purification and partial characterization of an α-chymotrypsin-like protease of rat peritoneal mast cells.Biochimie 61:653–662

    PubMed  Google Scholar 

  • Goodenough, D.A. 1979. Lens gap junctions: A structural hypothesis for non-regulated low-resistance intercellular pathways.Invest. Ophthalmol. Vis. Sci. 18:1104–1122

    PubMed  Google Scholar 

  • Griffin, W.S.T., Wildenthal, K. 1978. Myofibrillar alkaline protease activity in rat heart and its responses to some interventions that alter cardiac size.J. Mol. Cell. Cardiol. 10:669–676

    PubMed  Google Scholar 

  • Gros, D.B., Nicholson, B.J., Revel, J.-P. 1983. Comparative analysis of the gap junction protein from rat heart and liver: Is there a tissue specificity of gap junctions?Cell 35:539–549

    PubMed  Google Scholar 

  • Henderson, D., Eibl, H., Weber, K. 1979. Structure and biochemistry of mouse hepatic gap junctions.J. Mol. Biol. 132:193–218

    PubMed  Google Scholar 

  • Hertzberg, E.L. 1980. Biochemical and immunological approaches to the study of gap junctional communication.In Vitro 16:1057–1067

    PubMed  Google Scholar 

  • Hertzberg, E.L., Gilula, N.B. 1979. Isolation and characterization of gap junctions from rat liver.J. Biol. Chem. 254:2138–2147

    Google Scholar 

  • Hertzberg, E.L., Morganstern, R.A., Gilula, N.B. 1978. Isolation and characterization of gap junctions from rat. mouse and bovine liver.J. Cell Biol. 79:223a

    Google Scholar 

  • Huisman, W., Lanting, L., Doddema, H.J., Bouma, J.M.W., Gruber, M. 1974. Role of individual cathepsins in lysosomal protein digestion as tested by specific inhibitors.,Biochim. Biophys. Acta 370:297–307

    PubMed  Google Scholar 

  • Kensler, R.W., Goodenough, D.A. 1980. Isolation of mouse myocardial gap junctions.J. Cell Biol. 86:755–764

    PubMed  Google Scholar 

  • Kirschke, H., Langner, J. Wiederanders, B., Ansorge, S., Bohley, P. 1977. Cathepson L: A new proteinase from rat liver lysosomes.Eur. J. Biochem. 74:293–301

    PubMed  Google Scholar 

  • Koszalka, T.R., Miller, L.L. 1960. Proteolytic activity of rat skeletal muscle. I. Evidence for the existence of an enzyme active optimally at pH 8.5 to 9.0.J. Biol. Chem. 235:669–672

    PubMed  Google Scholar 

  • Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London) 227:680–686

    Google Scholar 

  • Libby, P., Goldberg, A.L. 1978. Leupeptin, a protease inhibitor, decreases protein degradation in normal and diseased muscles.Science 199:534–536

    PubMed  Google Scholar 

  • Libby, P., Goldberg, A.L. 1980a. Effects of chymostatin and other proteinase inhibitors on protein breakdown and proteolytic activities in muscle.Biochem. J. 188:213–220

    PubMed  Google Scholar 

  • Libby, P., Goldberg, A.L. 1980b. The control and mechanism of protein breakdown in striated muscle: Studies with selective inhibitors.In: Degradative Processes in Heart and Skeletal Muscle. K. Wildenthal, editor, pp. 201–222. North Holland Biomedical, Amsterdam

    Google Scholar 

  • Manjunath, C.K., Goings, G.E., Page, E. 1982a. Isolation and protein composition of gap junctions from rabbit hearts.Biochem. J. 205:189–194

    PubMed  Google Scholar 

  • Manjunath, C.K., Goings, G.E., Page, E. 1982b. Protein composition of cardiac gap junctions: Comparison between mammalian species and between junctions from rat heart and liver.J. Cell Biol. 95:88a

    Google Scholar 

  • Manjunath, C.K., Goings, G.E., Page, E. 1984a. Detergent sensitivity and splitting of isolated liver gap junctions.J. Membrane Biol. 78:147–155

    Google Scholar 

  • Manjunath, C.K., Goings, G.E., Page, E. 1984b. Cytoplasmic surface and intramembrane components of rat heart gap junctional proteins.Am. J. Physiol. 246:H865-H875

    Google Scholar 

  • Manjunath, C.K., Page, E. 1984. Cytoplasmic surface and intramembrane components of rat heart gap junctional protein.Biophys. J. 45:22

    Google Scholar 

  • McKee, E.E., Clark, M.G., Beinlich, C.J., Lins, J.A., Morgan, H.E. 1979. Neutral-alkaline proteases and protein degradation in rat heart.J. Mol. Cell. Cardiol. 11:1033–1051

    PubMed  Google Scholar 

  • Morgan, H.E., Chua, B., Beinlich, C.J. 1980. Regulation of protein degradation in heart.In: Degradative Processes in Heart and Skeletal Muscle. K. Wildenthal, editor, pp. 87–112. North Holland Biomedical. Amsterdam

    Google Scholar 

  • Murakami, U., Uchida, K. 1978. Purification and characterization of a myosin-cleaving protease from rat heart myofibrils.Biochim. Biophys. Acta 525:219–229

    PubMed  Google Scholar 

  • Nicholson, B.J., Hunkapiller, M.W., Grim, L.B., Hood, L.E., Revel, J.-P. 1981. Rat liver gap junction protein: Properties and partial sequence.Proc. Natl. Acad. Sci. USA 78:7594–7598

    PubMed  Google Scholar 

  • Nicholson, B.J., Takemoto, L.J., Hunkapiller, M.W., Hood, L.E., Revel, J.-P. 1983. Differences between liver gap junction protein and lens MIP 26 from rat: Implications for tissue specificity of gap junctions.Cell 32:967–978

    Article  PubMed  Google Scholar 

  • Noguchi, T., Kandatsu, M. 1976. Some properties of alkaline protease in rat muscle compared with that in peritoneal cavity cells.Agric. Biol. Chem. 40:927–933

    Google Scholar 

  • Pastan, I., Almqvist, S. 1966. Localization of rat thyroid alkaline protease to mast cells.Endocrinology 78:361–366

    PubMed  Google Scholar 

  • Reddy, M.K., Etlinger, J.D., Rabinowitz, M., Fischman, D.A., Zak, R. 1975. Removal of Z-lines and α-actinin from isolated myofibrils by a calcium-activated protease.J. Biol. Chem. 250:4278–4284

    PubMed  Google Scholar 

  • Sanada, Y., Yasogawa, N., Katunuma, N. 1978. Crystallization and amino acid composition of a serine protease from rat skeletal muscle.Biochem. Biophys. Res. Commun. 82:108–113

    PubMed  Google Scholar 

  • Uchida, K., Murakami, U., Hiratsuka, T. 1977. Purification of cardiac myosin from rat heart. Proteolytic cleavage and its inhibition.J. Biochem. (Tokyo) 82:469–476

    Google Scholar 

  • Waxman, L., Krebs, E.G. 1978. Identification of two protease inhibitors from bovine cardiac muscle.J. Biol. Chem. 253:5888–5891

    PubMed  Google Scholar 

  • Woodbury, R.G., Everitt, M., Sanada, Y., Katunuma, N., Lagunoff, D., Neurath, H. 1978a. A major serine protease in rat skeletal muscle: Evidence for its mast cell origin.Proc. Natl. Acad. Sci. USA 75:5311–5313

    PubMed  Google Scholar 

  • Woodbury, R.G., Gruzenski, G.M., Lagunoff D. 1978b. Immunofluorescent localization of a serine protease in rat small intestine.Proc. Natl. Acad. Sci. USA 75:2785–2789

    PubMed  Google Scholar 

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  1. Department of Medicine, The University of Chicago, 60637, Chicago, Illnois

    Chellakere K. Manjunath, Gwendolyn E. Goings & Ernest Page

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  1. Chellakere K. Manjunath
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  2. Gwendolyn E. Goings
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  3. Ernest Page
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Manjunath, C.K., Goings, G.E. & Page, E. Proteolysis of cardiac gap junctions during their isolation from rat hearts. J. Membrain Biol. 85, 159–168 (1985). https://doi.org/10.1007/BF01871268

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