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Oligomerization of immunoglobulin-g heavy and light-chains in vitro - a cell-free assay to study the assembly of the endoplasmic-reticulum

Academic Article
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Overview

authors

  • Watkins, J. D.
  • Hermanowski, A. L.
  • Balch, William E.

publication date

  • March 1993

journal

  • Journal of Biological Chemistry  Journal

abstract

  • A biochemical assay to study the assembly of the endoplasmic reticulum (ER) in a cell-free system is introduced. Incubation in vitro of ER vesicles containing only immunoglobulin gamma 1 heavy (H) chains with ER vesicles containing only K light (L) chains results in fusion and oligomerization of the H and L chains to form the H2L2 complex (immunoglobulin G). ER fusion/H2L2 oligomerization is time and temperature dependent and requires energy in the form of ATP. It is stimulated by the addition of cytosol and requires protease-sensitive components present on the membranes. The addition of guanosine 5'-O-(thiotriphosphate) inhibits membrane fusion and subsequent H2L2 oligomerization without affecting the assembly of H2L2 from detergent-solubilized pools, suggesting an important role for GTPases in vesicle recognition or fusion. The development of a rapid and quantitative assay to study the assembly of the ER in a cell-free system will allow us to identify components involved in the recognition, fusion, and post-fusion events critical for ER function in vivo.

subject areas

  • Adenosine Triphosphate
  • Animals
  • Biopolymers
  • Cell-Free System
  • Cells, Cultured
  • Cytosol
  • Endopeptidases
  • Endoplasmic Reticulum
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Light Chains
  • Intracellular Membranes
  • Membrane Fusion
  • Mice
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

PubMed ID

  • 8444894
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Additional Document Info

start page

  • 5182

end page

  • 5192

volume

  • 268

issue

  • 7

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