[14] The isolation and characterization of plasma membrane from cultured chicken embryo fibroblasts

1974 ◽  
pp. 162-168 ◽  
Author(s):  
James F. Perdue
Keyword(s):  
Plasma Membrane ◽  
Chicken Embryo ◽  
Isolation And Characterization ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

scholarly journals Membrane association of a 36,000-dalton substrate for tyrosine phosphorylation in chicken embryo fibroblasts transformed by avian sarcoma viruses.

1983 ◽  
Vol 97 (5) ◽  
pp. 1601-1611 ◽  
Author(s):  
K Radke ◽  
V C Carter ◽  
P Moss ◽  
P Dehazya ◽  
M Schliwa ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Chicken Embryo ◽  
Cytoskeletal Proteins ◽  
Immunofluorescent Staining ◽  
Transformed Cells ◽  
Light Density ◽  
Embryo Fibroblasts ◽  
Actin Cables ◽  
Avian Sarcoma ◽  
Chicken Embryo Fibroblasts

A cellular protein of 36,000 daltons becomes phosphorylated at tyrosine in chicken embryo fibroblasts transformed with avian sarcoma viruses. We have used cellular fractionation and immunofluorescence to locate the 36-kdalton protein in virus-transformed and uninfected chicken fibroblasts. The 36-kdalton protein in transformed cells fractionated mainly with high-speed particulate material, and in density gradient separations, the 36-kdalton protein was found in association with light density membranes together with most of the plasma membrane marker. Increasing the concentration of salt or adding ion chelators solubilized some of the 36-kdalton protein that otherwise was pelletable with high g forces. Based on these data, we conclude that this protein is peripherally or indirectly attached to light density membranes, including plasma membranes. Indirect immunofluorescent staining of the 36-kdalton protein in fixed cells revealed that it was located inside the cell in an extensive reticulum apposed to surface membranes. The same pattern of staining was found in both uninfected and virus-transformed cells. Pretreatment of cells with nonionic detergents before fixation altered or abolished 36-kdalton staining. The 36-kdalton protein appeared to be excluded from regions of the cells where actin cables were present. The pattern of staining observed with the anti-36-kdalton antibody was similar, but not identical, to that observed with antiserum against nonerythroid spectrin. Thus, the data obtained by biochemical fractionation and by immunofluorescent staining indicate that the 36-kdalton protein is found in a reticulum at the inner surface of the plasma membrane, possibly in association with cytoskeletal proteins.

scholarly journals HSP47: a tissue-specific, transformation-sensitive, collagen-binding heat shock protein of chicken embryo fibroblasts.

1991 ◽  
Vol 11 (8) ◽  
pp. 4036-4044 ◽  
Author(s):  
K Hirayoshi ◽  
H Kudo ◽  
H Takechi ◽  
A Nakai ◽  
A Iwamatsu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Heat Shock ◽  
Amino Acid Sequence ◽  
Heat Shock Protein ◽  
Chicken Embryo ◽  
Noncoding Region ◽  
Isolation And Characterization ◽  
Embryo Fibroblasts ◽  
Retention Signal ◽  
Chicken Embryo Fibroblasts

We report the isolation and characterization of a cDNA clone encoding HSP47, a transformation-sensitive heat shock protein that binds to collagen. A cDNA library was prepared from total RNA isolated from heat-shocked chicken embryo fibroblasts and screened by using oligonucleotide mixtures prepared on the basis of the N-terminal amino acid sequence of biochemically purified HSP47. The cDNA insert contained 3,278 bp, which encoded a 15-amino-acid signal peptide and a mature protein coding region consisting of 390 amino acid residues; it also included part of the 5' noncoding region and a long 3' noncoding region. The deduced amino acid sequence revealed an RDEL sequence at the C terminus, which is a variant of the KDEL retention signal for retention of proteins in the endoplasmic reticulum. Northern (RNA) blot analyses and nuclear run-on assays established that the induction of HSP47 by heat shock and its suppression after transformation of chicken embryo fibroblasts by Rous sarcoma virus are regulated at the transcriptional level. A homology search revealed that this protein belongs to the serpin family, the superfamily of plasma serine protease inhibitors. Although structurally homologous to the serpins, HSP47 lacks the active site thought to be essential for the inhibition of proteases and does not appear to bind to intracellular proteases. HSP47 is the first heat shock protein found to be a member of the serpin superfamily. Conversely, it is the first serpin family member that is not secreted from cells, which could be explained by acquisition of the RDEL retention signal during evolution.

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