Selective surface radioiodination of keratinocytes in primary culture labels a 59 kD keratin and other surface proteins.
Murine keratinocytes were vectorially labeled in order to identify and characterize surface proteins. Freshly trypsinized keratinocytes were obtained from neonatal BALB/c mice and primary cultures were established. Cells were radiolabeled with iodogen and 125I immediately after harvesting and after 4, 24, 48, and 72 h in culture. Cells were solubilized with 1% sodium dodecyl sulfate (SDS) and reducing agents (total extract), or sequentially extracted with: (a) 1% Triton X-100 (membrane/cytosol fraction); (b) 2 M NaCl (salt fraction); and (c) 1% sodium dodecyl sulfate (SDS; cytoskeleton fraction). Extracts were analyzed by 1- or 2-D polyacrylamide gel electrophoresis (PAGE), followed by autoradiography (AR). The 59 kD acidic murine keratin was identified by immunoblot analysis with monoclonal antibody AE1 and a human polyclonal anti-59 kD keratin autoantibody designated Cascas-42. Total extracts contained up to ten labeled proteins ranging from 10 to 180 kD. Eight of these proteins were present in the membrane/cytosol fraction (10, 12, 18, 30, 38, 41, 66, and 130 kD). Intense labeling of a 59 kD cytoskeletal protein was consistently seen; this protein was identified as the 59 kD acidic murine keratin (Moll's catalogue no. 10) by immunoblot analysis of extracted proteins separated by 1-D and 2-D PAGE. A 180 kD protein had variable solubility characteristics, fractionating with either the cytoskeleton or membrane/cytosol as a function of time. No labeled proteins were detected in the salt extract, and neither actin nor other major cytoskeletal proteins were radiolabeled. These studies demonstrate the cell-surface disposition of at least ten keratinocyte proteins, and suggest that the 59 kD murine acidic keratin has a surface-exposed domain.
Dugan
E M
EM
Department of Dermatology, Johns Hopkins Hospital, Baltimore, Md. 21205.
Labib
R S
RS
Anhalt
G J
GJ
Diaz
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D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000032:analysis; D003600:Cytosol / Q000032:analysis; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D015603:Keratinocytes / Q000032:analysis*; D007633:Keratins / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis*; D051379:Mice; D008970:Molecular Weight; D014508:Urea / Q000031:analogs & derivatives
D000911:Antibodies, Monoclonal; D003598:Cytoskeletal Proteins; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; C012385:1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril; D007633:Keratins; D014508:Urea