Identification of a vimentin-reactive Peptide associated with ocular lens membranes as cytokeratin.
We wished to identify a 44-kD peptide isolated from lens membrane fractions which has apparent immunoreactivity to antivimentin and to antiphakinin. Urea-soluble proteins from rat lens membrane fractions were separated by two-dimensional electrophoresis, transferred to polyvinylidene fluoride membranes and probed with monoclonal antibodies to vimentin or cytokeratins, or a polyclonal antibody to phakinin. The monoclonal antibody to vimentin recognized the expected protein (M(r) = 56 kD, pI = 5.1) and several smaller and more acidic peptides including a 44-kD spot with a pI of 4.9. A similar pattern of immunoreactivity was found with commercially available purified vimentin. The polyclonal antibody to phakinin recognized the expected protein (M(r) = 52 kD, pI = 5.0) and several smaller and more acidic peptides, including a 44-kD spot with a pI of 4.9. Matrix-assisted laser desorption ionization mass spectroscopy analysis of the tryptic digest of the spot corresponding to 44 kD and pI of 4.9 identified neither vimentin nor phakinin but did identify peptides derived from ovalbumin (added to samples as an internal standard) and cytokeratin 1. Antibodies which recognize cytokeratin 1 reacted weakly with a 44-kD peptide which comigrated with the 44-kD vimentin-immunoreactive peptide in adjacent lanes of single-dimension SDS polyacrylamide gels. The 44-kD acidic (pI = 4.9) peptide which reacts with a monoclonal antibody to vimentin may be derived from cytokeratin 1.
Brooks
J
J
Truman State University, Kirksville College of Osteopathic Medicine, Kirksville, Mo., USA.
Fleschner
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D000818:Animals; D000911:Antibodies, Monoclonal; D002462:Cell Membrane / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D005136:Eye Proteins / Q000032:analysis; D007381:Intermediate Filament Proteins / Q000032:analysis; D007526:Isoelectric Point; D007633:Keratins / Q000032:analysis*; D007908:Lens, Crystalline / Q000737:chemistry*; D008565:Membrane Proteins / Q000032:analysis*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010455:Peptides / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014746:Vimentin / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D005136:Eye Proteins; D007381:Intermediate Filament Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D010455:Peptides; D014746:Vimentin; C084808:phakinin; D007633:Keratins