Identification and immunohistochemical localization of a tryptophan binding protein in nuclear envelopes of rat liver.
A tryptophan binding protein which was identified by binding studies has been purified to apparent homogeneity from rat liver nuclear envelopes. Two affinity matrices, namely, concanavalin A-agarose and tryptophan-agarose, were utilized for purification of the binding protein. Findings with lectin affinity chromatography suggested that the binding entity was a glycoprotein since it could be eluted off the column with methyl alpha-D-mannopyranoside (0.2 M). Eluates from both columns, when electrophoresed separately (under denaturing conditions) on polyacrylamide gels, revealed the presence of a protein with an apparent molecular weight of approximately 33,000-34,000 which is the same as that observed when covalently bound (i.e., crosslinked) [3H]-tryptophan is analyzed on polyacrylamide gels under denaturing conditions and then autoradiographed. Polyclonal antibodies raised against the binding protein recognized polypeptides with molecular weights of 64,000 and 33,000-34,000 when analyzed by the Western blot technique, suggesting that the protein was probably a dimer. Immunohistochemical studies revealed that the antigen is localized in the nuclear membranes, thereby corroborating our biochemical premise that the binding protein was present in the nuclear envelopes.
Department of Pathology, George Washington University Medical Center, Washington, DC 20037.
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RN
Kurl
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E
Verney
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Sidransky
D000818:Animals; D002352:Carrier Proteins / Q000032:analysis*; D002458:Cell Fractionation; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005629:Frozen Sections; D008099:Liver / Q000378:metabolism*; D008297:Male; D009685:Nuclear Envelope / Q000378:metabolism*; D009687:Nuclear Proteins / Q000032:analysis*; D051381:Rats; D014364:Tryptophan / Q000378:metabolism*
D002352:Carrier Proteins; D009687:Nuclear Proteins; D014364:Tryptophan