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.
Kurl
R N
RN
Department of Pathology, George Washington University Medical Center, Washington, DC 20037.
Verney
E
E
Sidransky
H
H
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