Pyridoxal phosphate as a probe of the cytoplasmic domains of transmembrane proteins: application to the nicotinic acetylcholine receptor.
A novel procedure has been developed to specifically label the cytoplasmic domains of transmembrane proteins with the aldehyde pyridoxal 5-phosphate (PLP). Torpedo californica acetylcholine receptor (AcChR) vesicles were loaded with [3H]pyridoxine 5-phosphate ([3H]PNP) and pyridoxine-5-phosphate oxidase, followed by intravesicular enzymatic oxidation of [3H]PNP at 37 degrees C in the presence of externally added cytochrome c as a scavenger of possible leaking PLP product. The resulting Schiff's bases between PLP and AcChR amino groups were reduced with NaCNBH3, and the pyridoxylated proteins were analyzed by fluorography. The four receptor subunits were labeled whether the reaction was carried out on the internal surface or separately designed to mark the external one. On the other hand, the relative pyridoxylation of the subunits differed in both cases, reflecting differences in accessible lysyl residues in each side of the membrane. Proteinase K treatment of labeled AcChR vesicles generated a peptide of 13 kDa that could be detected with anti-PLP antibodies only when the pyridoxylation was carried out on the internal surface of the vesicles. Even though there are no large differences in the total lysine content among the subunits and there are two copies of the alpha-subunit, internal surface labeling by PLP was greatest for the highest molecular weight (delta) subunit, reinforcing the concept that the four receptor subunits are transmembranous and may protrude into the cytoplasmic face in a fashion [Strader, C. D., & Raftery, M. A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5807-5811] that is proportional to their subunit molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)
Perez-Ramirez
B
B
Division of Molecular Biology and Biochemistry, School of Basic Life Sciences, University of Missouri, Kansas City 64110.
Martinez-Carrion
M
M
D000345:Affinity Labels; D000818:Animals; D002463:Cell Membrane Permeability; D003593:Cytoplasm / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005615:Freezing; D006868:Hydrolysis; D008565:Membrane Proteins / Q000032:analysis*; D010447:Peptide Hydrolases; D011732:Pyridoxal Phosphate / Q000032:analysis* / Q000138:chemical synthesis; D011734:Pyridoxaminephosphate Oxidase / Q000302:isolation & purification; D011978:Receptors, Nicotinic / Q000032:analysis* / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D014101:Torpedo
D000345:Affinity Labels; D008565:Membrane Proteins; D011978:Receptors, Nicotinic; D011732:Pyridoxal Phosphate; D011734:Pyridoxaminephosphate Oxidase; D010447:Peptide Hydrolases