Purification of p100, a protein antigenically related to the signal transducing G proteins Gt and Gi. Evidence for an adaptin-like protein.
A 100-kDa protein, termed p100, cross-reacts with antisera raised against a synthetic peptide corresponding to the carboxyl-terminal decapeptide of the alpha-subunit of the retinal G protein Gt. p100 is abundantly expressed in liver and, on subcellular fractionation of rat liver homogenates, is distributed between the cytosolic and microsome fractions (Traub, L. M., Evans, W. H., and Sagi-Eisenberg, R. (1990) Biochem. J. 272, 453-458; Udrisar, D., and Rodbell, M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6321-6325). We have now purified p100 to near-homogeneity from rat liver microsomes. The protein was purified approximately 500-fold by ATP extraction followed by a series of four chromatographic steps. Similar to partially purified p100, on two-dimensional electrophoresis, the final preparation contained a major series of five immunoreactive 100-kDa charge isoforms. Partial amino terminus amino acid sequencing of the purified protein revealed that p100 is a previously unidentified protein. Further analysis of the soluble form of p100 showed the protein migrated with an apparent molecular weight of approximately 110,000 on gel filtration, indicating that the soluble protein occurs as a monomeric polypeptide. The soluble form of p100 was also partially purified from rat liver cytosol and amino acid sequencing yielded the same amino-terminal sequence as obtained from the microsome-associated form. The amino-terminal sequence of p100 exhibits significant similarity to the deduced amino-terminal amino acid sequences of both alpha- and gamma-adaptins. Using the amino-terminal sequence from p100, we have raised antipeptide polyclonal antisera. The antisera reacted specifically with the purified 100-kDa protein on immunoblots. With the purified protein and specific antisera now available, it will be possible to explore the physiological role of p100.
Traub
L M
LM
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Sagi-Eisenberg
R
R
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002853:Chromatography, Liquid; D003429:Cross Reactions; D003600:Cytosol / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D000939:Epitopes; D019204:GTP-Binding Proteins / Q000276:immunology* / Q000302:isolation & purification* / Q000378:metabolism; D006868:Hydrolysis; D008862:Microsomes, Liver / Q000737:chemistry*; D008969:Molecular Sequence Data; D051381:Rats; D011919:Rats, Inbred Strains; D012689:Sequence Homology, Nucleic Acid; D015398:Signal Transduction
D000939:Epitopes; D019204:GTP-Binding Proteins