Hydroxylamine cleavage of proteins in polyacrylamide gels.
A modification of the hydroxylamine cleavage of proteins is presented in which proteins were cleaved while immobilized in the matrix of a polyacrylamide gel. The reaction under these conditions retains its high specificity for Asn-Gly bonds and has the advantage that the gel matrix, acting as a carrier, facilitates simultaneous treatment of many samples, and contributes to a high recovery efficiency (60-90%) of the cleavage products. The cleavage is performed with individual protein bands excised from dried slab gels after detection by staining, autoradiography, or fluorography. The procedure can be easily combined with other techniques to further characterize the cleavage fragments. Also a two-dimensional version of the cleavage method was developed, which allows rapid recognition of interrelationships between proteins in a complicated mixture. The versatility of the procedure is demonstrated in a number of applications. Highly related strains of murine leukemia viruses were easily distinguished from one another by the unique cleavage patterns of their gag- and env-precursor polypeptides. Comparing the env-precursor gPr82env synthesized in the presence or absence of tunicamycin with its cell-free synthesized counterpart, revealed the presence of an amino-terminal signal sequence. Cleavage patterns of pro-opiomelanocortin (POMC) from three different species revealed a high degree of homology between rat and mouse POMC, whereas Xenopus POMC was very different. Regions to which carbohydrates are attached could be identified by comparing glycosylated and unglycosylated forms of POMC. Combining the hydroxylamine cleavage procedure with immunological characterization of the fragments showed a small but significant difference between the amino-terminal sequences of the recombinant transforming protein P120 of Abelson murine leukemia virus and of its parent molecule Pr65gag of Moloney murine leukemia virus.
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006898:Hydroxylamines; D051379:Mice; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis*; D010908:Pituitary Hormones, Anterior / Q000302:isolation & purification; D011333:Pro-Opiomelanocortin; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification*; D051381:Rats; D014764:Viral Proteins / Q000302:isolation & purification; D014771:Virion / Q000235:genetics; D014981:Xenopus
D006898:Hydroxylamines; D010455:Peptides; D010908:Pituitary Hormones, Anterior; D011498:Protein Precursors; D011506:Proteins; D014764:Viral Proteins; D011333:Pro-Opiomelanocortin