PubMed1981
A procedure is described that is designed to identify the primary site of cross-linking by bisimidoesters of a component of an interacting protein system. It is based on the mutually exclusive nature of acetylation and amidination. The procedure has been applied to the regulatory protein of lactose synthase, alpha-lactalbumin. A sample of bovine alpha-lactalbumin was acetylated with a trace amount of high specific activity [3H]acetic anhydride to produce a population of protein molecules essentially all of which contain 0 to 1 acetyl group; partial labeling of all 13 amino groups was obtained. This material was mixed with bovine colostrum galactosyltransferase in the presence of Mn2+, UDP-glucose, and N-acetylglucosamine, at pH 8.0, to promote complex formation and was cross-linked with dimethyl 3,3'-dithiobispropionimidate. Covalently cross-linked alpha-lactalbumin-galactosyltransferase (1:1) complex with characteristic enzymic and other properties was purified from the reaction mixture, and the distribution of [3H]acetyl label on each amino group of the alpha-lactalbumin component was determined, using procedures similar to those described in previous differential labeling studies (Richardson, R., and Brew, K. (1980) J. Biol. Chem. 255, 3377-3385). In comparison with the original labeled sample used for cross-linking, the specific activity of tritium label in 10 amino groups showed little change, whereas the labeling of three groups was changed markedly. The acetyl moiety on the epsilon-amino groups of lysines 5 and 108 showed major decreases in specific activity while that of lysine 114 was greatly increased. Similar results were obtained when the cross-linking was performed under different conditions of temperature, and cross-linker concentration. As the changes in lysines 5 and 114 are similar to those observed in differential labeling, they are attributed to alterations in the affinity for galactosyltransferase resulting from acetylation of these groups. In contrast, lysine 108, which is not sufficiently close to the interaction site to be perturbed in differential labeling studies but is greatly decreased in tritium content in the cross-linked complex, appears to represent the major site through which alpha-lactalbumin is cross-linked to galactosyltransferase as a result of the exclusion of protein molecules acetylated in this position from covalent cross-linking. Studies with a homologous series of bisimidoesters indicate that lysine 108 is situated 6.1 to 7.3 A degrees from an amino group on galactosyltransferase in the cross-linked complex. The general utility of the procedure and the nature of the interaction site in lactose synthase are discussed.