Gel Electrophoretic Isolation, in the Hundred Microgram Range, of Recombinant SDS-Syntaxin from Sea Urchin EGG Cortical Vesicles

摘要

Recombinant urchin syntaxin [Xa cut], electrophorsed at pH 9.0 (25 deg C) or 10.2 (0 deg C) in a discontinuous Tris-chloride-glycinate buffer system in the presence of 0.03% SDS in the catholyte, exhibits a multicomponent poattern in gels of a polyacrylamide concentration of 12% and 3% crosslinking. The position in the pattern of the syntaxin band was identified by reference to electropherograms of a previous study (OP.Baclund, pers. comm). The complexiity of the protein composition of the preparation was reduced by selective stacking of proteins with mobilities greater than that of syntaxin. This provides a gel pattern consisting of two bands with mobilities close to that identified as syntaxin, as well as a minor, more slowly migrating, contaminant. The two major components are designed as S1 and S2, the latter being the larger species. In the absence of SDS, the preparation exhibits two pairs of protein components. Three of the proteins are charge isomers, i.e., of equal size, differing only in net charge, assumed to be forms of S1, while the fourth component is larger and is assumed to be S2. Aliquots of the preparation, containing 150 #mu#g of portein were loaded on a cylindrical polyacrylamide gel of 18 mm diameter, and separated S1 and S2 were existed in a position defined by their charracteristic values of relative mobility (R_f). Two or three gel slices, corresponding in R_f to S1 or S2, were pooled and laoded onto a Stacking Gel (5% polyacrylamide, 20% crosslinked) of 18 mm diameter, equipped with a collection chamber of 200 #mu#L volume. The protein was electroeluted from the gel slices and concentrated into a stack by electrophoresis. The stack, marked by bromphenolblue, was allowed to migrate into the collection chamber, was collected and analyzed by protein assay and re-electrophoresis. Re-electrophoresis of S1 shows that it consists of at least three components. Recovered S1 constitutes 47% of the preparation, based on protein assay, S2 4%. S1, isolated from SDS-PAGE, exhibits an apparent M_r of 22.7 kDa, S2 one of 34.5 kDa, similar to the value of 32.6 kDa expected from the structure of syntaxin. The absence of S2 from the electroeluate re-electrophoresed at 0 deg C and their molecular weight relationship suggest a proteolytic transformation of S2 to S1.