Acrosome Biogenesis in the Hamster: RESULTS(2)
Structural Comparison of AM29 and AM22
The polypeptide composition of the ALM fraction has been characterized previously . It contains major low-molecular-mass bands of 29 kDa and 22 kDa (Fig. 2). A monoclonal antibody prepared against the ALM fraction, termed anti-AM29/22, recognized both the 29-kDa (AM29) and 22-kDa (AM22) bands and a set of adjacent minor bands by immunoblot analysis (Fig. 3, lane 1); the other polypeptides of the ALM fraction did not react with anti-AM29/22. Parallel blots stained with normal ascites fluid exhibited no immunostained bands (Fig. 3, lane 2). To explore the antigenic relationships of these proteins, AM22 was purified by preparative SDS-PAGE and used to prepare a polyclonal antiserum. By immunoblot analysis, polyclonal anti-AM22 stained the same set of polypeptides recognized by monoclonal anti-AM29/22 (Fig. 3, lane 3); preim-mune serum reacted with no polypeptides in the ALM fraction (Fig. 3, lane 4). These results demonstrate that AM29, AM22, and the set of adjacent polypeptides are an antigen-ically related family.
To identify structural relationships of AM29 and AM22, the polypeptides were purified by SDS-PAGE (Fig. 4A, lanes 1 and 3) and compared by peptide mapping. V8 digestion of AM29 and AM22 generated an identical set of four polypeptide bands of 20 kDa, 16 kDa, 13 kDa, and 12 kDa (Fig. 4A, lanes 2 and 4), demonstrating a structural similarity of AM29 and AM22. buy levaquin online
FIG. 2. Coomassie blue dye-stained PVDF blot of the ALM fraction separated by SDS-PAGE on a 7.5%-15% gradient gel demonstrates that polypeptides of 29 kDa (AM29) and 22 kDa (AM22) are major polypeptides of the acrosomal fraction. Molecular weight markers (x 10“3) are at left.
FIG. 3. Immunoblot analysis of ALM fraction separated on 7.5%-15% gradient gel; 5 jxg of protein was loaded per lane. Lane 1, immunostained with polyclonal anti-AM22, reveals staining of both AM29 and AM22 and an adjacent set of minor polypeptide bands. Lane 2, stained with preim-mune serum, shows no immunoreactive bands. Lane 3, immunostained with monoclonal anti-AM29/22, shows the same set of immunoreactive bands as noted in lane 1. Lane 4, immunostained with normal ascites fluid, shows no immunoreactive bands. Molecular weight markers (x 10“3) are at left.