Acrosome Biogenesis in the Hamster: RESULTS(3)
The structural relationship of AM29 and AM22 was also examined by N-terminal amino acid sequencing (Fig. 4B). Intact AM29 was N-blocked; however, intact AM22 was sequenced through 17 residues. The V8 protease-generated polypeptide fragments of 20 kDa, 16 kDa, and 12 kDa for both AM29 and AM22 also yielded N-terminal sequence data. The N-terminal sequence of each V8 protease-generated polypeptide fragment was distinct from intact AM22, indicating that internal sequences were obtained. The 20-kDa and 12-kDa polypeptides from both AM29 and AM22 exhibited identical N-terminal sequences. The 16-kDa bands of AM29 and AM22 were also identical to one another in N-terminal sequence but differed from the 20-kDa and 12-kDa fragments. These data demonstrate that AM29 and AM22 share sequence homology and may originate from a common parent molecule. ventolin inhalers
The N-terminus sequence of AM22 and of the 16-kDa V8 protease fragment exhibited no homologies to sequences in the blastp database (NCBI BLAST program). However, the N-terminal sequence of the 12-kDa and 20-kDa peptides exhibited sequence homology to the mouse acrosomal protein mSP-10, the murine homologue of human SP-10 . Over their first 10 amino acids, a 80% residue identity, or 90% similarity including conservative amino acid substitutions, was noted with amino acids 170-179 of mSP-10 (Fig. 4B).
FIG. 4. A) Peptide maps of AM29 and AM22 using V8 protease. SDS-PAGE-purified AM 29 (lanes 1 and 2) and AM22 (lanes 3 and 4) were incubated in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of V8 protease and then fractioned by SDS-PAGE on 15% acrylamide gels, transferred to PVDF membranes, and stained with Coomassie blue dye. Both AM29 and AM22 exhibit an identical pattern of V8 protease-generated polypeptides of 20, 16, 13, and 12 kDa (lanes 2 and 4). Molecular weight markers (X 10 3) are at left. B) N-terminal sequences of intact AM22 and V8-digest polypeptides of both AM29 and AM22. Apparent molecular mass of V8 protease generated polypeptides are given in parentheses. Single letter codes are given, “X” denotes that no definitive sequence was obtained. “‘Deduced sequence of mouse SP-10 residues 170-182 . The GenBank accession number for the mSP-10 cDNA sequence is U31992. Single underlined letters represent conservative amino acid substitution between mSP-10 and hamster AM29 and/or AM22; double underlined residue represents difference between mSP-10 and hamster AM29 and/or AM22.