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Painter et al. 10.1073/pnas.0306339101. |
Fig. 4. Schematic diagram of the reproductive tract of A. californica. Following ovulation, eggs are transported from the ovotestis (which would be at the far right) through the small hermaphroditic duct (SHD) to the accessory genital mass (AGM), where they are fertilized and packaged into a cordon. Packaging occurs as the eggs are transported past (or through) the three exocrine components of the AGM: the mucous, winding, and albumen glands. The albumen gland makes up the bulk of the structure. The accessory genital mass is connected to the common genital aperture (CGA; far left) by the large hermaphroditic duct, which is composed of the red hemiduct (RHD; the oviduct) and the white hemiduct (WHD; the copulatory duct). The exocrine atrial gland is associated with the oviduct and secretes into the large hermaphroditic duct.
Fig. 5. Final RP-HPLC purification of attractin from albumen gland extracts of four species. (A) A. brasiliana attractin. (B) A. vaccaria attractin. (C) A. fasciata attractin. (D) A. depilans attractin. Albumen glands were removed from A. brasiliana, A. vaccaria, A. fasciata, and A. depilans, frozen on dry ice, and subsequently extracted at 4°C in 0.1% heptafluorobutyric acid (HFBA) using a Polytron homogenizer and sonicated. The extract was centrifuged for 20 min at 48,000 ´g (4°C), and the supernatant was purified on C18 Sep-Pak Vac cartridges (Waters). Peptides were eluted with 50% acetonitrile/0.1% HFBA and lyophilized. The lyophilizate was resuspended in 0.1% HFBA and applied to a semipreparative Vydac C18 RP-HPLC column (10 × 250 mm). The column was eluted with a two-step linear gradient of 0.1% HFBA and 100% acetonitrile/0.1% HFBA (0 to 10% acetonitrile/0.1% HFBA in 5 min; 10 to 34% acetonitrile/0.1% HFBA in 85 min). The column eluate was monitored by absorbance at 215 nm, and 1-min (1.75-ml) fractions were collected. For chemical characterization studies, fractions containing attractin were pooled, lyophilized, reduced with 2-mercaptoethanol, alkylated with 4-vinylpyridine, and purified on an analytical Vydac C18 RP-HPLC column (4.6 × 250 mm), and 1-min (1-ml) fractions were collected. The same gradient conditions were used, except that 0.1% trifluoroacetic acid (TFA) was the counter-ion. Fractions indicated by solid bars were characterized by sequence analysis and used for endoproteinase Glu-C, endoproteinase Lys-C, and CNBr digestion. For bioassays, fractions containing A. californica or A. vaccaria attractin were not reduced and alkylated.
Fig. 6. Mass spectrum of A. vaccaria attractin. The predicted mass position of reduced and alkylated full-length A. vaccaria attractin with six 4-vinylpyridines (6,199 + 636 = 6,835 Da) is indicated with an arrow. (Inset) A series of higher mass peaks with a spacing of 163 Da.
Fig. 7. RP-HPLC fractionation of A. brasiliana and A. vaccaria egg cordon eluates. (A) Eluate of A. brasiliana egg cordons (19 ml volume) laid by 14 animals. Fractions were eluted with a linear gradient of 0.1% HFBA and acetonitrile/0.1% HFBA, and the peak indicated by the solid bar was analyzed by N-terminal sequence analysis. (B) Eluate of A. vaccaria egg cordons (19 ml volume) laid by 10 animals. Fractions were eluted and pooled for N-terminal sequence analysis as in A.