Embryo growth of oviparous animals depends upon utilization of nutritive proteins, primarily the glycoprotein vitellin (Vt). These proteins are usually extraovarian in origin and accumulate in the oocyte through receptor-mediated endocytosis. This event is well characterized for both insects and vertebrates. In contrast, the mechanisms of yolk protein utilization are not understood. In this study, the German cockroach Blattella germanica was used as a model insect system to explore the components that initiate and regulate Vt processing during early embryo development. In B. germanica, Vt processing is initiated at days 3-4 postoviposition at 30$\sp\circ$C. The yolk of freshly oviposited eggs assayed in vitro for protease was devoid of activity but protease specific activity increased dramatically during embryo development. This activity correlated temporally with Vt processing in vivo suggesting that the protease is necessary for Vt processing. The protease was purified from yolk at day 6 postoviposition by gel filtration and affinity chromatography and classified as a cysteine protease. Its molecular weight, estimated by SDS-PAGE and immunoblotting, was approximately 29 kDa. Its pH optimum was 5.5, within the pH range of yolk granules. The purified protease degraded Vt in vitro yielding peptides of the same apparent molecular weights as Vt processed in vivo. Acidification of day 0 yolk in vitro induced protease activity suggesting that a latent protease is present in eggs early in embryo development. The latent protease activity was purified from yolk at day 0 postoviposition by successive use of gel filtration, Mono Q FPLC, and hydrophobic interaction chromatography. The purified latent protease had a molecular weight of approximately 40 kDa and could be activated in vitro into a cysteine protease of 29 kDa. The conversion depended on acidification and was blocked by the cysteine protease inhibitor E-64, suggesting the activation is autocatalytic. Kinetic studies showed that activation occurred by intermolecular catalysis. The pH-activity and inhibitor sensitivity profile of the in vitro-activated protease matched those of the protease suggesting that the latent protease is the proenzyme of the protease. Active site derivatization of the 40 kDa proprotease revealed that its conversion to the 29 kDa protease in vivo occurs as Vt processing begins, suggesting that Vt processing is regulated through the protease. The proenzyme activation and pH optimum data of the purified protease emphasize that yolk granule acidification is an important cellular factor for the regulation of Vt processing by B. germanica in vivo. A murine polyclonal antibody against purified proprotease was made monospecific by affinity column chromatography. Using this antibody, the proprotease was detected in fat bodies and ovaries of vitellogenic females by immunoblotting. This result is consistent with the hypothesis that the protease precursor is synthesized extraovarily, probably in the fat body.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-9025 |
| Date | 01 January 1995 |
| Creators | Liu, Xiaodong |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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