ENZYME REGULATION AT FERTILIZATION OF SEA URCHIN EGGS

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The long term goal of this work is to understand regulation of enzyme activity upon fertilization, using sea urchin oocytes as a model system for studying activation. The proposed research will investigate an apparent activation of enzymes at fertilization, particularly glucose-6-phosphate dehydrogenase (G6PDH). This enzyme change is not seen in cell homogenates but is seen in vitro as a binding change, in permeabilized cells as an activity change and in vivo as an activity change. Comprehension of this phenomena will lead to greater understanding of egg activation at fertilization in particular and could be applicable to other cases of signal transduction, e.g., the action of growth factors on cells, in general. The objectives are to (1) identify the structural elements associated with G6PDH using crosslinking reagents or affinity chromatography, (2) characterize these proteins and their cDNAs, (3) use partial amino acid sequences to prepare antibodies and study the cytological locale of these proteins and any changes in locale and (4) also use the antibodies to examine any chemical modifications as assessed with Western blots. The research will also ascertain (5) whether the dissociation might be affected by small molecules produced or destroyed at fertilization and as assessed by enriching the permeabilization medium for endogenous small molecules. An ancillary but related goal is to extend new technologies for measuring enzyme activity in vivo by using caged substrates to ascertain (6) whether amino acid catabolism is an important energy source for development. All oocytes, even those of mammals, have yolk but the role of this yolk in supporting early development is not yet appreciated. This work with caged substrates will extend this technology and also provide new insights into the energetics of development.