The overal objective of the research to be conducted is to achieve an understanding of how the unique alkyl resorcinols and pyrones (which are the membrane lipids in encysting cells and cysts) are synthesized, are inserted into the bilayer and how they function in the membrane. The Beta-ketoacyl synthases of vegetative and encysting cells of Azotobacter vinelandii will be purified and compared with respect to their products, kinetics and immunological relationship. The objective is to test the theory that resorcinol synthesis during encystment requires a new form of the enzyme which can catalyze the formation of saturated acyl chains of C25 to C30. The vegetative cell synthase will only catalyze the formation of C16 chains. If different, the DNA coding for the encystment enzyme will be cloned and a test will be made of its ability to hybridize with nRNA templates of the vegetative Beta-ketoacyl-ACP synthase. If positive, attempts will be made to sequence cloned DNA of enzyme. The encystment enzyme DNA will be used as a probe to establish any relationship between its sequence of those derived from plants which also produce long chain alkyl resorcinols. The symmetry of phospholipid degradation in membranes of encysting cells will be studied using trinitrophenylation of phosphotidydlethanolanine to study that P-lipid concentration on each face of the lipid bilayer. The insertion of resorcinols into the changing bilayer will be observed in a like manner using diazobenzene sulfonate. The extrinsic proteins of the membrane will be identified by 14C labeling and an attempt will be made to correlate changes in protein species with shifts in lipid bilayer composition. Glucose and calcium transport in cells, cysts, and germinating cysts will be studied as a test of the effects of radical changes in lipid bilayer composition on the function of the membrane's intrinsic proteins.
