Studies on the cyclic AMP-phosphodiesterases and other aspects of the aggregation of Dictyostelium discoideum

• Main Author: McDonald, Charles John
• Published: University of Leicester 1983
• Subjects: 572; Biochemistry
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.348765

Membrane bound cyclic AMP phosphodiesterase (mPDE) is prematurely induced by cyclic nucleotides during early development of D.discoideum. Factors affecting this induction were studied, including transcriptional inhibitors and an inhibitor of cyclic AMP dependant protein kinases (indomethacin). Amoebae secrete a form of PDE (SPDE) which was chosen as a molecular marker for the induction due to its ease of purification. In preparations free of the protein inhibitor (PDE-I) multiple forms (A, B and C) of sPDE were studied. sPDE-A was purified to homogeneity by column chromatography and consists of one subunit, P50. Antibody, raised against pure sPDE-A, immunoprecipitated all three forms of sPDE. sPDE-B was correlated with equimolar amounts of p50 and p52 and is a dimer. sPDE-C was correlated with P52 and p50 in an approximate 2/l ratio and is thus probably a trimer. The formula [(P50)x + (P52)y] z, may describe the subunit arrangements of all observed forms of sPDE. P50 and P52 were both immunoprecipitated by the antibody and peptide mapping revealed that P50 and P52 are very similar but P52 contained 24% more carbohydrate than P50. The inhibitor of protein glycosylation tunicamycin (TM), blocked the appearance of sPDE, mPDE; and P50 and P52. TM reversibly blocked development but small aggregates did form at the same time as normal aggregation. TM prevented the appearance of Contact sites A as well as PDE but cell surface cyclic AMP binding sites developed normally, a finding compatible with the observed small-scale aggregation event. Addition of exogenous PDE to TM inhibited amoebae restored the ability to chemotact normally towards cyclic AMP; thus the primary block in aggregation caused by TM is to prevent the appearance of PDE.