Direct evidence for extracellular cyclic amp phospho di esterase induction and phospho di esterase inhibitor repression by exogenous cyclic amp in dictyostelium purpureum
Tsang, A.S.; Coukell, M.B.
European Journal of Biochemistry 95(2): 419-426
1979
ISSN/ISBN: 0014-2956 Accession: 005165203
The phosphodiesterase inhibitor of D. purpureum was purified from buffered suspensions of starved amoebae and identified on 2-dimensional electrophoretic gels. The polypeptide associated with the inhibitor activity has an apparent MW of 50,000 and an isoelectric point of about pH 4.0. To determine whether exogenous c[cyclic]AMP stimulates the synthesis de novo of one or both of the extracellular phosphodiesterases of D. purpureum, pre-aggregative cells were incubated with 14C-labeled protein hydrolysate in the presence and absence of 1 mM cAMP. The extracellular proteins of the labeled suspensions were separated by 2-dimensional gel electrophoresis and the relative amount of label incorporated into the polypeptides was analyzed by autoradiography. The polypeptides associated with the phosphodiesterase-2 activity were labeled in the cAMP-treated suspension, but not in the control. The polypeptide associated with the phosphodiesterase-1 activity was not labeled under these conditions. Apparently exogenous cAMP induces the synthesis de novo of phosphodiesterase-2, but not phosphodiesterase-1, during the early development of D. purpureum. Under the same conditions, there was little or no incorporation of labeled amino acids into the polypeptide associated with the phosphodiesterase inhibitor activity in the presence or absence of cAMP. When the extracellular proteins, which accumulated in the cell suspensions during the first 10 h of starvation, were separated on 2-dimensional gels and then stained with Coomassie blue, the inhibitor polypeptide was missing from the cAMP-treated suspension. Possibly exogenous cAMP represses the synthesis of the phosphodiesterase inhibitor, or perhaps, interferes with the processing of precursor inhibitor molecules.