Studies of cell-surface glorin receptors, glorin degradation, and glorin-induced cellular responses during development of Polysphondylium violaceum

Abstract

The chemoattractant mediating cell aggregation in the slime mold Polysphondylium violaceum is N-propionyl-γ- l-glutamyl- l-ornithine-δ-lactam ethylester (glorin). Here we examine the binding properties of tritiated glorin to intact P. violaceum cells. Scatchard analysis of binding data yielded slightly curvilinear plots with K d values in the range of 20 and 100 n M. The number of glorin receptors increased from 35,000 in the vegetative stage to 45,000 per cell during aggregation. Later, during culmination receptor numbers decreased to undetectable levels (<1000). The receptor binding kinetics show binding equilibrium within 30 s at 0 °C, and ligand dissociation occurs from two kinetically distinct receptors whose half-times were 2 s for 72% of the bound glorin and 28 s for the remainder. The enzymatic degradation of glorin did not affect binding data during incubations of up to 1 min at 0 °C. Two glorinase activities were observed. An ornithine δ-lactam cleaving activity with a K m of ca. 10 −4 M and a propionic acid removing activity ( K m 10 −5 M), both of which were detected mainly on the cell surface. Cleavage of the lactam occurred at a higher rate than removal of propionic acid. Lactam-cleaved glorin showed no chemotactic activity nor did it bind to cell-surface glorin receptors. Cell-surface-bound glorinase activity and glorin-induced cGMP synthesis were developmentally regulated, peaking at aggregation. In the most sensitive stage half-maximal responses (cGMP synthesis, chemotaxis, light-scattering) were elicited in the 10–100 n M range. Neither cAMP synthesis nor glorin-induced glorin synthesis was observed. Guanine nucleotides specifically modulated glorin receptor binding on isolated membranes, and, conversely, glorin modulated GTPγS binding to membrane preparations. Our results support the notion that glorin mediates chemotactic cell aggregation in P. violaceum acting via cell-surface receptors, G-proteins, and cGMP accumulation.