De Wit R J, van Bemmelen M X, Penning L C, Pinas J E, Calandra T D, Bonner J T
Cell Biology and Genetics Unit, University of Leiden, The Netherlands.
Exp Cell Res. 1988 Dec;179(2):332-43. doi: 10.1016/0014-4827(88)90272-8.
The chemoattractant mediating cell aggregation in the slime mold Polysphondylium violaceum is N-propionyl-gamma-L-glutamyl-L-ornithine-delta-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 Kd values in the range of 20 and 100 nM. 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 (less than 1000). The receptor binding kinetics show binding equilibrium within 30 s at 0 degrees 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 degrees C. Two glorinase activities were observed. An ornithine delta-lactam cleaving activity with a Km of ca. 10(-4) M and a propionic acid removing activity (Km 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 nM 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 GTP gamma S 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.
在黏菌紫多头绒泡菌中，介导细胞聚集的趋化因子是N-丙酰基-γ-L-谷氨酰-L-鸟氨酸-δ-内酰胺乙酯（格洛林）。在此，我们研究了氚标记的格洛林与完整的紫多头绒泡菌细胞的结合特性。对结合数据进行Scatchard分析得到的曲线略呈非线性，解离常数（Kd）值在20至100 nM范围内。格洛林受体的数量从营养阶段的每个细胞35,000个增加到聚集阶段的每个细胞45,000个。随后，在发育阶段后期，受体数量降至检测不到的水平（少于1000个）。受体结合动力学表明，在0℃下30秒内达到结合平衡，配体从两种动力学不同的受体上解离，其中72%结合的格洛林的半衰期为2秒，其余的为28秒。在0℃下孵育长达1分钟的过程中，格洛林的酶促降解不影响结合数据。观察到两种格洛林酶活性。一种鸟氨酸δ-内酰胺裂解活性，其米氏常数（Km）约为10⁻⁴ M，以及一种丙酸去除活性（Km为10⁻⁵ M），这两种活性主要在细胞表面检测到。内酰胺的裂解速率高于丙酸的去除速率。内酰胺裂解的格洛林既没有趋化活性，也不与细胞表面的格洛林受体结合。细胞表面结合的格洛林酶活性和格洛林诱导的环鸟苷酸（cGMP）合成受到发育调控，在聚集时达到峰值。在最敏感阶段，在10 - 100 nM范围内引发半最大反应（cGMP合成、趋化性、光散射）。未观察到环磷酸腺苷（cAMP）合成或格洛林诱导的格洛林合成。鸟嘌呤核苷酸特异性调节分离膜上的格洛林受体结合，相反，格洛林调节鸟苷-5'-O-（3-硫代三磷酸）（GTPγS）与膜制剂的结合。我们的结果支持这样一种观点，即格洛林通过细胞表面受体、G蛋白和cGMP积累，介导紫多头绒泡菌中的趋化性细胞聚集。
