Kishore B K, Fuming L u, Maldague P, Tulkens P M, Courtoy P J
Unité de Pharmacologie Cellulaire et Moléculaire, Université Catholique de Louvain, Brussels, Belgium.
Lab Invest. 1996 Jun;74(6):1025-37.
In the companion paper, we report that a single injection of poly-D-glutamic acid causes an acute lysosomal storage condition and apparently impairs the lysosomal fission dynamics. The present paper addresses the mechanisms of these two alterations using a combination of in vivo and in vitro biochemical approaches. After a single intravenous injection, 14C-poly-D-glutamic acid was rapidly cleared from the plasma and appeared in the urine. Yet, a small but sizable fraction of the injected polymer was taken up by the kidney cortex through a saturable process (Kuptake, 150 mg/kg body wt; uptakemax 96 micrograms/g cortex). Analytical subcellular fractionation of cortex homogenates demonstrated that at initial stages, the 14C label was predominantly associated with subcellular particles of intermediate size and low equilibrium density, and was therefore slowly transferred to larger particles equilibrating at high density, then codistributing with the lysosomal hydrolases. At a concentration of 10 mg/ml (equivalent to its estimated concentration in lysosomes), poly-D-glutamic acid formed micronic aggregates ( > or = 10 microns) when brought to solution at pH < or = 6 in relation to its decreased ionization (pKa of lateral chains approximately equal to 4.25). Finally, 1 day after the injection of poly-D-glutamic acid, the activities of several lysosomal enzymes (hexosaminidase, cathepsin B, acid sphingomyelinase, and sulfatase B), but not of all of them (eg, acid phosphatase), were increased in the kidney cortex. We propose that poly-D-glutamic acid reaches lysosomes by adsorptive endocytosis and becomes concentrated within these organelles because its withstands hydrolysis until it forms aggregates or precipitates, causing a decrease in the fluidity or the deformability ("gelling") of the lysosomal matrix. This should alter the dynamics of intercommunication of these organelles by impairing their fission without a proportionate effect on their fusion properties. In addition, the data suggest that the presence of poly-D-glutamic acid directly or indirectly slows down the degradation of several lysosomal enzymes.
在配套论文中，我们报道单次注射聚-D-谷氨酸会导致急性溶酶体贮积状况，并明显损害溶酶体分裂动力学。本文使用体内和体外生化方法相结合的方式探讨这两种改变的机制。单次静脉注射后，14C-聚-D-谷氨酸迅速从血浆中清除并出现在尿液中。然而，一小部分但数量可观的注入聚合物通过可饱和过程被肾皮质摄取（摄取量，150mg/kg体重；最大摄取量96μg/g皮质）。对皮质匀浆进行亚细胞分级分离分析表明，在初始阶段，14C标记主要与中等大小和低平衡密度的亚细胞颗粒相关，因此缓慢转移到高密度平衡的较大颗粒中，然后与溶酶体水解酶共分布。当在pH≤6的条件下溶解时，相对于其电离度降低（侧链的pKa约等于4.25），聚-D-谷氨酸在浓度为10mg/ml（相当于其在溶酶体中的估计浓度）时形成微米级聚集体（≥10微米）。最后，注射聚-D-谷氨酸1天后，肾皮质中几种溶酶体酶（己糖胺酶、组织蛋白酶B、酸性鞘磷脂酶和硫酸酯酶B）的活性增加，但并非所有酶（如酸性磷酸酶）的活性都增加。我们提出聚-D-谷氨酸通过吸附性胞吞作用进入溶酶体并在这些细胞器内浓缩，因为它能抵抗水解直至形成聚集体或沉淀物，导致溶酶体基质的流动性或可变形性（“胶凝”）降低。这应该会通过损害溶酶体的分裂而改变这些细胞器相互通讯的动力学，而对其融合特性没有相应影响。此外，数据表明聚-D-谷氨酸的存在直接或间接减缓了几种溶酶体酶的降解。
