Angelides K J
J Neurochem. 1980 Oct;35(4):949-62. doi: 10.1111/j.1471-4159.1980.tb07094.x.
Highly purified "heavy" synaptic vesicles were isolated from rat heart by differential centrifugation. Because of the high intravesicular concentrations of proteins, catecholamine, and ATP, resealed vesicle ghosts were prepared and used to study the detailed kinetics of catecholamine transport. ATP stimulated the uptake of l-norepinephrine and was saturable with a Km for l-norepinephrine at 3.3 microM and 1.8 mM for ATP. The ghosts also accumulated 5-hydroxytryptamine and l-epinephrine via an ATP-dependent mechanism. Uptake was stereospecific for the l-form. A functional catecholamine transporter could be solubilized by the detergent octyl-glucoside and incorporated into phospholipid vesicles, which, after detergent removal, generated proteoliposomes that accumulated l-norepinephrine. Reserpine- and l-propranolol-sensitive accumulation against a concentration gradient is achieved by artificially creating a pH gradient across the membrane, and lends further support to the idea that at least the initial phase of catecholamine transport is driven by the trans-membrane pH gradient created by the proton-translocating ATPase.
通过差速离心从大鼠心脏中分离出高度纯化的“重”突触小泡。由于小泡内蛋白质、儿茶酚胺和ATP的浓度很高，制备了重新封闭的小泡空壳，并用于研究儿茶酚胺转运的详细动力学。ATP刺激去甲肾上腺素的摄取，对去甲肾上腺素的Km值为3.3微摩尔，对ATP为1.8毫摩尔，呈饱和状态。空壳还通过ATP依赖机制积累5-羟色胺和肾上腺素。摄取对L型具有立体特异性。功能性儿茶酚胺转运体可以用去污剂辛基葡糖苷溶解，并整合到磷脂小泡中，去除去污剂后，产生积累去甲肾上腺素的蛋白脂质体。通过人为地在膜上建立pH梯度，实现了对利血平和L-普萘洛尔敏感的逆浓度梯度积累，这进一步支持了至少儿茶酚胺转运的初始阶段是由质子转运ATP酶产生的跨膜pH梯度驱动的这一观点。
