Shingles R, McCarty R E
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218-2685, USA.
Anal Biochem. 1995 Jul 20;229(1):92-8. doi: 10.1006/abio.1995.1383.
A comparison of plasma membrane vesicles prepared by a freeze/thaw method was made with vesicles prepared by extrusion through a 100-nm polycarbonate filter. Based on ATPase measurements in the presence or absence of detergent, plasma membrane vesicles were approximately 30% right-side-out in freeze/thaw vesicles, whereas vesicles produced by extrusion were approximately 80% right-side-out. Chloroplast inner-envelope membrane vesicles were loaded with a membrane-impermeant, pH-sensitive fluorophore, pyranine, by either freeze/thaw or extrusion techniques. ATP-linked proton transport activity was considerably lower in vesicles prepared by extrusion compared to vesicles prepared by freeze/thaw. However, total ATPase activity measured as ADP release from ATP was equivalent in both preparations of vesicles. These results suggest that the inner-envelope vesicles produced by extrusion were predominantly oriented right-side-out. Inner-envelope vesicles were loaded internally with phosphate to study proton-linked transport of 3-phosphoglycerate. Vesicle acidification by 3-phosphoglycerate addition was similar in both freeze/thaw and extruded vesicle preparations, indicating that metabolite transport by the phosphate translocator is both functional and bidirectional. These results indicate that extrusion can be used as a method to produce proteoliposomes which are competent for transport studies.
对通过冻融法制备的质膜囊泡与通过100纳米聚碳酸酯滤膜挤出法制备的囊泡进行了比较。基于在有或没有去污剂存在的情况下进行的ATP酶测量，冻融囊泡中的质膜囊泡约30%为外翻取向，而通过挤出法产生的囊泡约80%为外翻取向。通过冻融或挤出技术，将一种膜不透性、对pH敏感的荧光团吡喃装载到叶绿体内膜囊泡中。与通过冻融制备的囊泡相比，通过挤出制备的囊泡中与ATP相关的质子转运活性要低得多。然而，以ATP中ADP释放量衡量的总ATP酶活性在两种囊泡制剂中是相当的。这些结果表明，通过挤出产生的内膜囊泡主要为外翻取向。向内膜囊泡内部装载磷酸盐以研究3-磷酸甘油酸的质子偶联转运。在冻融和挤出囊泡制剂中，添加3-磷酸甘油酸引起的囊泡酸化相似，这表明磷酸盐转运体介导的代谢物转运既有功能又具有双向性。这些结果表明，挤出法可作为一种制备适用于转运研究的蛋白脂质体的方法。
