Wheatly M G, Weil J R, Douglas P B
Department Of Biological Sciences, Wright State University, Dayton, Ohio 45435, USA.
Am J Physiol. 1998 Mar;274(3):R725-34. doi: 10.1152/ajpregu.1998.274.3.R725.
Procedures were developed to isolate basolateral membrane vesicles (BLMV) from gill, hepatopancreas, and antennal gland of intermolt freshwater crayfish, Procambarus clarkii. Individual procedures involved a discontinuous sucrose gradient (gill), a 65% sucrose cushion (hepatopancreas), or differential centrifugation (antennal gland). BLMV were visualized, characterized (37 degrees C), and tested for osmotic reactivity with a view to using them for Ca2+ uptake studies. Mean diameters of BLMV were 159 nm (gill), 363 nm (hepatopancreas), and 226 nm (antennal gland). Enrichments of basolateral membranes and mitochondria in BLMV were, respectively, 18- and 1.7-fold for gill, 9- and 0.4-fold for hepatopancreas, and 10- and 1-fold for antennal gland. Apical contamination was negligible in BLMV. Percentages of resealing of vesicles as inside out, right side out, or leaky/sheets were 17:27:56% (gill), 14:26:60% (hepatopancreas), and 21:39:40% (antennal gland). Vesicles exhibited osmotic reactivity, as indicated by a linear relationship between vesicular 45Ca2+ uptake and osmolality. Nonspecific 45Ca2+ binding was 20% in gill, 39% in hepatopancreas, and 31% in antennal gland. Data were compared with published values for marine crustaceans.
已开发出从淡水螯虾克氏原螯虾蜕壳间期的鳃、肝胰腺和触角腺中分离基底外侧膜囊泡（BLMV）的方法。具体方法包括不连续蔗糖梯度离心法（用于鳃）、65%蔗糖垫层法（用于肝胰腺）或差速离心法（用于触角腺）。对BLMV进行了可视化、表征（37℃）以及渗透压反应测试，以便将其用于钙离子摄取研究。BLMV的平均直径分别为159纳米（鳃）、363纳米（肝胰腺）和226纳米（触角腺）。BLMV中基底外侧膜和线粒体的富集倍数，鳃分别为18倍和1.7倍，肝胰腺分别为9倍和0.4倍，触角腺分别为10倍和1倍。BLMV中的顶端污染可忽略不计。囊泡外翻、正位和渗漏/片状重封的百分比分别为17:27:56%（鳃）、14:26:60%（肝胰腺）和21:39:40%（触角腺）。囊泡表现出渗透压反应，囊泡对45Ca2+的摄取与渗透压之间呈线性关系即证明了这一点。非特异性45Ca2+结合在鳃中为20%，在肝胰腺中为39%，在触角腺中为31%。已将数据与已发表的海洋甲壳类动物的数据进行了比较。
