Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles.
Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles.
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冻融和高压放电使大分子摄取到回肠刷状缘囊泡中。
DOI：
10.1152/ajpgi.1987.252.6.g723
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Malmstrom,K
中科院分区：
文献类型：
--
作者：
Donowitz,M;Emmer,E;McCullen,J;Reinlib,L;Cohen,ME;Rood,RP;Madara,J;Sharp,GW;Murer,H;Malmstrom,K
高压放电或一个循环的冻融显示瞬时透化兔回肠刷状缘膜囊泡的大分子。通过快速过滤技术测定用作囊泡渗透性标记物的放射性标记大分子右旋糖酐（mol wt 70，000）的摄取，摄取定义为与囊泡相关的底物，并在囊泡与0.1%皂苷孵育后释放。在电击（2,000 V）后立即或在一个冻融循环开始时加入的葡聚糖与对照相比吸收约8倍;采用这两种技术，吸收到囊泡中后的葡聚糖浓度与孵育培养基中的浓度相似，表明达到了平衡。ATP也被吸收到冻融囊泡中，而对照囊泡中没有显著的吸收。囊泡渗透性的增加是可逆的，根据Na依赖性D-葡萄糖摄取减少时，研究5，但不是15分钟后电击，并没有显着减少后完成一个循环的冻融。此外，腺苷3 '，5'-环磷酸和Ca 2 +-钙调素依赖性蛋白激酶活性在对照囊泡和暴露于高压放电或冻融的囊泡中相似。此外，与[32 P]ATP冻融的囊泡表现出增加的磷酸化与nonfrozen囊泡相比，而冻融并没有改变囊泡蛋白的考马斯亮蓝染色判断。这些技术应允许肠膜囊泡用于研究细胞内控制的运输过程，例如，研究蛋白激酶调节运输。
High-voltage discharge or one cycle of freeze-thawing are shown to transiently permeabilize rabbit ileal brush-border membrane vesicles to macromolecules. Uptake of the radiolabeled macromolecule dextran, mol wt 70,000, used as a marker for vesicle permeability, was determined by a rapid filtration technique, with uptake defined as substrate associated with the vesicle and releasable after incubation of vesicles with 0.1% saponin. Dextran added immediately after electric shock (2,000 V) or at the beginning of one cycle of freeze-thawing was taken up approximately eightfold compared with control; with both techniques, the concentration of dextran after being taken up into the vesicles was similar to that in the incubation medium, suggesting attainment of equilibrium. ATP also was taken up into freeze-thawed vesicles, whereas there was no significant uptake into control vesicles. The increase in vesicle permeability was reversible, based on Na-dependent D-glucose uptake being decreased when studied 5 but not 15 min after electric shock, and was not significantly decreased after completion of one cycle of freeze-thawing. In addition, adenosine 3',5'-cyclic monophosphate and Ca2+-calmodulin-dependent protein kinase activity were similar in control vesicles and vesicles exposed to high-voltage discharge or freeze-thawing. Also, vesicles freeze-thawed with [32P]ATP demonstrated increased phosphorylation compared with nonfrozen vesicles, while freeze-thawing did not alter vesicle protein as judged by Coomassie blue staining. These techniques should allow intestinal membrane vesicles to be used for studies of intracellular control of transport processes, for instance, studies of protein kinase regulation of transport.
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DOI：
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发表时间：
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