Garson K, Matlashewski G J, Adeli K, Robert L S, Altosaar I
Anal Biochem. 1983 Oct 15;134(2):512-6. doi: 10.1016/0003-2697(83)90332-9.
A rapid method for obtaining large quantities of developing groats suitable for the isolation of highly intact polysomes has been developed. Developing spikelets were harvested directly from oat panicles into liquid nitrogen and then quickly passed through a dehuller. Chaff was removed by air aspiration and the resultant groats were collected directly back into liquid nitrogen. Approximately 250 g of groats could be isolated each man-hour by the above method. In comparison, only 10 g of endosperm could be collected by squeezing it out of spikelets using an endosperm mangle. Membrane-bound polysomes extracted from the immature groats were compared to those extracted from endosperm. The largest polysomes discernable as unique peaks on sucrose gradients were ten-mers and nine-mers for groats and endosperm, respectively. Polysomes isolated from both starting materials stimulated similar incorporations of [35S]methionine into trichloroacetic acid-insoluble products during in vitro translations in wheat germ extract. Both polysome preparations directed the synthesis of similar high-molecular-weight proteins. Based on these criteria, polysomes from both preparations were found to be of similar intactness, although the groat starting material was much more readily obtained. The polysome classes having the maximum absorbance peak for endosperm and groat polysomes were six-mers and eight-mers, respectively.
已开发出一种快速方法，可获得大量适合分离高度完整多核糖体的发育中去壳燕麦粒。将发育中的小穗直接从燕麦穗收获到液氮中，然后快速通过脱壳机。通过气吸去除谷壳，所得去壳燕麦粒直接收集回液氮中。通过上述方法，每人每小时可分离出约250克去壳燕麦粒。相比之下，使用胚乳压榨机从小穗中挤出胚乳，只能收集到10克胚乳。将从未成熟去壳燕麦粒中提取的膜结合多核糖体与从胚乳中提取的多核糖体进行比较。在蔗糖梯度上可分辨为独特峰的最大多核糖体，去壳燕麦粒的是十聚体，胚乳的是九聚体。在小麦胚芽提取物的体外翻译过程中，从两种起始材料中分离的多核糖体刺激了类似的[35S]甲硫氨酸掺入三氯乙酸不溶性产物的过程。两种多核糖体制剂都指导了类似的高分子量蛋白质的合成。基于这些标准，发现两种制剂的多核糖体完整性相似，尽管去壳燕麦粒起始材料更容易获得。胚乳和去壳燕麦粒多核糖体具有最大吸光度峰的多核糖体类别分别是六聚体和八聚体。
