Department of Biochemistry and Biophysics, University of California, Davis, California 95616.
Plant Physiol. 1978 Feb;61(2):154-7. doi: 10.1104/pp.61.2.154.
Large numbers of vacuoles (10(6)-10(7)) have been isolated from Sorghum bicolor protoplasts and analyzed for the cyanogenic glucoside dhurrin. Leaves from light-grown seedlings were incubated for 4 hours in 1.5% cellulysin and 0.5% macerase to yield mesophyll protoplasts which then were recovered by centrifugation, quantitated by a hemocytometer, and assayed for cyanogenic glucosides. Mature vacuoles, released from the protoplasts by osmotic shock, were purified on a discontinuous Ficoll gradient and monitored for intactness by their ability to maintain a slightly acid interior while suspended in an alkaline buffer as indicated by neutral red stain. Cyanide analysis of the protoplasts and the vacuoles obtained there from yielded equivalent values of 11 mumoles of cyanogenic glucoside dhurrin per 10(7) protoplasts or 10(7) vacuoles. This work supports an earlier study from this laboratory which demonstrated that the vacuole is the site of accumulation of the cyanogenic glucoside in Sorghum.
从高粱原生质体中分离出大量（10^6-10^7）个空泡，并分析其中的氰苷葡萄糖苦马豆素。将光照培养的幼苗叶片在 1.5%纤维素酶和 0.5%离析酶中孵育 4 小时，以产生叶肉原生质体，然后通过离心回收，用血球计数器定量，并测定氰苷葡萄糖苦马豆素的含量。通过渗透压冲击从原生质体中释放出成熟的空泡，在不连续的菲可尔梯度上进行纯化，并通过中性红染色监测其在碱性缓冲液中保持略微酸性内部的能力来监测其完整性。对原生质体和从中获得的空泡进行氰化物分析，得到的氰苷葡萄糖苦马豆素含量相等，每 10^7 个原生质体或 10^7 个空泡中含有 11 微摩尔氰苷葡萄糖苦马豆素。这项工作支持了本实验室的早期研究，该研究表明空泡是高粱中氰苷葡萄糖苦马豆素积累的部位。
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