Development of a quantitative assay for mycobacterial endogenous arabinase and ensuing studies of arabinase levels and arabinan metabolism in Mycobacterium smegmatis
Development of a quantitative assay for mycobacterial endogenous arabinase and ensuing studies of arabinase levels and arabinan metabolism in Mycobacterium smegmatis
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DOI：
10.1128/aem.72.4.2601-2605.2006
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发表时间：
2006-04-01
影响因子：
4.4
通讯作者：
McNeil, MR
中科院分区：
文献类型：
--
作者：
Dong, X;Bhamidi, S;McNeil, MR
治疗结核分枝杆菌或M. smeginatis与乙胺丁醇的组合导致阿拉伯聚糖合成的抑制和先前形成的阿拉伯聚糖从细胞壁的大量损失。已证明阿拉伯聚糖的损失是由于内源性阿拉伯糖酶的作用。为了更好地理解这一现象，开发了内源性阿拉伯糖酶的定量测定。使用该测定法，确定了M.丝霉孢菌显示出显著量的内源性阿拉伯糖酶活性。令人惊讶的是，用乙胺丁醇处理仅使内源性阿拉伯糖酶活性的量产生微小变化。内源阿拉伯糖酶存在于细胞壁中，并且一致地，M.仅在缓冲液中的细胞壁导致阿拉伯聚糖的释放，模拟乙胺丁醇对整个细胞的作用。为了确定细胞壁阿拉伯聚糖是否快速翻转，在培养的早期对数期通过补料[C-14]葡萄糖标记阿拉伯聚糖，然后用非放射性葡萄糖“追逐”。培养物生长到对数生长后期后，大部分标记的阿拉伯聚糖仍留在细胞壁中。因此，在细胞壁中有活性阿拉伯糖酶，但除非存在乙胺丁醇，否则阿拉伯聚糖不能迅速除去。正在使用所述测定法纯化内源性阿拉伯糖酶，以帮助进一步辨别其生物学功能。
Treatment of either Mycobacterium tuberculosis or M. smeginatis with ethambutol results both in inhibition of arabinan synthesis and in copious loss of previously formed arabinan from the cell wall. The loss of arabinan has been shown to be due to the action of an endogenous arabinase. To better understand this phenomenon, a quantitative assay for endogenous arabinase was developed. Using the assay it was determined that various subcellular fractions of M. smegmatis showed significant amounts of endogenous arabinase activity. Surprisingly, treatment with ethambutol yielded only minor changes in the amounts of endogenous arabinase activities. Endogenous arabinase was present in the cell wall, and consistently, incubation of the M. smegmatis cell wall in only buffer resulted in the release of arabinan, mimicking the effect of ethambutol on whole cells. To determine if cell wall arabinan is rapidly turned over, the arabinan was labeled in the early log phase of culture by feeding [C-14] glucose, followed by a "chase" with nonradioactive glucose. Most of the labeled arabinan remained in the cell wall after the culture was grown to late log phase. Thus, there is active arabinase in the cell wall, but arabinan is not rapidly removed unless ethambutol is present. Purification of the endogenous arabinase, using the assay described, is ongoing to help further discern its biological function.