Dong Xu, Bhamidi Suresh, Scherman Michael, Xin Yi, McNeil Michael R
Mycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
Appl Environ Microbiol. 2006 Apr;72(4):2601-5. doi: 10.1128/AEM.72.4.2601-2605.2006.
Treatment of either Mycobacterium tuberculosis or M. smegmatis 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 [(14)C]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.
用乙胺丁醇处理结核分枝杆菌或耻垢分枝杆菌，既会抑制阿拉伯聚糖的合成，也会导致细胞壁中先前形成的大量阿拉伯聚糖流失。阿拉伯聚糖的流失已被证明是由于内源性阿拉伯糖苷酶的作用。为了更好地理解这一现象，开发了一种内源性阿拉伯糖苷酶的定量测定方法。使用该测定方法确定，耻垢分枝杆菌的各种亚细胞组分显示出大量的内源性阿拉伯糖苷酶活性。令人惊讶的是，用乙胺丁醇处理后，内源性阿拉伯糖苷酶活性的量仅有微小变化。内源性阿拉伯糖苷酶存在于细胞壁中，而且一致的是，仅在缓冲液中孵育耻垢分枝杆菌细胞壁会导致阿拉伯聚糖的释放，这与乙胺丁醇对整个细胞的作用相似。为了确定细胞壁阿拉伯聚糖是否快速周转，在培养的对数早期通过加入[（14）C]葡萄糖标记阿拉伯聚糖，然后用非放射性葡萄糖进行“追踪”。当培养物生长到对数后期时，大多数标记的阿拉伯聚糖仍留在细胞壁中。因此，细胞壁中存在活性阿拉伯糖苷酶，但除非存在乙胺丁醇，阿拉伯聚糖不会被快速去除。使用所述测定方法对内源性阿拉伯糖苷酶进行纯化的工作正在进行中，以帮助进一步了解其生物学功能。
