Molecularly cloned mammalian glucosamine-6-phosphate deaminase localizes to transporting epithelium and lacks oscillin activity.
Molecularly cloned mammalian glucosamine-6-phosphate deaminase localizes to transporting epithelium and lacks oscillin activity.
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DOI：
10.1096/fasebj.12.1.91
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发表时间：
1998
期刊：
影响因子：
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通讯作者：
Herman Wolosker;D. Kline;Y. Bian;S. Blackshaw;A. Cameron;T. Fralich;R. Schnaar;S. Snyder
中科院分区：
文献类型：
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作者：
Herman Wolosker;D. Kline;Y. Bian;S. Blackshaw;A. Cameron;T. Fralich;R. Schnaar;S. Snyder
葡萄糖胺-6-磷酸脱氨酶（GNPDA）催化葡萄糖胺-6-磷酸转化为果糖-6-磷酸，该反应在生理条件下进行以形成果糖-6-磷酸。虽然在1956年首次在哺乳动物组织中发现，但这种酶以前没有在哺乳动物组织中进行过分子表征，尽管已经克隆了细菌GNPDA。最近，从精子提取物中纯化并克隆了一种与细菌GNPDA相似的蛋白。有人提出，这种蛋白质是在精子提取物中发现的引起细胞中钙振荡的因子;因此，这种蛋白质被命名为“钙蛋白”。“我们证明，克隆的Bclarin具有强大的GNPDA活性，并且可以解释哺乳动物组织提取物中的所有此类活性，从而证明Bclarin是葡糖胺6-磷酸脱氨酶的哺乳动物形式。原位杂交和免疫组织化学定位GNPDA选择性地与高能量需求的组织，如在近曲小管的肾脏和小肠的运输上皮细胞的顶端区;神经元（但不是神经胶质），特别是在大脑中的神经末梢;和运动精子。重组GNPDA和GNPDA纯化到同质从仓鼠精子不能提高细胞内钙离子注入小鼠卵细胞在一个广泛的浓度范围内的条件下，精子提取物引起明显的钙振荡。因此，精子提取物的钙释放或钙结合蛋白活性是由于GNPDA以外的物质。由于GNPDA是唯一的酶连接己糖胺系统与糖酵解途径，我们建议，它提供了一个来源的能量来源的磷酸糖的形式，从cataline的己糖胺中发现的糖蛋白，糖脂，和唾液酸含大分子。GNPDA可以调节己糖胺储存的证据来自我们的观察，即GNPDA转染到HEK-293细胞中降低了唾液酸的细胞水平。
Glucosamine-6-phosphate deaminase (GNPDA) catalyzes the conversion of glucosamine-6-phosphate to fructose-6-phosphate, a reaction that under physiological conditions proceeds to the formation of fructose-6-phosphate. Though first identified in mammalian tissues in 1956, the enzyme has not previously been molecularly characterized in mammalian tissues, although a bacterial GNPDA has been cloned. Recently, a protein displaying similarity to bacterial GNPDA was purified and cloned from sperm extract. It was proposed that this protein was the factor, found in sperm extracts, that causes calcium oscillations in cells; thus, the protein was named 'oscillin.' We demonstrate that oscillin is the mammalian form of glucosamine 6-phosphate deaminase by showing that cloned oscillin has a robust GNPDA activity and can account for all such activity in mammalian tissues extracts. In situ hybridization and immunohistochemistry localize GNPDA selectively to tissues with high energy requirements such as the apical zone of transporting epithelia in the proximal convoluted tubules of the kidney and the small intestine; to neurons (but not glia) and especially to nerve terminals in the brain; and to motile sperm. Recombinant GNPDA and GNPDA purified to homogeneity from hamster sperm fail to elevate intracellular calcium when injected into mouse eggs over a wide range of concentrations under conditions in which sperm extracts elicit pronounced calcium oscillations. Thus, the calcium-releasing or oscillin activity of sperm extracts is due to a substance other than GNPDA. Since GNPDA is the sole enzyme linking hexosamine systems with glycolytic pathways, we propose that it provides a source of energy in the form of phosphosugar derived from the catabolism of hexosamines found in glycoproteins, glycolipids, and sialic acid-containing macromolecules. Evidence that GNPDA can regulate hexosamine stores comes from our observation that transfection of GNPDA into HEK-293 cells reduces cellular levels of sialic acid.