| Preparation of HeLa Total Membranes and Assay of Lipid-inhibition of Serine Palmitoyltransferase Activity
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Author:
Date:
2020-06-20
[Abstract] Serine palmitoyltranferase (SPT) is a pyridoxal 5′ phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of de novo synthesis of sphingolipids. SPT activity is homeostatically regulated in response to increased levels of sphingolipids. This homeostatic regulation of SPT is mediated through small ER membrane proteins termed the ORMDLs. Here we describe a procedure to assay ORMDL dependent lipid inhibition of SPT activity. The assay of SPT activity using radiolabeled L-serine was developed from the procedure established by the Hornemann laboratory. The activity of SPT can also be measured using deuterated L-serine but it requires mass spectrometry, which consumes money, time and instrumentation. The ORMDL dependent lipid inhibition of SPT activity can be ...
[摘要] [摘要] 丝氨酸Palmitoyltranferase （SPT）是吡哆醛5 ' 磷酸（PLP）依赖酶催化第一和限速步骤中从头合成鞘脂。SPT活动是Homeostatically调控响应水平的提高鞘脂。这SPT的稳态调节是通过小ER膜蛋白介导称为ORMDLs。在这里，我们描述了一种方法用放射性标记的L-丝氨酸以测定SPT活性的SPT活性。测定的ORMDL依赖性抑制脂质从由规定的程序被开发Hornemann 实验室。 SPT的活性也可以使用氘化的L-丝氨酸进行测定，但需要进行质谱分析，这会耗费金钱，时间和仪器。可以在细胞和无细胞系统中研究ORMDL依赖性脂质对SPT活性的抑制作用。在这里，我们提供了详细的协议来测量存在短链（C8-神经酰胺）或长链神经酰胺（C24-神经酰胺）时SPT活性。该协议的最大优势之一我们通过在HeLa细胞膜中提供外源鞘氨醇和24：1酰基辅酶A通过内源性神经酰胺合酶生成长链神经酰胺来实现这一目标。需要精密的仪器。
[背景 ] 丝氨酸palmit oyltranferase （SPT）是一种多亚基酶是在真核生物和原核生物一些广泛表达（花田等人，1997; Ikushiro 。等人，2001; Hornemann 等人，2007）.The ...
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| Purification of Starch Granules from Arabidopsis Leaves and Determination of Granule-Bound Starch Synthase Activity
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Author:
Date:
2014-12-05
[Abstract] Starch constitutes the most important carbon reserve in plants and is composed of branched amylopectin and linear amylose. The latter is synthesized exclusively by the Granule-Bound Starch Synthase (GBSS, EC 2.4.1.21). Here we report a readily reproducible, specific and highly sensitive protocol, which includes the isolation of intact starch granules from Arabidopsis thaliana leaves and the subsequent determination of GBSS activity. We have applied this method to study GBSS activity in diurnal cycles in vegetative growth and during the photoperiodic transition to flowering in Arabidopsis (Tenorio et al., 2003; Ortiz-Marchena et al., 2014).
[摘要] 淀粉构成植物中最重要的碳储量，由支链支链淀粉和线性直链淀粉组成。 后者仅由颗粒结合的淀粉合酶（GBSS，EC 2.4.1.21）合成。 在这里我们报告一个容易重现，具体和高度敏感的协议，其中包括完整的淀粉颗粒从拟南芥叶中分离和随后的GBSS活动的确定。 我们已经应用这种方法来研究营养生长的日变化周期中和在拟南芥中的光周期过渡到开花期间的GBSS活性（Tenorio等人，2003; Ortiz-Marchena& em> et al。，2014）。
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