TREHALOSE-6-PHOSPHATE SYNTHASE PHOSPHATASE COMPLEX FROM BAKERS-YEAST - PURIFICATION OF A PROTEOLYTICALLY ACTIVATED FORM
TREHALOSE-6-PHOSPHATE SYNTHASE PHOSPHATASE COMPLEX FROM BAKERS-YEAST - PURIFICATION OF A PROTEOLYTICALLY ACTIVATED FORM
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DOI：
10.1099/00221287-137-2-323
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发表时间：
1991-02-01
期刊：
影响因子：
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通讯作者：
VUORIO, O
中科院分区：
文献类型：
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作者：
LONDESBOROUGH, J;VUORIO, O
从面包酵母中纯化出一种约800 kDa的具有海藻糖-6-磷酸合成酶(TPS)和海藻糖-6-磷酸磷酸酶(TPP)活性的蛋白质。该TPS/P复合体含有57、86和93 kDa的多肽。86和93 kDa的多肽似乎都来自天然酶中至少115 kDa的多肽。还纯化了一个TPS激活子(58 kDa亚基的二聚体)。它使UDP-葡萄糖(3倍)和葡萄糖6-磷酸(G6P)(4.5倍)的米氏常数降低，并使5 mM-UDP-葡萄糖/10 mM-G6P的TPS活性增加约3倍。不影响TPP活性。TPS/P的纯化包括内源蛋白分解步骤，使TPS活性提高约3倍，并取消了对TPS激活剂的要求，但不改变TPP活性。伴随着这种激活，抗血清识别的一簇约115 kDa的SDS-PAGE条带的分子质量比纯TPS/P减少了约20 kDa，但57 kDa的条带没有变化。磷酸盐抑制TPS活性(K(I)约5 mM)，但使TPP活性增加约6倍(K(A)约4 mM)。磷酸盐(6 MM)促进G6P和UDP-葡萄糖合成海藻糖，减少6-磷酸海藻糖的积累。
A protein of about 800 kDa with trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) activity was purified from bakers' yeast. This TPS/P complex contained 57, 86 and 93 kDa polypeptides. The 86 and 93 kDa polypeptides both appeared to be derived from a polypeptide of at least 115 kDa in the native enzyme. A TPS-activator (a dimer of 58 kDa subunits) was also purified. It decreased the Michaelis constants for both UDP-glucose (three-fold) and glucose 6-phosphate (G6P) (4.5-fold), and increased TPS activity at 5 mM-UDP-glucose/10 mM-G6P about three-fold. It did not affect TPP activity. The purification of TPS/P included an endogenous proteolytic step that increased TPS activity about three-fold and abolished its requirement for TPS-activator, but did not change TPP activity. This activation was accompanied by a decrease of some 20 kDa in the molecular mass of a cluster of SDS-PAGE bands at about 115 kDa recognized by antiserum to pure TPS/P, but by no change in the 57 kDa band. Phosphate inhibited TPS activity (K(i) about 5 mM), but increased TPP activity about six-fold (K(a) about 4 mM). Phosphate (6 mM) stimulated the synthesis of trehalose from G6P and UDP-glucose and decreased the accumulation of trehalose 6-phosphate.