THE CONTROL OF GLYCOGEN-METABOLISM IN YEAST .2. A KINETIC-STUDY OF THE 2 FORMS OF GLYCOGEN-SYNTHASE AND OF GLYCOGEN-PHOSPHORYLASE AND AN INVESTIGATION OF THEIR INTERCONVERSION IN A CELL-FREE-EXTRACT
THE CONTROL OF GLYCOGEN-METABOLISM IN YEAST .2. A KINETIC-STUDY OF THE 2 FORMS OF GLYCOGEN-SYNTHASE AND OF GLYCOGEN-PHOSPHORYLASE AND AN INVESTIGATION OF THEIR INTERCONVERSION IN A CELL-FREE-EXTRACT
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DOI：
10.1111/j.1432-1033.1988.tb14135.x
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发表时间：
1988-06-15
期刊：
影响因子：
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通讯作者：
HERS, HG
中科院分区：
文献类型：
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作者：
FRANCOIS, J;HERS, HG
糖原合酶和糖原磷酸化酶的两种相互转化形式，一种活性（a）或另一种活性较低（B），主要存在于已经在35 ℃的限制性温度下预孵育的热敏腺苷酸环化酶缺陷突变体中。C，在存在或不存在葡萄糖的情况下。在葡萄糖存在下孵育细胞后，糖原磷酸化酶的活性至少降低20倍，但这种残余活性具有与在没有葡萄糖存在下孵育后获得的酶的活性形式相同的动力学性质;这表明B型可能是完全无活性的，葡萄糖处理后测得的低活性必须归因于残留量的磷酸化酶a相比之下，这两种形式的糖原合酶的动力学性质是非常不同的。当在不存在葡萄糖6-磷酸的情况下测量时，两种形式的酶对UDP-Glc具有相似的亲和力，但它们的Vmax基本上不同。葡萄糖6-磷酸对合成酶a没有影响，但增加了合成酶B的Vmax和Km;然而，这些影响在很大程度上被硫酸盐和无机磷酸盐抵消，后者也具有增加a型Km的特性，而不影响Vmax。据估计，在底物和效应物的生理浓度下，合酶a的活性比合酶B高约20倍。当凝胶过滤在没有葡萄糖存在下预温育的细胞提取物，然后在30 ℃温育时，C，磷酸化酶逐渐完全失活和合成酶被部分激活，这些反应是几倍更快，在糖原合成酶的情况下，在10 mM葡萄糖6-磷酸的存在下，更完整。当在葡萄糖存在下预温育的细胞的凝胶过滤提取物在30 ℃温育时，C在ATP-Mg和EGTA的存在下，磷酸化酶被激活，合酶被灭活;这两个反应中的第一个被微摩尔浓度的Ca 2+刺激了几倍，而这两个反应都被10 mM葡萄糖6-磷酸完全抑制，只有轻微和不规则的环AMP刺激。
Two interconvertible forms of glycogen synthase and glycogen phosphorylase, one active (a) or the other less active (b), were predominantly present in a thermosensitive adenylate-cyclase-deficient mutant that had been preincubated at the restrictive temperature of 35.degree. C, either in the presence or in the absence of glucose. Glycogen phosphorylase was at least 20-fold less active active after incubation of the cells in the presence of glucose, but this residual activity had kinetic properties identical to those of the active form of enzyme, obtained after incubation in the absence of glucose; this suggests that the b form might be completely inactive and that the low activity measured after glucose treatment must be attributed to a residual amount of phosphorylase a. By contrast, the kinetic properties of the two forms of glycogen synthase were very different. When measured in the absence of glucose 6-phosphate, the two forms of enzyme had a similar affinity for UDP-Glc but different essentially by their Vmax. Glucose 6-phosphate had no effect on synthase a, but increased both Vmax and Km of synthase b; these effects, however, were in great part counteracted by sulfate and by inorganic phosphate, the latter also having the property of increasing the Km of the a form, without affecting Vmax. It was estimated that at physiological concentrations of substrates and effectors, synthase a was about 20-fold more active than synthase b. When an extract of cells that had been preincubated in the absence of glucose was gel-filtered and then incubated at 30.degree. C, phosphorylase was progressively fully inactivated and synthase was partially activated; these reactions were severalfold faster and, in the case of glycogen synthase, more complete in the presence of 10 mM glucose 6-phosphate. When a gel-filtered extract of cells that had been preincubated in the presence of glucose was incubated at 30.degree. C in the presence of ATP-Mg and EGTA, phosphorylase became activated and synthase was inactivated; the first of these two reactions was severalfold stimulated by micromolar concentrations of Ca2+, whereas both reactions were completely inhibited by 10 mM glucose 6-phosphate and only slightly and irregularly stimulated by cyclic AMP.