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Regulation of cyclic AMP phosphodiesterase from Mucor rouxii by phosphorylation and proteolysis. Interrelationship of the activatable and insensitive forms of the enzyme.

机译:通过磷酸化和蛋白水解调节来自Mucor rouxii的环状AMP磷酸二酯酶。酶的可激活形式和不敏感形式的相互关系。

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摘要

DEAE-cellulose chromatography of mycelial extracts of Mucor rouxii grown to mid-exponential phase resolves two types of low-Km cyclic AMP phosphodiesterase (EC 3.1.4.17; PDE): PDE I, highly activatable (4-6-fold) by phosphorylation or proteolysis, and PDE II, unresponsive to activation. The enzymic profile of PDE activity obtained from germlings shows only PDE I activity, whereas PDE activity from mycelia grown to stationary phase is eluted from the DEAE-cellulose column at the position of PDE II, and like PDE II is unresponsive to activation. Endogenous proteolysis or controlled trypsin treatment transforms PDE I into PDE II. The insensitive forms of PDE exhibit a slightly smaller sedimentation coefficient than the activatable forms, as judged by sucrose-gradient centrifugation. The basal activity of the highly activatable form of PDE is elevated almost to the value in the presence of trypsin on storage at 4 degrees C in the absence of proteinase inhibitors. Benzamidine, leupeptin, antipain or EGTA prevents the activation produced by storage. PDE I remains strongly activatable by phosphorylation and proteolysis after resolution by polyacrylamide-gel electrophoresis.
机译:生长到中指数期的Mucor rouxii菌丝体提取物的DEAE纤维素色谱法可分离两种类型的低Km环状AMP磷酸二酯酶(EC 3.1.4.17; PDE):PDE I,可通过磷酸化高度活化(4-6倍)或蛋白水解和PDE II对激活无反应。从种苗中获得的PDE活性的酶学特征仅显示PDE I活性,而从生长到固定相的菌丝体的PDE活性则从DEAE-纤维素柱上的PDE II位置洗脱下来,并且像PDE II一样对激活没有反应。内源性蛋白水解或受控的胰蛋白酶处理可将PDE I转化为PDE II。通过蔗糖梯度离心法可以判断,不敏感的PDE形式的沉降系数比可激活的形式小。在没有蛋白酶抑制剂的情况下,在4°C下储存胰蛋白酶时,高度可活化形式的PDE的基础活性几乎提高到该值。苄am,亮肽素,抗痛药或EGTA可以防止储存引起的活化。通过聚丙烯酰胺凝胶电泳拆分后,PDE I仍可通过磷酸化和蛋白水解强烈激活。

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