Strigácová J, Chovanec P, Liptaj T, Hudecová D, Turský T, Simkovic M, Varecka L
Department of Biochemistry and Microbiology, Slovak University of Technology, Bratislava.
Arch Microbiol. 2001 Jan;175(1):32-40. doi: 10.1007/s002030000235.
Glutamic acid decarboxylase (GAD) activity was measured in homogenates of conidia and both submerged and aerial mycelia of Trichoderma viride. The GAD activity in conidia had a temperature optimum at 30 degrees C and a pH optimum at pH 4. GAD was stimulated by EDTA (2 mM) and was insensitive to treatment with calmodulin antagonists calmidazolium (10 microM) or phenothiazine neuroleptics (60 microM). Cyclosporin A (up to 300 microM) partially inhibited GAD in the homogenate, but not in the supernatant obtained after centrifuging the homogenate. Attempts to release GAD activity from the homogenate using high ionic strength, detergents, or urea failed. Freezing-thawing led to the partial increase of activity in the conidial homogenate. These results indicate that GAD is a membrane-bound enzyme. The highest specific activity of GAD was present in the mitochondrial/vacuolar organellar fraction. Germination of conidia in the submerged culture led to a temporary decrease in GAD activity. After prolonged cultivation, the activity displayed quasi-oscillatory changes. The stationary state was characterized by a high GAD activity. The presence of gamma-aminobutyric acid in the submerged mycelia was demonstrated. In surface culture in the dark, GAD activity increased in a monophasic manner until conidia formation. The illumination of dark-cultivated mycelia by a white-light pulse caused a dramatic increase in GAD activity. Light-induced changes were not observed in mutants with delayed onset of conidiation. In the dark or upon illumination by light pulse, the increase of GAD activity preceded the appearance of conidia. Thus, GAD activity in T. viride is closely associated with its developmental status and may represent a link between differentiation events and energy metabolism.
测定了绿色木霉分生孢子以及深层和气生菌丝体匀浆中的谷氨酸脱羧酶（GAD）活性。分生孢子中的GAD活性在30℃时具有最佳温度，在pH 4时具有最佳pH值。GAD受到EDTA（2 mM）的刺激，对钙调蛋白拮抗剂氯咪唑（10 microM）或吩噻嗪类抗精神病药物（60 microM）的处理不敏感。环孢菌素A（高达300 microM）部分抑制匀浆中的GAD，但对匀浆离心后获得的上清液中的GAD没有抑制作用。尝试使用高离子强度、去污剂或尿素从匀浆中释放GAD活性均未成功。冻融导致分生孢子匀浆中的活性部分增加。这些结果表明GAD是一种膜结合酶。GAD的最高比活性存在于线粒体/液泡细胞器部分。深层培养中分生孢子的萌发导致GAD活性暂时降低。长时间培养后，活性呈现准振荡变化。稳定状态的特征是GAD活性高。证明了深层菌丝体中存在γ-氨基丁酸。在黑暗中的表面培养中，GAD活性以单相方式增加直至分生孢子形成。用白光脉冲照射黑暗培养的菌丝体会导致GAD活性急剧增加。在分生孢子形成延迟的突变体中未观察到光诱导的变化。在黑暗中或受到光脉冲照射时，GAD活性的增加先于分生孢子的出现。因此，绿色木霉中的GAD活性与其发育状态密切相关，可能代表了分化事件与能量代谢之间的联系。
