Kupper U, Niedermann D M, Schilling B C, Lerch K
Biochemisches Institut, Universität Zürich, Switzerland.
Pigment Cell Res. 1990 Oct;3(4):207-13. doi: 10.1111/j.1600-0749.1990.tb00291.x.
The effects of 3'-5' cyclic AMP and ATP upon tyrosinase induction in Neurospora crassa were examined. Northern analysis of total cellular RNA revealed rapid de novo synthesis of protyrosinase after addition of these substances to stationary-phase mycelia. The maturation of protyrosinase in crude extracts of mycelia was followed by Western analysis. Polyclonal rabbit antiserum directed against the denatured carboxyl-terminal extension of protyrosinase does recognize the proform and several intermediate forms of different molecular weight but not mature tyrosinase. Disruption of ATP-induced mycelia in sodium phosphate buffer (pH 6.0) demonstrate processing at the carboxyl-terminal end of protyrosinase. The activity assays revealed that protyrosinase is an inactive precursor and that at least two active forms of slightly different molecular weight are present in crude extracts. Maturation of protyrosinase thus involves specific and sequential proteolytic cleavage at the carboxyl-terminus. These results suggest the presence of a tyrosinase activator in Neurospora crassa mycelia, which is kept apart from protyrosinase in the intact mycelium.
研究了3'-5'环磷酸腺苷（cAMP）和三磷酸腺苷（ATP）对粗糙脉孢菌中酪氨酸酶诱导的影响。对总细胞RNA进行的Northern分析显示，将这些物质添加到静止期菌丝体后，原酪氨酸酶迅速从头合成。通过Western分析追踪菌丝体粗提物中原酪氨酸酶的成熟过程。针对原酪氨酸酶变性羧基末端延伸的多克隆兔抗血清确实能识别原形式和几种不同分子量的中间形式，但不能识别成熟的酪氨酸酶。在磷酸钠缓冲液（pH 6.0）中破坏ATP诱导的菌丝体，证明原酪氨酸酶在羧基末端进行了加工。活性测定表明，原酪氨酸酶是一种无活性的前体，粗提物中存在至少两种分子量略有不同的活性形式。因此，原酪氨酸酶的成熟涉及羧基末端特异性和顺序性的蛋白水解切割。这些结果表明，粗糙脉孢菌菌丝体中存在一种酪氨酸酶激活剂，在完整的菌丝体中它与原酪氨酸酶是分开的。
