Kupper U, Niedermann D M, Travaglini G, Lerch K
Biochemisches Institut der Universität Zürich, Switzerland.
J Biol Chem. 1989 Oct 15;264(29):17250-8.
A precursor form of Neurospora crassa tyrosinase has been identified by Western transfer from crude protein extracts and by immunoprecipitation of in vitro translated tyrosinase mRNA. The molecular weight of protyrosinase (75,000) exceeds that of mature tyrosinase (46,000) by about 50%. In order to deduce the primary structure and the nature of the extension, the tyrosinase gene was cloned. Poly(A) RNA isolated from tyrosinase-induced cultures of N. crassa was used as a template for cDNA synthesis, primed by a tyrosinase-specific, 32-fold degenerate heptadecanucleotide. Based on this sequence, a unique 21-mer was synthesized and used to screen a cDNA library constructed from tyrosinase-enriched mRNA. A partial genomic DNA library from wild-type strain TS and a genomic library from strain OR were screened using a 400-base pair nick-translated SalI fragment from a tyrosinase-positive cDNA clone as hybridization probe. The DNA sequences obtained revealed the presence of two allelic forms of this enzyme. The coding regions are interrupted by two short introns, of 52 and 99 base pairs. The encoded proteins differ in 3 out of 621 amino acid residues. A comparison of the deduced amino acid sequence with the known primary structure of mature tyrosinase alleles (Rüegg, C., Ammer, D., and Lerch, K. (1982) J. Biol. Chem. 257, 6420-6426) showed that the enzyme is synthesized as a precursor. Protyrosinase exceeds the mature protein by 213 amino acids at its carboxyl terminus. The possible involvement of carboxyl-terminal processing in enzyme activation is discussed.
粗糙脉孢菌酪氨酸酶的前体形式已通过从粗蛋白提取物进行蛋白质印迹转移以及体外翻译的酪氨酸酶mRNA的免疫沉淀得以鉴定。前酪氨酸酶的分子量（75,000）比成熟酪氨酸酶（46,000）大约高出50%。为了推导其一级结构和延伸部分的性质，克隆了酪氨酸酶基因。从粗糙脉孢菌酪氨酸酶诱导培养物中分离的聚腺苷酸RNA用作cDNA合成的模板，由酪氨酸酶特异性的、32倍简并的十七聚体寡核苷酸引发。基于该序列，合成了一个独特的21聚体并用于筛选由富含酪氨酸酶的mRNA构建的cDNA文库。使用来自酪氨酸酶阳性cDNA克隆的400个碱基对的缺口平移SalI片段作为杂交探针，筛选来自野生型菌株TS的部分基因组DNA文库和来自菌株OR的基因组文库。获得的DNA序列揭示了该酶存在两种等位基因形式。编码区被两个短内含子打断，分别为52和99个碱基对。编码的蛋白质在621个氨基酸残基中有3个不同。将推导的氨基酸序列与成熟酪氨酸酶等位基因的已知一级结构（吕格，C.，阿默，D.，和勒奇，K.（1982年）《生物化学杂志》257，6420 - 6426）进行比较表明，该酶作为前体合成。前酪氨酸酶在其羧基末端比成熟蛋白多213个氨基酸。讨论了羧基末端加工在酶激活中的可能作用。
