Suiko M, Fernando P H, Sakakibara Y, Nakajima H, Liu M C, Abe S, Nakatsu S
Department of Biological Resource Sciences, Miyazaki University, Japan.
Nucleic Acids Symp Ser. 1992(27):183-4.
In vitro tyrosine sulfation of recombinant proteins would be a valuable tool in converting those proteins expressed in prokaryotic vectors to their natural form. For this purpose tyrosylprotein sulfotransferase (TPST), the enzyme responsible for tyrosine sulfation of proteins, was characterized from a bovine liver Golgi preparation. TPST was active in a acidic environment with a pH optimum of 6.25, and displayed a stimulation by the Mn2+, with the optimum activity in the presence of 5mM MnCl2. TPST was able to sulfate recombinant hirudin variant 1 (rHV-1) expressed in Escherichia coli and the C-terminal hirudin fragment 54-65 but not the N-terminal hirudin fragment 1-15 by using 3'-phosphoadenosine 5'-phosphosulfate (PAPS), indicating its specificity for the naturally sulfated tyrosine 63. Comparison of the reaction kinetics on synthetic peptides showed that the bovine liver TPST has a higher affinity and reaction rates for those peptides with a aspartyl residue on the N-terminal side of the tyrosine when compared with a glutamyl residue.
重组蛋白的体外酪氨酸硫酸化将是一种将原核载体中表达的蛋白转化为其天然形式的有价值工具。为此，从牛肝高尔基体提取物中鉴定了负责蛋白酪氨酸硫酸化的酶——酪氨酰蛋白硫酸转移酶（TPST）。TPST在酸性环境中具有活性，最适pH为6.25，在Mn2+存在下表现出刺激作用，在5mM MnCl2存在时活性最佳。TPST能够利用3'-磷酸腺苷5'-磷酸硫酸酯（PAPS）对在大肠杆菌中表达的重组水蛭素变体1（rHV-1）和水蛭素C末端片段54-65进行硫酸化，但不能对水蛭素N末端片段1-15进行硫酸化，这表明其对天然硫酸化的酪氨酸63具有特异性。对合成肽反应动力学的比较表明，与谷氨酰残基相比，牛肝TPST对酪氨酸N末端侧带有天冬氨酰残基的肽具有更高的亲和力和反应速率。
