Glembotski C C, Eipper B A, Mains R E
J Biol Chem. 1984 May 25;259(10):6385-92.
A secretory granule associated enzymatic activity from the rat anterior pituitary that can convert the synthetic peptide D-Tyr-Val-Gly into D-Tyr-Val-NH2 is described. Due to the presence of inhibitory activity in the cytosol and rough endoplasmic reticulum/Golgi apparatus fractions, the alpha-amidation activity is barely detectable in crude rat anterior pituitary homogenates. The alpha-amidation activity was primarily soluble, displayed a pH optimum of about 7.0, and showed a requirement for molecular oxygen. The activity was stimulated 7-fold by the addition of optimal concentrations of copper. Of the cofactors tested, only reduced ascorbate produced a severalfold stimulation of activity. Studies with varied D-Tyr-Val-Gly or varied monoiodo-D-Tyr-Val-Gly demonstrate Michaelis Menten kinetics with indistinguishable kinetic constants for both peptides. Upon addition of ascorbate, the apparent Km and Vmax for the synthetic substrate, as estimated from Lineweaver-Burk and Eadie-Hofstee plots, increased by 30- and 60-fold, respectively. Several alpha-melanotropin- and gamma-melanotropin-related peptides with COOH-terminal glycine residues were effective competitive inhibitors of the reaction while the corresponding alpha-amidated peptides were very poor inhibitors. The rat anterior pituitary alpha-amidation activity appears to be very similar to the alpha-amidation activity in rat intermediate and neural pituitary, and mouse anterior pituitary tumor cells.
本文描述了一种来自大鼠垂体前叶的分泌颗粒相关酶活性，该活性可将合成肽D-酪氨酸-缬氨酸-甘氨酸转化为D-酪氨酸-缬氨酸-NH2。由于细胞质和粗面内质网/高尔基体部分存在抑制活性，在大鼠垂体前叶粗匀浆中几乎检测不到α-酰胺化活性。α-酰胺化活性主要为可溶性，最适pH约为7.0，且需要分子氧。加入最佳浓度的铜可使该活性提高7倍。在所测试的辅因子中，只有还原型抗坏血酸能使活性产生几倍的刺激。对不同的D-酪氨酸-缬氨酸-甘氨酸或不同的单碘-D-酪氨酸-缬氨酸-甘氨酸进行的研究表明，两者均呈现米氏动力学，且两种肽的动力学常数无法区分。加入抗坏血酸后，根据Lineweaver-Burk和Eadie-Hofstee图估算，合成底物的表观Km和Vmax分别增加了30倍和60倍。几种具有COOH末端甘氨酸残基的α-促黑素和γ-促黑素相关肽是该反应的有效竞争性抑制剂，而相应的α-酰胺化肽则是非常弱的抑制剂。大鼠垂体前叶的α-酰胺化活性似乎与大鼠垂体中间叶和神经垂体以及小鼠垂体前叶肿瘤细胞中的α-酰胺化活性非常相似。
