Murthy A S, Mains R E, Eipper B A
J Biol Chem. 1986 Feb 5;261(4):1815-22.
Extracts of bovine neurointermediate pituitary secretory granules and frozen bovine neurointermediate pituitary contain multiple forms of peptidylglycine alpha-amidating monooxygenase (PAM) activity differing in apparent molecular weight and in charge. Metal chelate affinity chromatography, substrate affinity chromatography, and gel filtration resulted in the purification of two forms of amidation activity from frozen bovine neurointermediate pituitary: PAM-A, apparent molecular weight 54,000, was purified 7,000-fold and PAM-B, apparent molecular weight 38,000, was purified 21,000-fold. Enzyme activity of similar molecular weights was observed in the starting material. Purified PAM-A and PAM-B correspond to two of the three charge forms present in crude extracts, and both exhibited optimal activity at alkaline pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of PAM-B revealed the presence of two bands with apparent molecular weights of 42,000 and 37,000; autoradiography of 125I-labeled PAM-B revealed only the same two bands, and 125I-labeled PAM-B co-eluted with enzyme activity during gel filtration. PAM-A was still heterogeneous based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The properties of purified PAM-A and PAM-B were very similar to those of amidation activity in crude extracts: activity was reduced upon removal of molecular oxygen; activity was stimulated by the addition of CuSO4 and eliminated by the addition of diethyldithiocarbamate; activity was stimulated by the addition of ascorbate, with optimal levels of ascorbate increasing as the concentration of peptide substrate was increased. In the presence of 1.25 mM ascorbate, PAM-B exhibited a Km of 7.0 microM for D-Tyr-Val-Gly and a Vmax of 84 nmol/micrograms/h.
牛神经垂体中间部分分泌颗粒提取物和冷冻的牛神经垂体中间部分含有多种形式的肽基甘氨酸α-酰胺化单加氧酶（PAM）活性，其表观分子量和电荷不同。金属螯合亲和层析、底物亲和层析和凝胶过滤从冷冻的牛神经垂体中间部分纯化出了两种酰胺化活性形式：PAM-A，表观分子量为54,000，纯化了7000倍；PAM-B，表观分子量为38,000，纯化了21,000倍。在起始材料中观察到了类似分子量的酶活性。纯化的PAM-A和PAM-B对应于粗提物中存在的三种电荷形式中的两种，并且两者在碱性pH下均表现出最佳活性。PAM-B的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示存在两条带，表观分子量分别为42,000和37,000；125I标记的PAM-B的放射自显影仅显示相同的两条带，并且在凝胶过滤过程中125I标记的PAM-B与酶活性共洗脱。基于十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，PAM-A仍然是异质的。纯化的PAM-A和PAM-B的性质与粗提物中的酰胺化活性非常相似：去除分子氧后活性降低；添加硫酸铜可刺激活性，添加二乙基二硫代氨基甲酸盐可消除活性；添加抗坏血酸可刺激活性，随着肽底物浓度的增加，抗坏血酸的最佳水平也增加。在存在1.25 mM抗坏血酸的情况下，PAM-B对D-酪氨酸-缬氨酸-甘氨酸的Km为7.0 microM，Vmax为84 nmol/微克/小时。
