Glembotski C C
Arch Biochem Biophys. 1985 Sep;241(2):673-83. doi: 10.1016/0003-9861(85)90594-6.
In previous studies we have demonstrated a secretory granule-associated peptide alpha-amidation activity in rat anterior, intermediate, and posterior pituitary. This activity is capable of converting 125I-labeled synthetic D-Tyr-Val-Gly to labeled D-Tyr-Val-NH2, and requires ascorbic acid, CuSO4, and molecular oxygen for optimal activity. Because of the requirement for peptides with COOH-terminal glycine residues, and cofactor requirements similar to monooxygenases such as dopamine beta-monooxygenase, we have proposed that the alpha-amidating enzyme be named peptidylglycine alpha-amidating monooxygenase, or PAM. The present study focused on (i) verifying that PAM could utilize a physiologically relevant peptide substrate, and (ii) demonstrating the retention of the cofactor requirements with purification of PAM. PAM (Mr = 50,000) was partially purified from rat anterior pituitary secretory granules and was shown to be capable of converting alpha-N-acetyl-ACTH(1-14) to alpha-N-acetyl-ACTH(1-13)NH2 (alpha-melanocyte stimulating hormone) and ACTH(9-14) to ACTH(9-13)NH2. The optimal rates for these conversions were dependent on ascorbic acid and CuSO4. Kinetic analyses, using the model compound D-Tyr-Val-Gly as the peptide substrate, demonstrated that, compared to the crude granule extract, the partially purified enzyme displayed increased apparent affinities for both the peptide substrate and ascorbate. These analyses also showed that the Km for D-Tyr-Val-Gly was dependent on the concentration of ascorbate, while the Km for ascorbate was constant over a wide range of D-Tyr-Val-Gly concentrations. The results presented here indicate that PAM can alpha-amidate physiologically relevant peptides related to alpha MSH, and performs the reaction in an ascorbate-dependent fashion. Retention of the ascorbate and copper requirements with purification further support the hypothesis that these cofactors are important requirements for the COOH-terminal alpha-amidation of neuro and endocrine peptides.
在先前的研究中，我们已经证明大鼠垂体前叶、中叶和后叶存在一种与分泌颗粒相关的肽α-酰胺化活性。这种活性能够将125I标记的合成D-酪氨酸-缬氨酸-甘氨酸转化为标记的D-酪氨酸-缬氨酸-NH2，并且需要抗坏血酸、硫酸铜和分子氧以达到最佳活性。由于需要具有COOH末端甘氨酸残基的肽，以及与单加氧酶（如多巴胺β-单加氧酶）相似的辅因子需求，我们提议将α-酰胺化酶命名为肽基甘氨酸α-酰胺化单加氧酶，即PAM。本研究着重于：（i）验证PAM是否能利用生理相关的肽底物；（ii）证明随着PAM的纯化，其对辅因子的需求得以保留。从大鼠垂体前叶分泌颗粒中部分纯化得到了PAM（Mr = 50,000），结果表明它能够将α-N-乙酰-促肾上腺皮质激素（1-14）转化为α-N-乙酰-促肾上腺皮质激素（1-13）NH2（α-黑素细胞刺激素），并将促肾上腺皮质激素（9-14）转化为促肾上腺皮质激素（9-13）NH2。这些转化的最佳速率取决于抗坏血酸和硫酸铜。以模型化合物D-酪氨酸-缬氨酸-甘氨酸作为肽底物进行动力学分析表明，与粗颗粒提取物相比，部分纯化的酶对肽底物和抗坏血酸盐的表观亲和力均有所增加。这些分析还表明，D-酪氨酸-缬氨酸-甘氨酸的Km值取决于抗坏血酸盐的浓度，而抗坏血酸盐的Km值在较宽的D-酪氨酸-缬氨酸-甘氨酸浓度范围内保持恒定。此处呈现的结果表明，PAM能够对与α-黑素细胞刺激素相关的生理相关肽进行α-酰胺化，并以依赖抗坏血酸 的方式进行反应。随着纯化过程中抗坏血酸和铜需求的保留，进一步支持了这些辅因子是神经肽和内分泌肽COOH末端α-酰胺化的重要需求这一假说。
