May V, Eipper B A
J Biol Chem. 1985 Dec 25;260(30):16224-31.
The intermediate lobe of the pituitary contains the alpha-amidated peptide alpha-melanotropin and high levels of a copper and ascorbate-dependent peptidylglycine alpha-amidating monooxygenase (PAM) capable of converting peptides terminating in -X-Gly into amidated products (-X-NH2). As reported previously, the ability of cultured intermediate pituitary cells to produce alpha-amidated alpha-melanotropin declined rapidly. A decline in PAM activity assayed in vitro under optimized conditions failed to account quantitatively for the lack of production of alpha-amidated product, while a 100-fold decline in cellular levels of ascorbate could account for the lack of production of alpha-amidated product. Incubation of intermediate pituitary cultures with ascorbate partially restored the ability of the cells to produce alpha-amidated product without significantly increasing the level of PAM activity. In intermediate pituitary cultures made competent to produce alpha-melanotropin by addition of ascorbate, the actual extent of amidation occurring was modulated by the presence of specific secretagogues (bromocriptine or corticotropin-releasing factor). Cultured anterior pituitary cells showed a similar rapid 3-fold decline in PAM activity assayed in vitro under optimized conditions. Cellular levels of ascorbate also declined rapidly to levels 100-fold below those in the intact anterior pituitary. The addition of ascorbate to the anterior pituitary cultures rapidly restored the enzyme activity assayed in vitro to the levels in the initial cell suspension. Thus, production of amidated product peptide may be regulated by cellular levels of ascorbate, by cellular levels of PAM activity, and by the concentration of specific secretagogues to which the cells are exposed.
垂体中间叶含有α-酰胺化肽α-促黑素以及高水平的一种依赖铜和抗坏血酸的肽基甘氨酸α-酰胺化单加氧酶（PAM），该酶能够将以-X-Gly结尾的肽转化为酰胺化产物（-X-NH2）。如先前报道，培养的垂体中间叶细胞产生α-酰胺化α-促黑素的能力迅速下降。在优化条件下体外测定的PAM活性下降并不能定量解释α-酰胺化产物缺乏产生的原因，而细胞内抗坏血酸水平下降100倍可以解释α-酰胺化产物缺乏产生的原因。用抗坏血酸孵育垂体中间叶培养物可部分恢复细胞产生α-酰胺化产物的能力，而不会显著增加PAM活性水平。在通过添加抗坏血酸使垂体中间叶培养物有能力产生α-促黑素的情况下，实际发生的酰胺化程度受特定促分泌素（溴隐亭或促肾上腺皮质激素释放因子）的存在调节。在优化条件下体外测定，培养的垂体前叶细胞的PAM活性也迅速下降了3倍。细胞内抗坏血酸水平也迅速下降至完整垂体前叶水平以下100倍。向垂体前叶培养物中添加抗坏血酸可迅速将体外测定的酶活性恢复到初始细胞悬液中的水平。因此，酰胺化产物肽的产生可能受细胞内抗坏血酸水平、细胞内PAM活性水平以及细胞所接触的特定促分泌素浓度的调节。
