Tateishi K, Arakawa F, Misumi Y, Treston A M, Vos M, Matsuoka Y
First Department of Biochemistry, School of Medicine, Fukuoka University, Japan.
Biochem Biophys Res Commun. 1994 Nov 30;205(1):282-90. doi: 10.1006/bbrc.1994.2662.
Pancreastatin (PST) is processed from chromogranin A and the C-terminal amide of the peptide is an absolute requirement for biological activities. Human pancreatic carcinoma cells QGP-1 which produce both chromogranin A and PST were used to isolate cDNAs encoding two forms of peptidylglycine alpha-amidating monooxygenase (PAM). The two forms are a full length bifunctional enzyme and a variant lacking the transmembrane domain-coding region. When the cDNAs of these two forms were expressed in COS-7 cells, cells transfected with the predicted soluble form released into the culture medium a very much higher amidating activity which converts human chromogranin A-(273-302) to PST-29. The optimal pH for amidating activity was 5.4 and Cu2+, ascorbate and catalase were required as cofactors for the both forms of PAM. Km values for the membrane-bound and the soluble forms of PAM were 15.7 +/- 3.1 microM and 12.4 +/- 1.6 microM, respectively. These results demonstrate that both forms of PAM can function in the posttranslational processing of chromogranin A to PST in the environment of a secretory vesicle.
胰抑制素（PST）由嗜铬粒蛋白A加工而成，该肽的C末端酰胺是其生物活性的绝对必需条件。利用产生嗜铬粒蛋白A和PST的人胰腺癌细胞QGP-1分离编码两种形式肽基甘氨酸α-酰胺化单加氧酶（PAM）的cDNA。这两种形式分别是全长双功能酶和缺少跨膜结构域编码区的变体。当这两种形式的cDNA在COS-7细胞中表达时，转染了预测的可溶性形式的细胞向培养基中释放出非常高的酰胺化活性，该活性可将人嗜铬粒蛋白A-（273-302）转化为PST-29。酰胺化活性的最适pH为5.4，两种形式的PAM都需要Cu2+、抗坏血酸和过氧化氢酶作为辅因子。膜结合型和可溶性型PAM的Km值分别为15.7±3.1μM和12.4±1.6μM。这些结果表明，在分泌小泡的环境中，两种形式的PAM都可以在嗜铬粒蛋白A向PST的翻译后加工中发挥作用。
