Eipper B A, Perkins S N, Husten E J, Johnson R C, Keutmann H T, Mains R E
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Biol Chem. 1991 Apr 25;266(12):7827-33.
The production of alpha-amidated peptides from their glycine-extended precursors is a two-step process involving the sequential action of two catalytic domains encoded by the bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) precursor. The NH2-terminal third of the PAM precursor contains the first enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), a copper, molecular oxygen, and ascorbate-dependent enzyme. The middle third of the PAM precursor contains the second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The COOH-terminal third of the PAM precursor encodes a transmembrane domain and a hydrophilic domain that may form a cytoplasmic tail. Antisera to a peptide within the PAL domain were used to identify a 50-kDa protein as the major form of PAL in bovine neurointermediate pituitary granules. This 50-kDa PAL protein was purified and found to begin at Asp434 of bPAM, indicating that it could arise through endoproteolytic cleavage of the bPAM precursor at Lys432-Lys433. With alpha-N-acetyl-Tyr-Val-alpha-hydroxyglycine as the substrate, PAL exhibits a pH optimum of 5.0; enzymatic activity is inhibited by high concentrations of salt but is relatively resistant to thiol reagents and urea. PAL activity is inhibited by EDTA and restored by a number of divalent metals, including Cd2+, Cu2+, Zn2+, and Ca2+. Kinetic studies using alpha-N-acetyl-Tyr-Val-alpha-hydroxyglycine indicate that PAL has a Km of 38 microM and a turnover number of 220/s. Expression vectors encoding only the soluble PHM domain or the PAM precursor from which the PHM domain had been deleted were constructed. hEK293 cells transfected with the PHM vector exhibited a 10-fold increase in secretion of PHM activity with no PHM activity detectable in control or transfected cells. hEK293 cells transfected with the PAL vector exhibited a 2-fold increase in secretion of PAL activity and a 15-fold increase in cellular PAL activity. Most of the PAL activity produced by the transfected cells remained membrane-associated.
从其甘氨酸延伸前体生成α-酰胺化肽是一个两步过程，涉及由双功能肽基甘氨酸α-酰胺化单加氧酶（PAM）前体编码的两个催化结构域的顺序作用。PAM前体的NH2末端三分之一包含第一种酶，肽基甘氨酸α-羟化单加氧酶（PHM），一种依赖铜、分子氧和抗坏血酸的酶。PAM前体的中间三分之一包含第二种酶，肽基-α-羟基甘氨酸α-酰胺化裂解酶（PAL）。PAM前体的COOH末端三分之一编码一个跨膜结构域和一个可能形成细胞质尾巴的亲水区。针对PAL结构域内一种肽的抗血清被用于鉴定一种50 kDa的蛋白质，它是牛神经垂体中间叶颗粒中PAL的主要形式。这种50 kDa的PAL蛋白被纯化，发现它从bPAM的Asp434开始，这表明它可能是通过bPAM前体在Lys432-Lys433处的内蛋白水解裂解产生的。以α-N-乙酰-Tyr-Val-α-羟基甘氨酸为底物时، PAL的最适pH为5.0；酶活性受到高浓度盐的抑制，但相对耐受硫醇试剂和尿素。PAL活性受到EDTA的抑制，并被多种二价金属恢复，包括Cd2+、Cu2+、Zn2+和Ca2+。使用α-N-乙酰-Tyr-Val-α-羟基甘氨酸的动力学研究表明，PAL的Km为38 μM，周转数为220/s。构建了仅编码可溶性PHM结构域或已缺失PHM结构域的PAM前体的表达载体。用PHM载体转染的hEK293细胞分泌的PHM活性增加了10倍，在对照细胞或转染细胞中均未检测到PHM活性。用PAL载体转染的hEK293细胞分泌的PAL活性增加了2倍，细胞内PAL活性增加了15倍。转染细胞产生的大部分PAL活性仍与膜相关。
