Peptidyl-alpha-hydroxyglycine alpha-amidating lyase. Purification, characterization, and expression.
Peptidyl-alpha-hydroxyglycine alpha-amidating lyase. Purification, characterization, and expression.
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发表时间：
1991-04
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通讯作者：
B. Eipper;S. Perkins;E. Husten;R. C. Johnson;H. Keutmann;R. Mains
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作者：
B. Eipper;S. Perkins;E. Husten;R. C. Johnson;H. Keutmann;R. Mains
从甘氨酸延伸前体生产α -酰胺肽是一个两步过程，涉及由双功能肽基甘氨酸酰胺化单加氧酶（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-羟化单加氧酶（PHM），一种铜、分子氧和抗坏血酸依赖酶。PAM前体的中间三分之一含有第二种酶，肽基-羟甘氨酸-酰胺裂解酶（PAL）。PAM前体的cooh末端三分之一编码可形成细胞质尾部的跨膜结构域和亲水结构域。利用PAL结构域内肽的抗血清鉴定了一种50 kda的蛋白是牛垂体神经中间体颗粒中PAL的主要形式。这个50 kda的PAL蛋白被纯化并发现始于bPAM的Asp434，表明它可能通过bPAM前体Lys432-Lys433的蛋白内水解裂解产生。以α - n -乙酰基- tyr1 - val - α -羟基甘氨酸为底物，PAL的最佳pH值为5.0；酶活性受高浓度盐的抑制，但对硫醇试剂和尿素具有相对的抗性。PAL活性被EDTA抑制，并被一些二价金属恢复，包括Cd2+， Cu2+， Zn2+和Ca2+。用α - n -乙酰基- tyrl - val - α -羟基甘氨酸进行动力学研究表明，PAL的Km为38微米，周转率为220/s。构建了只编码可溶PHM结构域或删除PHM结构域的PAM前体的表达载体。转染了PHM载体的hEK293细胞分泌的PHM活性增加了10倍，而在对照组和转染的细胞中没有检测到PHM活性。转染PAL载体的hEK293细胞分泌PAL活性增加2倍，细胞PAL活性增加15倍。大多数由转染细胞产生的PAL活性仍然与膜相关。
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.