IDENTIFICATION IN PITUITARY TISSUE OF A PEPTIDE ALPHA-AMIDATION ACTIVITY THAT ACTS ON GLYCINE-EXTENDED PEPTIDES AND REQUIRES MOLECULAR-OXYGEN, COPPER, AND ASCORBIC-ACID
IDENTIFICATION IN PITUITARY TISSUE OF A PEPTIDE ALPHA-AMIDATION ACTIVITY THAT ACTS ON GLYCINE-EXTENDED PEPTIDES AND REQUIRES MOLECULAR-OXYGEN, COPPER, AND ASCORBIC-ACID
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DOI：
10.1073/pnas.80.16.5144
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发表时间：
1983-01-01
期刊：
影响因子：
--
通讯作者：
GLEMBOTSKI, CC
中科院分区：
文献类型：
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作者：
EIPPER, BA;MAINS, RE;GLEMBOTSKI, CC
能够产生α-半乳糖苷酶的酶活性，在大鼠垂体前叶、中间叶和神经垂体以及牛垂体中间叶的分泌颗粒中鉴定了其甘氨酸延伸前体的酰胺化肽产物。高水平的该α-半乳糖苷酶的内源性抑制剂在组织匀浆中也发现了酰胺化活性。α-通过加入二价金属离子螯合剂如二乙基二硫代氨基甲酸酯、邻菲咯啉和EDTA完全抑制酰胺化活性;加入Cu后酰胺化活性恢复到高于对照水平。α-酰胺化反应需要分子O2的存在。在测试的各种辅因子中，抗坏血酸是α-谷氨酰胺的最有效的刺激剂。酰胺化。α-酰胺化活性具有中性pH最佳值，并且在几次冻融循环后主要可溶。牛垂体中间颗粒相关活性的动力学研究表明，当D-Tyr-Val-Gly是不同的底物时，线性Lineweaver-Burk图;表观Km和Vmax随抗坏血酸的浓度而变化。α-半乳糖苷酶的底物特异性酰胺化活性似乎相当广泛; D-Tyr-Val-Gly向D-Tyr-Val-NH 2的转化通过加入多种甘氨酸延伸的肽而被抑制。
An enzymatic activity capable of producing an .alpha.-amidated peptide product from its glycine-extended precursor was identified in secretory granules of rat anterior, intermediate and neural pituitary and bovine intermediate pituitary. High levels of endogenous inhibitors of this .alpha.-amidation activity have also been found in tissue homogenates. The .alpha.-amidation activity is totally inhibited by addition of divalent metal ion chelators such as diethyldithiocarbamate, o-phenanthroline and EDTA; .alpha.-amidation activity is restored to above control levels upon addition of Cu. The .alpha.-amidation reaction requires the presence of molecular O2. Of the various cofactors tested, ascorbic acid was the most potent stimulator of .alpha.-amidation. The .alpha.-amidation activity has a neutral pH optimum and is primarily soluble following several cycles of freezing and thawing. Kinetic studies with the bovine intermediate pituitary granule-associated activity demonstrated a linear Lineweaver-Burk plot when D-Tyr-Val-Gly was the varied substrate; the apparent Km and Vmax varied with the concentration of ascorbic acid. The substrate specificity of the .alpha.-amidation activity appears to be quite broad; the conversion of D-Tyr-Val-Gly into D-Tyr-Val-NH2 is inhibited by the addition of a variety of glycine-extended peptides.