Yasothornsrikul S, Toneff T, Hwang S R, Hook V Y
Department of Medicine and Center for Molecular Genetics, University of California, San Diego, La Jolla 92093-0822, USA.
J Neurochem. 1998 Jan;70(1):153-63. doi: 10.1046/j.1471-4159.1998.70010153.x.
Conversion of prohormones and neuropeptide precursors to smaller, biologically active peptides requires specific proteolytic processing at paired basic residues, which generates intermediate peptides with NH2 and COOH termini extended with Lys or Arg residues. These basic residues are then removed by aminopeptidase and carboxypeptidase activities, respectively. Among the proteases involved in prohormone processing, the basic residue aminopeptidase activity has not been well studied. This report demonstrates arginine and lysine aminopeptidase activities detected with Arg-methylcoumarinamide (Arg-MCA) and Lys-MCA substrates in neurosecretory vesicles of bovine adrenal medulla [chromaffin granules (CG)], which contain endoproteolytic processing enzymes co-localized with [Met]enkephalin and other neuropeptides. These arginine and lysine aminopeptidase activities showed many similarities and some differences. Both arginine and lysine aminopeptidase activities were stimulated by the reducing agent beta-mercaptoethanol (beta-ME) and inhibited by p-hydroxymercuribenzoate, suggesting involvement of reduced cysteinyl residues. The arginine aminopeptidase activity was stimulated by NaCl (150 mM), but the lysine aminopeptidase activity was minimally affected. Moreover, characteristic beta-ME/NaCl-stimulated Arg-MCA cleaving activity and beta-ME-stimulated Lys-MCA cleaving activity were detected only in CG and not in other subcellular fractions; these findings indicate the localization of these particular basic residue aminopeptidase activities to secretory vesicles. The arginine and lysine aminopeptidase activities showed pH optima at 6.7 and 7.0, respectively. Km(app) values for the arginine and lysine aminopeptidase activities were 104 and 160 microM, respectively. Inhibition by the aminopeptidase inhibitors bestatin, amastatin, and arphamenine was observed for Arg-MCA and Lys-MCA cleaving activities. Inhibition by the metal ion chelators indicated that metalloproteases were involved; Co2+ stimulated the arginine aminopeptidase activity but was less effective in stimulating lysine aminopeptidase activity. In addition, the lysine aminopeptidase activity was partially inhibited by Ni2+ and Zn2+ (1 mM), whereas the arginine aminopeptidase activity was minimally affected. These results demonstrate the presence of related arginine and lysine thiol metalloaminopeptidase activities in CG that may participate in prohormone processing.
激素原和神经肽前体转化为更小的、具有生物活性的肽需要在成对的碱性残基处进行特定的蛋白水解加工，这会产生NH2和COOH末端带有Lys或Arg残基延伸的中间肽。然后这些碱性残基分别被氨肽酶和羧肽酶活性去除。在参与激素原加工的蛋白酶中，碱性残基氨肽酶活性尚未得到充分研究。本报告展示了在牛肾上腺髓质的神经分泌囊泡[嗜铬颗粒（CG）]中，用精氨酸-甲基香豆素酰胺（Arg-MCA）和赖氨酸-甲基香豆素酰胺（Lys-MCA）底物检测到的精氨酸和赖氨酸氨肽酶活性，这些囊泡含有与[甲硫氨酸]脑啡肽和其他神经肽共定位的内切蛋白水解加工酶。这些精氨酸和赖氨酸氨肽酶活性表现出许多相似之处和一些差异。精氨酸和赖氨酸氨肽酶活性均受到还原剂β-巯基乙醇（β-ME）的刺激，并被对羟基汞苯甲酸抑制，表明还原型半胱氨酰残基参与其中。精氨酸氨肽酶活性受到NaCl（150 mM）的刺激，但赖氨酸氨肽酶活性受到的影响最小。此外，仅在CG中检测到特征性的β-ME/NaCl刺激的Arg-MCA切割活性和β-ME刺激的Lys-MCA切割活性，而在其他亚细胞组分中未检测到；这些发现表明这些特定的碱性残基氨肽酶活性定位于分泌囊泡。精氨酸和赖氨酸氨肽酶活性的最适pH分别为6.7和7.0。精氨酸和赖氨酸氨肽酶活性的Km（app）值分别为104和160 microM。观察到氨肽酶抑制剂贝司他汀、抑氨肽酶素和阿法美宁对Arg-MCA和Lys-MCA切割活性有抑制作用。金属离子螯合剂的抑制作用表明涉及金属蛋白酶；Co2+刺激精氨酸氨肽酶活性，但对刺激赖氨酸氨肽酶活性的效果较差。此外，赖氨酸氨肽酶活性受到Ni2+和Zn2+（1 mM）的部分抑制，而精氨酸氨肽酶活性受到的影响最小。这些结果证明CG中存在相关的精氨酸和赖氨酸硫醇金属氨肽酶活性，它们可能参与激素原加工。
