Camus A, Rose C, Schwartz J C
Unité de Neurobiologie et Pharmacologie (U. 109) de l'INSERM, Centre Paul Broca, Paris, France.
Neuroscience. 1989;29(3):595-602. doi: 10.1016/0306-4522(89)90133-4.
The participation of a serine endopeptidase, previously shown to be involved in endogenous cholecystokinin inactivation [Rose, Camus and Schwartz (1989) Neuroscience 29, 583-594], in the hydrolysis of various exogenous cholecystokinin peptides was studied with slices from rat cerebral cortex. In order to protect intermediate fragments from further degradation and mimick experimental conditions in this previous study, most experiments were performed in the presence of Thiorphan, an enkephalinase inhibitor, and bestatin, an aminopeptidase inhibitor, which did not significantly affect the rate of cholecystokinin-8 hydrolysis. All peptide fragments formed after incubation of cholecystokinin-8, non-sulphated cholecystokinin-8, cholecystokinin-6, cholecystokinin-5, cholecystokinin-4 or Asp-Tyr-Met-Gly-Trp were identified by isocratic high-performance liquid chromatography in several systems, fluorescence spectra and/or amino acid analysis. When identified, the appearing fragments were quantified by u.v. spectrophotometry and found to fully account for the substrate disappearance. The hydrolysis rate was higher for short cholecystokinin peptides than for the octapeptide and was, in all cases, diminished by 30-50% in the presence of diisopropyl fluorophosphate, a serine peptidase inhibitor. One of the main hydrolysis products of cholecystokinin-8, or its non-sulphated analogue, was cholecystokinin-5, whose formation was impaired in the presence of diisopropyl fluorophosphate. Cholecystokinin-5 itself was apparently a substrate for a serine peptidase leading to the formation of the tripeptide Gly-Trp-Met, later cleaved into Trp-Met and Trp. Hence a serine endopeptidase(s) appears to be responsible for cleavage of the two peptides bonds of the cholecystokinin-8 molecule where the carboxyl group is donated by a methionine residue.2+n addition,
一种丝氨酸内肽酶先前已被证明参与内源性胆囊收缩素的失活过程[罗斯、卡缪和施瓦茨（1989年），《神经科学》29卷，583 - 594页]，本研究利用大鼠大脑皮层切片研究了其对各种外源性胆囊收缩素肽的水解作用。为了保护中间片段不被进一步降解，并模拟先前研究中的实验条件，大多数实验是在硫磷酰胺（一种脑啡肽酶抑制剂）和贝司他汀（一种氨肽酶抑制剂）存在的情况下进行的，这两种抑制剂对胆囊收缩素 - 8的水解速率没有显著影响。通过在多个系统中进行等度高效液相色谱、荧光光谱和/或氨基酸分析，鉴定了胆囊收缩素 - 8、非硫酸化胆囊收缩素 - 8、胆囊收缩素 - 6、胆囊收缩素 - 5、胆囊收缩素 - 4或天冬酰胺 - 酪氨酸 - 蛋氨酸 - 甘氨酸 - 色氨酸孵育后形成的所有肽片段。鉴定出的片段通过紫外分光光度法定量，发现其完全解释了底物的消失情况。短的胆囊收缩素肽的水解速率高于八肽，并且在所有情况下，在丝氨酸肽酶抑制剂二异丙基氟磷酸存在时，水解速率降低30 - 50%。胆囊收缩素 - 8或其非硫酸化类似物的主要水解产物之一是胆囊收缩素 - 5，在二异丙基氟磷酸存在时其形成受到损害。胆囊收缩素 - 5本身显然是丝氨酸肽酶的底物，导致形成三肽甘氨酸 - 色氨酸 - 蛋氨酸，随后裂解为色氨酸 - 蛋氨酸和色氨酸。因此，一种丝氨酸内肽酶似乎负责裂解胆囊收缩素 - 8分子中两个肽键，其中羧基由甲硫氨酸残基提供。此外，
