Yamaguchi T, Carretero O A, Scicli A G
Hypertension Research Division, Henry Ford Hospital, Detroit, Mich. 48202.
Hypertension. 1991 Jan;17(1):101-6. doi: 10.1161/01.hyp.17.1.101.
We detected a novel vasoconstrictor in an arginine esterase fraction separated from fractions containing tonin and other esterases that were obtained from a rat submandibular gland extract. When tested on isolated rabbit aorta rings, the substance caused dose-related contractions that were slow in onset, long-lasting, and difficult to reverse by rinsing. The substance acts directly on vascular smooth muscle, since preincubation with plasma or intact endothelium is not required. The fact that the constrictor was destroyed by heat and incubation with pronase suggests that it is a protein. Molecular sieving indicates an estimated molecular weight of 24,000 Da. It has a neutral isoelectric point that is higher than the pI of tonin, from which it can be separated by anion exchange chromatography. A small amount of the vasoconstrictor was obtained by gel filtration and eluted from isoelectric focusing polyacrylamide gels. The purified substance showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was a potent vasoconstrictor; an estimated concentration of 2.5 nM induced contraction of isolated rabbit aorta rings ranging from 15% to 40% of the maximum contraction obtained by 60 mM KCl. Contraction was completely blocked by 1 mM (p-amidinophenyl)methanesulfonyl fluoride, a serine protease inhibitor. Contractile activity was not affected by hirudin, a thrombin inhibitor, but was completely inhibited by soybean trypsin inhibitor and blunted by aprotinin; thus it may be a trypsin-like serine protease. Purified vasoconstrictor preparation showed hydrolyzing activity on Pro-Phe-Arg-methyl-coumarin amide, a kallikrein substrate. We conclude that a novel vasoconstrictor serine protease is present in the rat submandibular gland.
我们在从大鼠下颌下腺提取物中获得的含有tonin和其他酯酶的组分中分离出的精氨酸酯酶组分中检测到一种新型血管收缩剂。在离体兔主动脉环上进行测试时，该物质引起剂量相关的收缩，起效缓慢、持续时间长且难以通过冲洗逆转。该物质直接作用于血管平滑肌，因为不需要与血浆或完整内皮进行预孵育。血管收缩剂被加热和与链霉蛋白酶孵育后被破坏，这一事实表明它是一种蛋白质。分子筛分析表明其估计分子量为24,000 Da。它具有中性等电点，高于tonin的pI，可通过阴离子交换色谱将其与tonin分离。通过凝胶过滤获得了少量的血管收缩剂，并从等电聚焦聚丙烯酰胺凝胶中洗脱。纯化后的物质在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上显示出一条带。它是一种强效血管收缩剂；估计浓度为2.5 nM时可诱导离体兔主动脉环收缩，收缩幅度为60 mM KCl诱导的最大收缩幅度的15%至40%。1 mM（对脒基苯基）甲磺酰氟（一种丝氨酸蛋白酶抑制剂）可完全阻断收缩。收缩活性不受凝血酶抑制剂水蛭素的影响，但被大豆胰蛋白酶抑制剂完全抑制，被抑肽酶减弱；因此它可能是一种类胰蛋白酶丝氨酸蛋白酶。纯化的血管收缩剂制剂对激肽释放酶底物Pro-Phe-Arg-甲基香豆素酰胺具有水解活性。我们得出结论，大鼠下颌下腺中存在一种新型血管收缩剂丝氨酸蛋白酶。
