Tyagi S C, Matsubara L, Weber K T
Department of Internal Medicine, University of Missouri-Columbia 65212.
Clin Biochem. 1993 Jun;26(3):191-8. doi: 10.1016/0009-9120(93)90025-2.
Measurement of collagenolytic activity is of interest to a wide variety of investigators using mammalian tissue. In order to develop a method that would quantitate active collagenase from microquantities of human tissue, we employed zymography to the heart and uterus of neonatal, adult, and postpartum rats. Collagenase rapidly cleaves native collagen into two fragments, which at 37 degrees C form gelatin. Gelatin can also be hydrolyzed by collagenase, but at a slower rate, and therefore we used gelatin to quantitate the amount of collagenase present in heart and uterine tissue and developed a method for the direct extraction of collagenase from small quantities of rat myocardium. Our method was found to be comparable with the chemical method reported by Masui et al. (Anal Biochem 1977; 17:215-21). The enzyme, which was not detected in normal adult rat cardiac tissue, was found to exist entirely in latent form and demonstrated typical properties of a mammalian collagenase/gelatinase after activation by trypsin and plasmin. We observed a 60-80% increase in collagenase activity after activation by these proteases and estimated that there is approximately 5 +/- 2 pg of procollagenase per microgram of normal adult rat left ventricle. Collagenolytic activity in the postpartum rat heart was found to be slightly (approximately 2-5%) reduced when compared to the adult heart but it was increased in the neonatal heart and postpartum uterus. This method allows for the rapid quantitative and qualitative measurement of collagenase activity in a variety of tissues containing collagenase/gelatinase activity. Our results indicate that most collagenase in the myocardium exists in latent form.
胶原酶活性的测定受到众多使用哺乳动物组织的研究人员的关注。为了开发一种能够从微量人体组织中定量活性胶原酶的方法，我们对新生、成年和产后大鼠的心脏和子宫进行了酶谱分析。胶原酶能迅速将天然胶原切割成两个片段，这两个片段在37摄氏度时会形成明胶。明胶也能被胶原酶水解，但速度较慢，因此我们用明胶来定量心脏和子宫组织中胶原酶的含量，并开发了一种从少量大鼠心肌中直接提取胶原酶的方法。我们发现我们的方法与Masui等人报道的化学方法相当（《分析生物化学》1977年；17:215 - 21）。在正常成年大鼠心脏组织中未检测到的这种酶，被发现完全以潜伏形式存在，并且在被胰蛋白酶和纤溶酶激活后表现出哺乳动物胶原酶/明胶酶的典型特性。我们观察到经这些蛋白酶激活后胶原酶活性增加了60 - 80%，并估计每微克正常成年大鼠左心室中大约有5±2皮克的前胶原酶。与成年心脏相比，产后大鼠心脏中的胶原酶活性略有降低（约2 - 5%），但在新生心脏和产后子宫中有所增加。这种方法能够对含有胶原酶/明胶酶活性的多种组织中的胶原酶活性进行快速的定量和定性测定。我们的结果表明心肌中的大多数胶原酶以潜伏形式存在。
