DIRECT EXTRACTION AND ESTIMATION OF COLLAGENASE(S) ACTIVITY BY ZYMOGRAPHY IN MICROQUANTITIES OF RAT MYOCARDIUM AND UTERUS

Measurement of collagenolytic activity is Of interest to a wide variet y of investigators using mammalian tissue. In order to develop a metho d that would quantitate active collagenase from microquantities of hum an tissue, we employed zymography to the heart and uterus of neonatal, adult, and postpartum rats. Collagenase rapidly cleaves native collag en into two fragments, which at 37-degrees-C form gelatin. Gelatin can also be hydrolyzed by collagenase, but at a slower rate, and therefor e we used gelatin to quantitate the amount of collagenase present in h eart and uterine tissue and developed a method for the direct extracti on 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 detec ted in normal adult rat cardiac tissue, was found to exist entirely in latent form and demonstrated typical properties of a mammalian collag enase/gelatinase after activation by trypsin and plasmin. We observed a 60-80%. increase in collagenase activity after activation by these p roteases and estimated that there is approximately 5 +/- 2 pg of proco llagenase per mug of normal adult rat left ventricle. Collagenolytic a ctivity in the postpartum rat heart was found to be slightly (approxim ately 2-5%) reduced when compared to the adult heart but it was increa sed in the neonatal heart and postpartum uterus. This method allows fo r the rapid quantitative and qualitative measurement of collagenase ac tivity in a variety of tissues containing collagenase/gelatinase activ ity. Our results indicate that most collagenase in the myocardium exis ts in latent form.