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 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.
Tyagi
S C
SC
Department of Internal Medicine, University of Missouri-Columbia 65212.
Matsubara
L
L
Weber
K T
KT
D000595:Amino Acid Sequence; D000818:Animals; D017364:Collagenases / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D009842:Oligopeptides / Q000378:metabolism; D051381:Rats; D014599:Uterus / Q000201:enzymology*
D009842:Oligopeptides; D017364:Collagenases