The extraction of a neutral metalloproteinase from the involuting rat uterus, and its action on cartilage proteoglycan

Abstract

1. Homogenates of rat uteri removed 1 and 2 days post partum were centrifuged at 6000 g. Both pellets and supernatants degraded Azocoll, a general proteinase substrate, at pH 7.5. More than 80% of the total activity was in the pellet fraction. 2. Part of the pellet activity was in a latent form. Trypsin and 4-aminophenylmercuric acetate (a thiol-blocking agent) both activated this latent form, indicating that it is an enzyme–inhibitor complex. An endogenous serine proteinase activated part of the latent enzyme during the assay. 3. The enzyme activity was low before parturition and after involution; it was highest during the first 2 days post partum, when the largest losses of uterine wet weight and matrix macromolecules occur. 4. Up to 70% of the enzyme in the pellets was extracted by heating at 60 degrees C for 4 min in 0.1 M-CaCl2/0.05 M-Tris/HCl, pH 7.5. Approx. 30% of the extracted enzyme was still latent. 5. The extracted enzyme was a metalloproteinase, since it was inhibited completely by 1,10-phenanthroline, but not by inhibitors of thiol or serine proteinases. 6. The enzyme was further purified 15–30-fold by gel chromatography and precipitation with (NH4)2SO4. The apparent molecular weight, estimated by gel filtration, was 24000 for the latent form and 12000 for the active form. The pH optimum was 7–7.5. 7. The enzyme also degraded cartilage proteoglycan. This activity was studied by viscometry and the products were analysed by analytical ultracentrifugation. The major product had a mol.wt. of approx. 100000. The sites of cleavage were in the protein core, since no free oligosaccharides were detected. 8. This neutral metalloproteinase is distinct from uterine collagenase and from a uterine metal-dependent endopeptidase that hydrolyses a heptapeptide related to collagen.

Evidence for metalloproteinase and metalloproteinase inhibitor imbalance in human osteoarthritic cartilage.

“…The present advance in measuring proteinases and TIMP in cartilage was based on finding a good extraction method and ways in which to selectively destroy either enzymes or inhibitor, so that the one would not interfere in the assay of the other. Extraction with guanidineHCl proved to be superior to heat extraction methods found useful for other tissues (19,31). The extracted activity is at least twice the activity detected by direct assay of homogenates, and there is good recovery ofenzymes added back to the homogenates.…”

“…TIMP assay. TIMP was assayed with a low M, metalloproteinase isolated from rat uteri at 1 d postpartum as previously described (13, 19) with slight modification. The enzyme was partially purified by chromatography on Ultrogel AcA 54 (LKB Instruments, Inc., Rockville, MD).…”

CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling

“…Because we observed DBA/1 CD44-null mice to display premature postpartum uterine and lactating mammary gland involution, we focused our attention on MMP-7/HB-EGF/ErbB4 localization and activity in these organs in wild-type and CD44 −/− animals. Heparan sulfate expression in the uterine epithelium varies during the estrous cycle, reaching a peak between 24 and 36 h postpartum (Yu and Woessner 2000), and closely mimics that of MMP-7 (Sellers and Woessner 1980;Wilson and Matrisian 1996;Woessner 1996). In the rat, MMP-7 proteolytic activity is present at term and its concentration rises to a peak between 24 and 36 h postpartum, which coincides with maximal glycoprotein and proteoglycan loss (Sellers and Woessner 1980).…”

Localization of collagenase in the growth plate of rachitic rats.

“…This method has been used in our laboratories for extracting collagenase and neutral metalloproteases from the rat uterus (17) and proved effective with the growth plate as well. No further attempts were made to optimize the extraction since six animals were required for each new protocol.…”