The purification and characterization of a glomerular-basement-membrane-degrading neutral proteinase from rat mesangial cells.
A neutral proteinase, capable of degrading gelatin, has been found in both an active and a latent form in the medium from the culture of rat mesangial cells. The latent form had an Mr of 80,000-100,000 and could be activated with either 4-aminophenylmercuric acetate or prolonged incubation at neutral pH. The active form of the enzyme was extensively purified. The estimated Mr of the purified enzyme on gel filtration was approximately 200,000, indicating that the active enzyme formed aggregates. However, analysis by SDS/polyacrylamide-gel electrophoresis under reducing conditions showed two protein bands, with Mr 68,000 and 66,000. Both proteins were found to contain proteolytic activity when run on SDS/substrate gels. The enzyme was inhibited by EDTA and 1,10-phenanthroline, but not by inhibitors for cysteine, serine or aspartic proteinases. The enzyme did not digest fibronectin, bovine serum albumin, proteoglycan or interstitial collagen. The enzyme degraded pepsin-solubilized placental type V collagen at 31 degrees C, whereas similarly solubilized type IV collagen was only degraded at higher temperatures. In addition, the neutral proteinase degraded native soluble type IV collagen. It also had activity on insoluble type IV collagen of glomerular basement membrane. The above properties suggest that the mesangial neutral proteinase belongs to the gelatinase group of metalloproteinases and that it may play a role in the normal turnover of extracellular glomerular matrix.
Davies
M
M
Department of Renal Medicine, University of Wales College of Medicine, Royal Infirmary, Cardiff, U.K.
Thomas
G J
GJ
Martin
J
J
Lovett
D H
DH
D000818:Animals; D001485:Basement Membrane / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018093:Gelatinases; D005920:Glomerular Mesangium / Q000201:enzymology*; D008666:Metalloendopeptidases / Q000302:isolation & purification* / Q000494:pharmacology; D008970:Molecular Weight; D010434:Pepsin A / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D051381:Rats; D013379:Substrate Specificity
D003094:Collagen; D010434:Pepsin A; D018093:Gelatinases; D008666:Metalloendopeptidases; C052706:neutral proteinase, cervical