Glomerular basement membrane degradation by endogenous cysteine proteinases in isolated rat glomeruli.
Recent in vitro and in vivo studies suggest that cysteine proteinases may play an important role in degradation of the glomerular basement membrane (GBM) by renal glomeruli. However, little information is available concerning the cysteine proteinases present in glomeruli, the distribution of cysteine proteinases in other areas of the kidney, or the potential role of endogenous glomerular cysteine proteinases in GBM degradation. Using well characterized fluorogenic substrates, we have documented the presence of the cysteine proteinases, cathepsins B, H, and L, in glomeruli (0.45 +/- 0.06, 0.39 +/- 0.05, and 0.66 +/- 0.14 mU/mg protein, mean +/- SEM, N = 8) and other fractions prepared from normal rat kidney. The presence of cysteine proteinases in glomeruli was verified by fluorescence microscopy. For each proteinase, the activity was: proportional to the amount of tissue protein and time of incubation; dependent on the presence of exogenously added dithiothreitol; and completely inhibited by the specific cysteine proteinase inhibitor, E-64. The pH optimum for cathepsin B (substrate: Z-Arg-Arg-HNMec) and L (substrate: Z-Phe-Arg-HNMec in the presence of Z-Phe-Phe-CHN2) was approximately pH 6.0 for both glomeruli and renal cortex; while that for cathepsin H (substrate: Arg-HNMec) was approximately 6.5. Incubation of sonicated glomeruli with 3H-GBM under conditions optimal for cysteine proteinase activity (pH 4.5, 1 mM EDTA, and 1 mM dithiothreitol, 37 degrees C) resulted in significant GBM degradation as measured by the release of non-sedimentable (10,000 x g, 10 min) radioactivity or hydroxyproline.(ABSTRACT TRUNCATED AT 250 WORDS)
Baricos
W H
WH
Department of Biochemistry, Tulane University Medical School, New Orleans, Louisiana.
Cortez
S L
SL
Le
Q C
QC
Zhou
Y W
YW
Dicarlo
R M
RM
O'Connor
S E
SE
Shah
S V
SV
D000818:Animals; D001485:Basement Membrane / Q000201:enzymology; D002403:Cathepsins / Q000378:metabolism*; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D066298:In Vitro Techniques; D007678:Kidney Glomerulus / Q000201:enzymology*; D007684:Kidney Tubules / Q000201:enzymology; D008297:Male; D008856:Microscopy, Fluorescence; D051381:Rats; D011919:Rats, Inbred Strains
D002403:Cathepsins; D003546:Cysteine Endopeptidases