title: Cysteine protease characteristics of the proteoglycanase activity from normal and pseudoxanthoma elasticum (PXE) fibroblasts.
abstract: Pseudoxanthoma elasticum (PXE) is an inherited disease characterized by calcified degenerative changes of elastin in the skin, eye, and vasculature. Previous studies suggested the abnormal presence of a protease from PXE fibroblasts that degrades sulfated proteoglycans. This study describes the use of a radioassay to quantitate proteoglycan degradation by proteases from normal and PXE fibroblasts. PXE protease had optimal activity at pH 6.0. Inhibition of activity by 5 mM diisopropylfluorophosphate, 5 mM phenylmethylsulfonylfluoride, and 0.1 mM HgCl2 was reversed by 10 mM dithiothreitol. Iodoacetamide (1 mM) irreversibly inhibited activity. Carbobenzyloxy-phenylalanyl-alanyl (0.1 mM) and carbobenzyloxy-lysyl-diazomethyl ketone (10 microM) inhibited the proteoglycanase activity. These data suggest that the PXE proteolytic proteoglycanase activity is a cysteine protease. After blocking activity with 5 mM EDTA, addition of 10 mM Mg++, Mn++, Cu++, or Co++ had little effect (less than 10%) on restoring activity, 10 mM CaCl2 restored approximately 70% recovery of the activity, and 10 mM ZnCl2 stimulated the activity to 500% of the initial level. Similar normal fibroblast samples contained little zinc-dependent activity and a substantial amount of calcium-dependent activity. Thus the distinction between the divalent ion requirements for proteoglycan degradation suggests that the PXE fibroblasts may produce a different cysteine protease than do normal fibroblasts.
mesh_terms: D002118:Calcium / Q000494:pharmacology; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000378:metabolism*; D004791:Enzyme Inhibitors; D005347:Fibroblasts / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases; D008970:Molecular Weight; D011509:Proteoglycans / Q000378:metabolism; D011561:Pseudoxanthoma Elasticum / Q000201:enzymology / Q000473:pathology*; D014357:Trypsin / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D011509:Proteoglycans; D010450:Endopeptidases; C022474:proteoglycan-degrading metalloendopeptidases; D014357:Trypsin; D003546:Cysteine Endopeptidases; D008666:Metalloendopeptidases; D015032:Zinc; D002118:Calcium