title: Purification and characterization of a high-molecular-weight protease, ingensin, from human placenta.
abstract: We purified a high-molecular-weight protease, ingensin, from extract of human placenta by successive DEAE-cellulose, hydroxyapatite, and high performance liquid chromatographies. The activity of ingensin was determined by using a synthetic substrate, succinyl-leucyl-leucyl-valyl-tyrosine-methylcoumarinamide (MCA). The purified ingensin, which gave a single band in 6.5% nondenaturing polyacrylamide gel electrophoresis, was activated by linoleic acid and sodium dodecyl sulfate (SDS). Maximum activity was observed at pH 9.5 in the presence of 0.06% SDS, but at pH 8.0 in the presence of linoleic acid. A subcellular fractionation study showed that a large amount of ingensin activity was present in the cytosol or microsome fraction rather than in the precipitate of low-speed centrifugation. The effect of protease inhibitors on the activated ingensin was also investigated.
mesh_terms: D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D009097:Multienzyme Complexes; D011480:Protease Inhibitors; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex