title: Purification and some properties of bound cysteine proteinase inhibitor from cornified cells of newborn rat epidermis.
abstract: Cysteine proteinase inhibitor exists in two forms in terminally differentiated keratinocytes. One is readily soluble in 20 mM sodium phosphate buffer but the other is bound to the plasma membrane and is poorly soluble. The cysteine proteinase inhibitor (CPI) from the membrane was extracted from cornified epidermal layers of 2-day-old rats and its properties were compared with those of soluble CPI. This CPI (bound CPI) was solubilized in alkaline 8 M urea containing 2-mercaptoethanol from the residual tissue exhaustively treated with buffered 4 M urea. CPI was separated from keratin by ammonium sulfate precipitation and purified by means of papain affinity chromatography, ion exchange column chromatography and gel filtration. Bound CPI had an Mr value of about 16,000, a pI value of 3.8 and was unstable at above 80 degrees C, while soluble CPI was of Mr 13,000 and stable at above 80 degrees C. Both CPIs were stable at 4 degrees C in the range of 3.0-9.0. Bound CPI contained half cystine and the ratio of acidic-to-basic amino acids was 3.18. Bound CPI inhibited rat liver cathepsins B, H, and L but did not inhibit the activity of noncysteine proteinases. Papain activity was inhibited by bound CPI at three sites, noncompetitively, and the Ki value was calculated to be 0.11 nM.
authors.forename: S
authors.initials: S
authors.lastname: Toku
authors.forename: K
authors.initials: K
authors.lastname: Fukuyama
authors.forename: W L
authors.initials: WL
authors.lastname: Epstein
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000378:metabolism*; D004817:Epidermis / Q000032:analysis*; D010206:Papain / Q000037:antagonists & inhibitors; D011480:Protease Inhibitors / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D011480:Protease Inhibitors; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D010206:Papain