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Properties of Partially Purified Mouse Epidermal Transglutaminase
Author(s) -
Nakayama Juichiro,
Osaki Mitsuhiko,
Nagae Shonosuke,
Asahi Masakazu,
Urabe Harukuni
Publication year - 1986
Publication title -
the journal of dermatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.9
H-Index - 65
eISSN - 1346-8138
pISSN - 0385-2407
DOI - 10.1111/j.1346-8138.1986.tb02973.x
Subject(s) - sephadex , chemistry , chromatography , size exclusion chromatography , enzyme assay , enzyme , dithiothreitol , trypsin , polyacrylamide gel electrophoresis , biochemistry , specific activity , isoelectric focusing , hydrophilic interaction chromatography , high performance liquid chromatography
Abstract Mouse epidermal transglutaminase was partially purified from the epidermis of newborn BALB/c mice. The epidermal homogenate was subjected to CM‐cellulose chromatography and then to gel filtration on Sephadex G‐150. Two separate peaks with enzyme activity were reproducibly observed in CM‐cellulose chromatography. Gel filtration on Sephadex G‐150 of the peak fractions in CM‐cellulose chromatography gave a single enzyme activity peak (molecular weight about 55,000). Native polyacrylamide gel electrophoresis at pH 4.5 showed two major bands with enzyme activity. The final enzyme preparation (Sephadex G‐150 fraction) had a pH optimum of 10.0 in glycine‐NaOH buffer. Calcium ion and SH‐protecting agent (dithiothreitol) were essential for enzyme activity. The enzyme was not inactivated by heating at 56°C for 45 min in the presence of calcium. Trypsin treatment of the enzyme enhanced the activity about threefold; this enhancement was blocked by trypsin inhibitor. The enzyme bound to lysine‐Sepharose 4B in the presence of casein in Tris‐acetate buffer, pH 6.0, at 37°C; 40% of the enzyme activity was eluted from this enzyme‐lysine‐Sepharose complex with 1 M NaCl.