Bovine stefin C, a new member of the stefin family.
Four low M(r) cysteine proteinase inhibitors with different pI values were isolated from bovine thymus using alkaline activation of the gland homogenate, affinity chromatography on carboxymethyl-papain-Sepharose, gel filtration on Sephadex G-50, ion-exchange chromatography on a DEAE-cellulose column and a fast protein liquid chromatography Mono Q column, and hydrophobic chromatography on a TSK Phenyl-5 PW column. One of the inhibitors was identified both as the monomeric and dimeric forms of stefin B. Two others, called cysteine proteinase inhibitor-1 and cysteine proteinase inhibitor-2, were N terminally blocked and most likely belong to the stefin family. The complete amino acid sequence of the last inhibitor, namely bovine stefin C, was determined. The inhibitor consisted of 101 amino acids and its M(r) was calculated to be 11,546. It exhibits considerable sequence homology with other inhibitors from the stefin family. It was identified as the first tryptophane-containing stefin and it had a prolonged N terminus. The four inhibitors had similar inhibitory activities on cysteine proteinases. They were fast-acting inhibitors of papain and cathepsin L (kass > or = 1.8 x 10(6) M-1 s-1) and formed very tight complexes with the enzymes (Ki < or = 180 pM). In contrast, they were relatively poor inhibitors of cathepsin B (Ki > 100 nM).
Turk
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B
Department of Biochemistry, J. Stefan Institute, Ljubljana, Slovenia.
Krizaj
I
I
Kralj
B
B
Dolenc
I
I
Popovic
T
T
Bieth
J G
JG
Turk
V
V
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D055313:Cystatin B; D015891:Cystatins / Q000302:isolation & purification / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D007700:Kinetics; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
C524994:CSTB protein, human; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D055313:Cystatin B; D010206:Papain