Differences in specificity for the interactions of stefins A, B and D with cysteine proteinases.
Four different stefin-type cysteine proteinase inhibitors have been isolated from porcine thymus and skin. Amino acid sequence determination revealed the presence of stefin A and stefin B type inhibitors and two new inhibitors, designated as porcine stefin D1 and stefin D2. Stefin D1 was identified as PLCPI, an inhibitor recently characterized from porcine polymorphonuclear leukocytes [Lenarcic et al. (1993) FEBS Lett. 336, 289-292]. Stefin A is composed of 101 amino acids and has an Mr of 11 391 while stefin B contains 98 amino acids, has an Mr of 11 174 and is N-terminally blocked. All inhibitors were found to be fast-acting inhibitors of papain, cathepsin L and cathepsin S (Ki = 0.009-0.161 nM). Stefins A and B also bind tightly and rapidly to cathepsin H (Ki = 0.027 and 0.069 nM, respectively), while stefins D1 and D2 have been shown to be very poor inhibitors of cathepsin H (Ki = 102-150 nM). The decreased affinity of these inhibitors toward cathepsin B (Ki = 2-1700 nM) was shown to be mainly due to the low second order association rate constants. The presence of a highly negatively charged N-terminus on stefin D1 constitutes a likely structural determinant of inhibitor specificity.
Lenarcic
B
B
Department of Biochemistry and Molecular Biology, J. Stefan Institute, Ljubljana, Slovenia. brigita.lenarcic@ijs.si
Krizaj
I
I
Zunec
P
P
Turk
V
V
D000595:Amino Acid Sequence; D000818:Animals; D002403:Cathepsins / Q000378:metabolism; D002417:Cattle; D055312:Cystatin A; D055313:Cystatin B; D015891:Cystatins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012879:Skin Physiological Phenomena; D013552:Swine; D013950:Thymus Gland / Q000502:physiology
C524994:CSTB protein, human; D055312:Cystatin A; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D010446:Peptide Fragments; C102233:stefin D protein, Sus scrofa; C026407:CSTA protein, human; D055313:Cystatin B; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D010206:Papain