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Originally published In Press as doi:10.1074/jbc.M001406200 on March 9, 2000
J. Biol. Chem., Vol. 275, Issue 20, 15572-15577, May 19, 2000
J Biol Chem, Vol. 275, Issue 20, 15572-15577, May 19, 2000
Saxiphilin, a Saxitoxin-binding Protein with Two Thyroglobulin
Type 1 Domains, Is an Inhibitor of Papain-like Cysteine
Proteinases*
Brigita
Lenar i §,
Gomathi
Krishnan¶,
Renata
Borukhovich¶,
Brian
Ruck¶,
Vito
Turk , and
Edward
Moczydlowski¶
From the Departments of Biochemistry and Molecular
Biology, J. Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia
and the Departments of ¶ Pharmacology and Cellular and
Molecular Physiology, Yale University School of Medicine,
New Haven, Connecticut 06520-8066
The type 1 domain of thyroglobulin is a protein
module (Thyr-1) that occurs in a variety of secreted and membrane
proteins. Several examples of Thyr-1 modules have been previously
identified as inhibitors of the papain family of cysteine proteinases.
Saxiphilin is a neurotoxin-binding protein from bullfrog and a homolog
of transferrin with a pair of such Thyr-1 modules located in the N-lobe. Saxiphilin is now characterized as a potent inhibitor of three
cysteine proteinases as follows: papain, human cathepsin B, and
cathepsin L. The stoichiometry of enzyme inhibition reveals that both
Thyr-1 domains of saxiphilin inhibit papain (apparent Ki = 1.72 nM), but only one of these
domains inhibits cathepsin B (Ki = 1.67 nM) and cathepsin L (Ki = 0.02 nM). Physical association of saxiphilin and papain blocked from turnover at the active-site cysteine residue can be detected by
cross-linking with glutaraldehyde. The rate of association of
saxiphilin and cathepsin B is strongly pH-dependent with an optimum at pH 5.2, reflecting control by at least two
H+-titratable groups. These results further demonstrate
that various Thyr-1 domains are selective inhibitors of cysteine
proteinases with utility in the study of protein interactions and degradation.
*
This work was supported by the Ministry of Science and
Technology of the Republic of Slovenia Grant J1-7422-0106 (to V. T.) and NIGMS Grant GM51172 from the National Institutes of Health (to
E. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, J. Stefan Institute, Jamova 39, 1000 Ljubljana,
Slovenia. Tel.: 386-1-4773623; Fax: 386-1-2573594; E-mail:
brigita.lenarcic@ijs.si.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 2000 by the American Society for Biochemistry and Molecular Biology.
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