PLANT PHYSIOLOGY , Vol 103, Issue 4 1227-1234, Copyright © 1993 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Proteolysis of the 85-Kilodalton Crystalline Cysteine Proteinase Inhibitor from Potato Releases Functional Cystatin Domains
T. A. Walsh and J. A. Strickland
Biotechnology Laboratory, DowElanco, 9330 Zionsville Road, Indianapolis, Indiana 46268-1054
The protein crystals found in potato (Solanum tuberosum L.) tuber cells
consist of a single 85-kD polypeptide. This polypeptide is an inhibitor of
papain and other cysteine proteinases and is capable of binding several
proteinase molecules simultaneously (P. Rodis, J.E. Hoff [1984] Plant
Physiol 74: 907-911). We have characterized this unusual inhibitor in more
detail. Titrations of papain activity with the potato papain inhibitor
showed that there are eight papain binding sites per inhibitor molecule.
The inhibition constant (Ki) value for papain inhibition was 0.1 nM.
Treatment of the inhibitor with trypsin resulted in fragmentation of the
85-kD polypeptide into a 32-kD polypeptide and five 10-kD polypeptides. The
32-kD and 10-kD fragments all retained the ability to potently inhibit
papain (Ki values against papain were 0.5 and 0.7 nM, respectively) and the
molar stoichiometries of papain binding were 2 to 3:1 and 1:1,
respectively. Other nonspecific proteinases such as chymotrypsin,
subtilisin Carlsberg, thermolysin, and proteinase K also cleaved the 85-kD
inhibitor polypeptide into functional 22-kD and several 10-kD fragments.
The fragments obtained by digestion of the potato papain inhibitor with
trypsin were purified by reverse-phase high-performance liquid
chromatography, and the N-terminal amino acid sequence was obtained for
each fragment. Comparison of these sequences showed that the fragments
shared a high degree of homology but were not identical. The sequences were
homologous to the N termini of members of the cystatin superfamily of
cysteine proteinase inhibitors. Therefore, the inhibitor appears to
comprise eight tandem cystatin domains linked by proteolytically sensitive
junctions. We have called the inhibitor potato multicystatin (PMC). By
immunoblot analysis and measurement of papain inhibitory activity, PMC was
found at high levels in potato leaves (up to 0.6 mg/g fresh weight tissue),
where it accumulated under conditions that induce the accumulation of other
proteinase inhibitors linked to plant defense. PMC may have a similar
defensive role, for example in protecting the plant from phytophagous
insects that utilize cysteine proteinases for dietary protein digestion.
