Original scientific paper

Purification and Characterization of Two Cysteine Proteinases from Potato Leaves and the Mode of Their Inhibition with Endogenous Inhibitors

Tatjana Popovič
Jože Brzin

Abstract

Two cysteine proteinases, PLCP-1 and PLCP-2, were purified from potato leaves (Solanum tuberosum
L.). SDS-PAGE of PLCP-2 gave a single band with Mr of 23400 and PLCP-1 gave a
doublet within the same Mr range. Isoelectric focusing of PLCP-2 revealed two bands with pI = 4.6
and 4.9. Both enzymes demonstrate pH optima and maximum stability at slightly acidic pH,
and strong inhibition by L-trans-epoxysuccionylleucylamido(4-guanidino)butane (E-64), cystatin
C and stefin A, enabling them to be assigned to the papain family of cysteine proteinases. PLCP-1
and PLCP-2 were inhibited by Kunitz-type cysteine proteinase inhibitors (PCPIs) and multicystatin,
all isolated from potato tubers. Among PCPIs, the strongest inhibitors were PCPI 9.4,
with Ki in the 10–8 M range, and PCPI 8.3 in the 10–7 M range, while Kis for PCPI 6.6 and
PCPI 5.4 were in the 10–6 M range. Multicystatin was the most potent inhibitor of both proteinases
with Ki of about 0.5 nmol dm–3. The stoichiometry of inhibition of both proteinases
with multicystatin was 1:4 (inhibitor : proteinase). The possible physiological significance of
these endogenous inhibitors, also present in potato leaves, is discussed. PLCP-1 and PLCP-2
could not be differentiated in terms of their Kis.

Keywords

cysteine proteinase; leaf; potato; Solanum tuberosum L.; potato Kunitz peptidase inhibitor-C; multicystatin

Hrčak ID:

12821

URI

https://hrcak.srce.hr/12821

Publication date:

11.3.2007.

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