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

Tatjana Popovič
Jože Brzin

Sažetak

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.

Ključne riječi

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

Hrčak ID:

12821

URI

https://hrcak.srce.hr/12821

Datum izdavanja:

11.3.2007.

Posjeta: 1.326 *