Purification and properties of an aspartic protease from potato tuber that is inhibited by a basic chitinase

Abstract

A protease was isolated from potato (Solanum tuberosum L. cv. Huinkul) tuber disks after 24 h of aeration when proteolysis is markedly increased. Purification was performed by ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography. A size of 40 kDa was estimated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and gel filtration, it is monomeric and its properties are consistent with those of aspartic proteinases (EC 3.4.23): it had a pH optimum between 4 and 5 and it was inhibited by pepstatin. Partial homology with other plant aspartic proteinases was observed in two sequenced tryptic fragments. It binds to Sepharose‐concanavalin A and can be eluted with α‐methyl mannoside, indicating that it is possibly glycosylated. Unlike other aspartic proteinases from Solanaceae that degrade pathogenesis‐related proteins, it is unable to cleave a basic chitinase from potato. Moreover, this aspartic protease is strongly inhibited by the basic chitinase; the 50% inhibition is obtained when the molar ratio approaches 1, the same as with pepstatin. The interaction between this aspartic protease and a new type of endogenous inhibitor may be an interesting starting point to study the regulation of these aspartic proteases during stress.

Protease inhibitor activity is associated to a basic chitinase from potato but not to an acidic one

“…However, it has significant similarity to two pathogenesis-related proteins, tobacco PRP-la and tomato PR leaf protein p14. Our results (Guevara et al, 1999) and those reported by Yamakawa et al (1998) suggest a new, still unknown physiological role for P25TI and its related proteins in plants.…”

“…However, in a previous work, we have described that a potato basic chitinase strongly inhibited the activity of an aspartic protease isolated from the same source (Guevara et al, 1999). These results indicate that at least one member of the family of plant proteins with chitinase activity may have an endogenous role in the regulation of proteases.…”

Antimicrobial activity of potato aspartic proteases (StAPs) involves membrane permeabilization

“…We have identified three potato (Solanum tuberosum) aspartic proteases, one from tubers (StAP1) (Guevara et al, 1999) and two from leaves (StAP2 and StAP3) (Guevara et al, 2001). Two of these isoforms, StAP1 and StAP3, have been purified and characterized.…”

“…Sterile disks (10 mm diameter, 2 mm thick) of potato tuber were prepared, washed extensively and suspended in sterile water (15 disks in 23 ml water) and aerated for 24 h at 25 uC in an orbital shaker at 60 cycles min 21 . StAP1 was purified from tubers using the protocol previously described by Guevara et al (1999).…”

BABA effects on the behaviour of potato cultivars infected by Phytophthora infestans and Fusarium solani

“…The nitrocellulose sheet was soaked for 2 h with a solution containing 100 mM Tris-HCl, pH 8.0 and 1% (w/v) BSA. The membrane was washed four times with 100 mM TrisHCl, pH 8.0 containing 150 mM NaCl and 0.05% (v/v) Tween 20 (TBST) and then incubated overnight with rabbit anti-StAP1 (1:10,000 v/v; Guevara et al 1999) in 100 mM Tris-HCl, pH 8.0, and 1% BSA. After four washes with TBST solution, the blot was allowed to react for 2 h with goat anti-rabbit antibody (1:10,000 v/v) labelled with alkaline phosphatase (Sigma).…”