Subtilisin Protein Inhibitor from Potato Tubers

T. A. Revina, A. S. Speranskaya, G. V. Kladnitskaya, A. B. Shevelev, and T. A. Valueva^*

Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow 119071, Russia; fax: (7-095) 954-2732; E-mail: valueva@inbi.ras.ru

^* To whom correspondence should be addressed.

Received May 18, 2004

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A protein with molecular weight of 21 kD denoted as PKSI has been isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii). The isolation procedure includes precipitation with (NH₄)₂SO₄, gel chromatography on Sephadex G-75, and ion-exchange chromatography on CM-Sepharose CL-6B. The protein effectively inhibits the activity of subtilisin Carlsberg (K_i = 1.67 ± 0.2 nM) by stoichiometric complexing with the enzyme at the molar ratio of 1 : 1. The inhibitor has no effect on trypsin, chymotrypsin, and the cysteine proteinase papain. The N-terminal sequence of the protein consists of 19 amino acid residues and is highly homologous to sequences of the known inhibitors from group C of the subfamily of potato Kunitz-type proteinase inhibitors (PKPIs-C). By cloning PCR products from the genomic DNA of potato, a gene denoted as PKPI-C2 was isolated and sequenced. The N-terminal sequence (residues from 15 to 33) of the protein encoded by the PKPI-C2 gene is identical to the N-terminal sequence (residues from 1 to 19) of the isolated protein PKSI. Thus, the inhibitor PKSI is very likely encoded by this gene.

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KEY WORDS: Kunitz-type proteinase inhibitors, subtilisin, potato tubers

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