Purification and Characterization of the 22-Kilodalton Potato Tuber Proteins

Abstract

Three abundant proteins of approximate molecular masses of 22, 23, and 24 kilodaltons were purified from potato (Solanum tuberosum L.) tubers by DEAE cellulose and CM-52 cellulose ion exchange column chromatography, electroelution, and high-pressure liquid chromatography (HPLC). Antibodies specific to the gel-purified 22-kilodalton protein were prepared. Immunoblot analysis showed that the 22-, 23-, and 24-kilodalton proteins are immunologically related and that these proteins are present in tubers and as higher molecular mass forms in leaves, but not in stems, roots, and stolons. The ratios of amino acid composition were compared among the three purified proteins, and the aminoterminal amino acid sequences were determined for these three proteins. All three proteins have identical amino-terminal sequences that match the deduced amino acid sequence of an abundant tuber protein cDNA.The three major tuber storage protein groups in potato (Solanum tuberosum L.) are the 40-kD glycoprotein, patatin, the 22-kD complex protein group, and the proteinase inhibitors. All three are developmentally regulated in a coordinate fashion during their growth, and accumulation of these three protein families is inhibited by gibberellic acid (5). Patatin, a glycoprotein that constitutes approximately 40% of the soluble protein in potato tubers, was purified by Racusen and Foote (15) by using DEAE cellulose and concanavalin A Sepharose chromatography. Patatin contains about 5% neutral sugar and 1% hexosamine. Isoelectric focusing detected 6 to 10 ionic forms of patatin in the tubers of all cultivars examined. Park et al.

Two Kunitz-Type Proteinase Inhibitors from Potato Tubers

“…One closely resembles the previously described cathepsin D inhibitor (10). The other is an inhibitor of the microbial proteinase subtilisin and appears to be the same as the abundant 22 kD protein isolated by Suh et al ( 19). This is the first Kunitz-type inhibitor of a microbial proteinase to be identified in a source other than from small grains.…”

“…3A). The amino-terminal sequence of PKI-2 is quite different from PKI-1 but is almost identical to that of a 22 kD protein also isolated from potato tubers (19), with only 2 of 21 residues being different. When compared to Kunitz-type inhibitors from other sources, PKI-2 bears more homology to the Kunitz-type subtilisin inhibitors from various grains than to PKI-1 (Fig.…”

“…To further define the differences between PKI-1 and 2, the purified proteins were tested for inhibitory activity against (20) showed that the sequence of an abundant cDNA clone prepared from tuber mRNA was related to the Kunitz inhibitor family and the deduced amino acid sequence of this clone is identical to that of several members of the 22 kD complex of proteins characterized by Suh et al (19). The inhibitory properties of these proteins have not been described, but a cathepsin D inhibitor recently characterized from tubers is a Kunitz-type inhibitor and is also quite abundant in tubers (7,10 Assays contained 10 mg proteinase and 50 yg inhibitor and were performed as described in "Materials and Methods."…”

“…Potato Kunitz inhibitor-1 (molecular weight 19,500, isoelectric point 6.9) is a potent inhibitor of the animal pancreatic proteinase trypsin, and its amino terminus has significant homology to a recently characterized cathepsin D Kunitz inhibitor from potato tubers (Mares et a!. [1989] (19,20). In this paper, we describe two serine proteinase inhibitors from potato tubers that have differing proteinase inhibitory profiles but are both Kunitztype inhibitors based on amino-terminal sequence homologies.…”

“…For example, a novel inhibitor of cathepsin D, an acid proteinase, has been isolated and characterized from potato tubers (10), and the amino acid sequence of this protein (and another closely related inhibitor, 16) is homologous to the Kunitz family of proteinase inhibitors. Also, the sequence of an abundant 22 kD protein from potato tubers has homology to Kunitz trypsin inhibitors (19,20). In this paper, we describe two serine proteinase inhibitors from potato tubers that have differing proteinase inhibitory profiles but are both Kunitztype inhibitors based on amino-terminal sequence homologies.…”