Purification, Characterization, and Sequencing of Two Cysteine Proteinase Inhibitors, Sca and Scb, from Sunflower (Helianthus annuus) Seeds.

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  • Purification, Characterization, and Sequencing of Two Cysteine Proteinase Inhibitors, Sea and Scb, from Sunflower (Helianthus annuus) Seeds

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Two proteinaceous cysteine proteinase inhibitors (cystatins) referred to as Sca and Scb were purified to homogeneity from the seeds of sunflower (Heliantas annuus) by gel filtration on Sephadex G-75 followed by a series of ion-exchange column chromatographies and reverse-phase HPLC (RP-HPLC). The isoelectric points (pI) of Sea and Scb were estimated to be 5.6 and 9.6, respectively. The inhibitory potencies of these two cystatins were examined with cysteine proteinases from various sources, such as plants, mammals, and bacteria. Papain was strongly inhibited by both Sca and Scb with K1 values of 5.6×10-9 and 1.7×10-10M, respectively. Sca and Scb were also found to be potent inhibitors of ficin (K1 values of 1.9×10-6 and 2.8×10-9M, respectively). Rat cathepsin H was inhibited strongly by Scb and slightly by Sea. Although rat cathepsins B and L were significantly inhibited by Seb, they were scarcely affected by Sea. Neither Sca nor Scb inhibited Arg-gingipain, an arginine-specific cysteine proteinase of Porphyromonas gingivalis. The complete amino acid sequences of the two inhibitors were determined by protein chemical methods. The proteins Sea and Scb consist of 83 and 101 amino acid residues with Mr of 9, 330 and 11, 187, respectively, and there are identical residues at 34 positions in the two sequences, that is at 42% of the residues compared. Comparison of their sequences with those of other cystatins revealed that Sea shares 59-73% identical residues with other phytocystatins, while Scb shows less identity to other phytocystatins, sharing only 28-38% identical residues. Furthermore, only 20-27% of the residues of both cystatins, Sca and Scb, are identical to those of the animal cystatins.

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