Isolation and characterization of isoinhibitors of the potato protease inhibitor I family from the latex of the rubber trees, Hevea brasiliensis.
Three isoinhibitors have been isolated to homogeneity from the C-serum of the latex of the rubber tree, Hevea brasiliensis clone RRIM 600, and named HPI-1, HPI-2a and HPI-2b. The three inhibitors share the same amino acid sequence (69 residues) but the masses of the three forms were determined to be 14,893+/-10, 7757+/-5, and 7565+/-5, respectively, indicating that post-translational modifications of the protein have occurred during latex collection. One adduct could be removed by reducing agents, and was determined to be glutathione, while the other adduct could not be removed by reducing agents and has not been identified. The N-termini of the inhibitor proteins were blocked by an acetylated Ala, but the complete amino acid sequence analysis of the deblocked inhibitors by Edman degradation of fragments from endopeptidase C digestion and mass spectrometry confirmed that the three isoinhibitors were derived from a single protein. The amino acid sequence of the protein differed at two positions from the sequence deduced from a cDNA reported in GenBank. The gene coding for the inhibitor is wound-inducible and is a member of the potato inhibitor I family of protease inhibitors. The inhibitor strongly inhibited subtilisin A, weakly inhibited trypsin, and did not inhibit chymotrypsin. The amino acid residues at the reactive site P(1) and P(1)(') were determined to be Gln45 and Asp46, respectively, residues rarely reported at the reactive site in potato inhibitor I family members. Comparison of amino acid sequences revealed that the HPI isoinhibitors shared from 33% to 55% identity (50-74% similarity) to inhibitors of the potato inhibitor I family. The properties of the isoinhibitors suggest that they may play a defensive role in the latex against pathogens and/or herbivores.
Sritanyarat
Wannapa
W
Department of Biochemistry, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand.
Pearce
Gregory
G
Siems
William F
WF
Ryan
Clarence A
CA
Wititsuwannakul
Rapepun
R
Wititsuwannakul
Dhirayos
D
D000595:Amino Acid Sequence; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D028482:Hevea / Q000737:chemistry*; D007840:Latex / Q000737:chemistry*; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000737:chemistry*
D004791:Enzyme Inhibitors; D007840:Latex; D011480:Protease Inhibitors