Characterization and molecular cloning of a proenzyme form of a ribosome-inactivating protein from maize. Novel mechanism of proenzyme activation by proteolytic removal of a 2.8-kilodalton internal peptide segment.
Ribosome-inactivating proteins (RIPs) are a widely distributed family of plant enzymes that are remarkably potent catalytic inactivators of eukaryotic protein synthesis. All RIPs described to date, including the A-chain of the plant cytotoxin ricin, are polypeptides of 25-32 kDa and share significant amino acid sequence homologies. We have characterized and cloned an RIP from maize (Zea mays). In contrast to previously described RIPs, we have found that maize RIP is synthesized and stored in the kernel as a 34-kDa inactive precursor (isoelectric point = 6.5). During germination, this neutral precursor is converted into a basic, active form (isoelectric point greater than 9) by limited proteolysis, which removes 25 amino acids (2.8 kDa) of net charge -6 from the center of the polypeptide chain. Additional processing also occurs at the amino and carboxyl termini of the polypeptide. The sequence of the internal processed region is unique and it is equivalent to an insertion centered around Thr-156 in the amino acid sequence of ricin toxin A-chain, i.e. in the center of the enzymatically active domain. The generation of an active enzyme by removal of a large amino acid segment from the middle of a precursor polypeptide chain represents a novel mechanism of proenzyme activation that is distinct from more conventional activation mechanisms involving NH2-terminal proteolytic processing. A two-chain active RIP (comprised of 16.5- and 8.5-kDa fragments that remain tightly associated) is produced from this processing event.
Walsh
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Agricultural Biotechnology Laboratory, DowElanco, Midland, Michigan 48674.
Morgan
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Hey
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D054788:Ribosome Inactivating Proteins; D012270:Ribosomes / Q000378:metabolism*; D016415:Sequence Alignment; D003313:Zea mays / Q000201:enzymology / Q000235:genetics*
D004792:Enzyme Precursors; D010940:Plant Proteins; D004247:DNA; D054788:Ribosome Inactivating Proteins