Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0277787 (stigma)
 13,352 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The three-dimensional structures of a series of 6-kDa trypsin inhibitors isolated from the stigma of the ornamental tobacco Nicotiana alata have been determined by 1H NMR spectroscopy combined with simulated annealing calculations. The proteins, T1-T4, are proteolytically cleaved from a 40.3-kDa precursor protein, NA-proPI, together with a chymotrypsin inhibitor, C1, the structure of which was reported recently [Nielsen, K.J., Heath, R.L., Anderson, M.A., & Craik, D.J. (1994) J. Mol. Biol. 242, 231-243]. Each of the proteinase inhibitors comprises 53 amino acids, including 8 cysteine residues which are linked to form 4 disulfide bridges. The proteins have a high degree of sequence identity and differ mainly in residues around the putative reactive sites. The structure of T1 was determined using a set of 533 interproton distance restraints derived from NOESY spectra, combined with 33 dihedral restraints derived from 3JNH-H alpha coupling constants and 16 hydrogen bonds. The structures of the remaining inhibitors (T2-T4) were deduced to be almost identical to T1, on the basis of their similar chemical shifts and 2D spectra. The current study demonstrates that the structures of the trypsin inhibitors (T1-T4) are similar to that previously found for the chymotrypsin inhibitor, C1. Despite differences in sequence, there is conservation in backbone geometry between the reactive site loops of the two classes of inhibitors. From this, it is clear that the nature of the side chain on the primary binding residue, rather than the backbone fold, is the main determinant of the enzyme specificities of these proteinase inhibitors.
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PMID:Structures of a series of 6-kDa trypsin inhibitors isolated from the stigma of Nicotiana alata. 757 34

A gene encoding a 40.3-kDa serine proteinase inhibitor (PI) precursor is expressed at high levels in the stigma of the ornamental tobacco, Nicotiana alata. The precursor is processed proteolytically in vivo to release five homologous proteinase inhibitors of approximately 6 kDa, as well as two flanking peptides. The five PIs have been purified from stigmas and identified by N-terminal sequencing, electrospray mass spectrometry and inhibition activity against chymotrypsin or trypsin. One of the PIs inhibits chymotrypsin and the other four are most active on trypsin. Cleavage occurs in a linker region (EEKKND) that is repeated six times in the precursor molecule. In the plant, the initial cleavage probably occurs between asparagine and the aspartate residues and ragged ends are formed by subsequent trimming. In vitro, the protease-sensitive linker region is selectively cleaved by the endoproteinases Asp-N, Glu-C and Lys-C to release fully active approximately 6-kDa PIs that are resistant to further proteolytic digestion. The precursor, produced by a recombinant baculovirus, inhibits chymotrypsin more effectively than trypsin. The stoichiometry of 2.6 trypsin molecules/1 precursor molecule indicates that processing is required to activate or expose all of the four trypsin inhibitory sites.
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PMID:Characterization of the protease processing sites in a multidomain proteinase inhibitor precursor from Nicotiana alata. 760 Nov 8

The three-dimensional structure and disulfide connectivities of a 6-kDa protein isolated from the stigma of the ornamental tobacco Nicotiana alata has been determined by 1H NMR spectroscopy combined with simulated annealing calculations. The protein, termed C1, is a chymotrypsin inhibitor and is one of five homologous proteinase inhibitors that are proteolytically cleaved from a 40.3-kDa precursor protein. The other four proteinase inhibitors (T1 to T4) contain reactive sites for trypsin. The three-dimensional structure of C1 is generally well defined and contains a triple stranded beta-sheet as the dominant secondary structural feature. Several turns and a short region of 3(10) helix are also present. The putative chymotrypsin reactive site is present on an exposed loop which is less defined than the rest of the protein. The overall shape of C1 is disc-like and the N and C termini are exposed, supporting the proposal that this protein results from post-translational processing of the 40.3-kDa precursor protein.
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PMID:The three-dimensional solution structure by 1H NMR of a 6-kDa proteinase inhibitor isolated from the stigma of Nicotiana alata. 808 44

A cDNA clone, NA-PI-II, encoding a protein with partial identity to proteinase inhibitor (PI) II of potato and tomato has been isolated from a cDNA library constructed from Nicotiana alata stigma and style mRNA. The cDNA encodes a polypeptide of 397 amino acids with a putative signal peptide of 29 amino acids and six repeated domains, each with a potential reactive site. Domains 1 and 2 have chymotrypsin-specific sites and domains 3, 4, 5, and 6 have sites specific for trypsin. In situ hybridization experiments demonstrated that expression of the gene is restricted to the stigma of both immature and mature pistils. Peptides with inhibitory activity toward chymotrypsin and trypsin have been isolated from stigmas of N. alata. The N-terminal amino acid sequence obtained from this protein preparation corresponds to six regions in the cDNA clone NA-PI-II. The purified PI protein preparation is likely to be composed of a mixture of up to five similar peptides of approximately 6 kD, produced in vivo by proteolytic processing of a 42-kD precursor. The PI may function to protect the reproductive tissue against potential pathogens.
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PMID:Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors. 845 2

NA-proPI is a 40.3-kDa multidomain precursor protein found in the stigma of the ornamental tobacco Nicotiana alata. It is selectively targeted to the vacuole and, as the plant matures, is processed to produce a series of five 6-kDa proteinase inhibitors (one chymotrypsin and four trypsin reactive sites) which are thought to play a vital role in plant protection against insect pests. A putative sixth domain with a chymotrypsin reactive site is likely to be formed by three disulfide bridges linking the N- and C-terminal fragments of NA-proPI. This domain contains two distinct structural elements: a 54-residue sequence with high identity to each of the five repeated PI domains, and a nonrepeated 25-residue sequence at the C-terminus which is proposed to contain a vacuolar targeting peptide. The structure of the putative sixth domain was predicted using a combination of secondary structure prediction and homology modeling based on the known structure of one of the intact domains. A 26-residue peptide corresponding to the nonrepeated C-terminal sequence and encompassing the putative vacuolar targeting sequence was synthesized and its structure determined using 1H NMR spectroscopy and simulated annealing calculations. The peptide was found to adopt an amphipathic helical structure. The calculations based on NOE data suggested that the helix is curved, with a hydrophobic concave face and a hydrophilic convex face. This curvature is consistent with an observed periodicity in backbone NH chemical shifts. The structure of the entire sixth domain was modeled by combining the experimentally determined structure of the putative vacuolar targeting peptide with the homology model of the PI domain. In this model the alpha-helix of the putative targeting peptide protrudes from the otherwise compact PI domain. This observation is consistent with the requirement for targeting sequences to be relatively exposed for recognition by the sorting apparatus. As there is no consensus on the structure of vacuolar targeting sequences, this study provides a valuable insight into their potential mechanism of interaction with the cellular sorting apparatus.
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PMID:Synthesis and structure determination by NMR of a putative vacuolar targeting peptide and model of a proteinase inhibitor from Nicotiana alata. 855 6

Reproductive and storage tissues of many plants produce large amounts of serine proteinase inhibitors (PIs). The ornamental tobacco, Nicotiana alata, produces a series of 6 kDa chymotrypsin and trypsin inhibitors that accumulate to up to 30% of soluble protein in the stigma. These inhibitors are derived by proteolytic processing of two closely related multidomain precursor proteins. Using immunogold electron microscopy, we find that the stigmatic PIs accumulate in both the central vacuole and in the extracellular mucilage. Labelling with antibodies specific for the C-terminal vacuolar targeting peptide (VTS) of each precursor confirms earlier biochemical data showing that the VTS is removed during passage through the secretory pathway. We have isolated and characterised the extracellular population of PIs, which are largely identical to PIs isolated from whole stigmas and are functional inhibitors of serine proteases. Subcellular fractionation of immature stigmas reveals that a sub-population of the PI precursor protein is proteolytically processed within the endoplasmic reticulum. This proteolysis results in the removal of the vacuolar sorting information, causing secretion of this PI population. We propose a novel mechanism whereby a single gene product may be simultaneously trafficked to two separate compartments mediated by proteolysis early in the secretory pathway.
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PMID:Dual location of a family of proteinase inhibitors within the stigmas of Nicotiana alata. 1705 91