Characterization of pNiXa, a serpin of Xenopus laevis oocytes and embryos, and its histidine-rich, Ni(II)-binding domain
| Autor: | F W, Sunderman, A H, Varghese, O S, Kroftova, S, Grbac-Ivankovic, J, Kotyza, A K, Datta, M, Davis, W, Bal, K S, Kasprzak |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
Embryo
Nonmammalian Pancreatic Elastase Sequence Homology Amino Acid Circular Dichroism Molecular Sequence Data Deoxyguanosine Xenopus Proteins Immunoenzyme Techniques Molecular Weight Xenopus laevis 8-Hydroxy-2'-Deoxyguanosine Nickel Endopeptidases Oocytes Animals Chymotrypsin Cattle Histidine Spectrophotometry Ultraviolet Amino Acid Sequence Carrier Proteins Trypsin Inhibitors Oxidation-Reduction Serpins Protein Binding |
| Zdroj: | Molecular reproduction and development. 44(4) |
| ISSN: | 1040-452X |
| Popis: | A Ni(II)-binding serpin, pNiXa, is abundant in Xenopus oocytes and embryos. Kinetic assays show that purified pNiXa strongly inhibits bovine alpha-chymotrypsin (Ki = 3 mM), weakly inhibits porcine elastase (K1 = 0.5 microM), and does not inhibit bovine trypsin. The reversible, slow-binding inhibition of alpha-chymotrypsin by pNiXa is unaffected by Ni(II). Ovochymase in egg exudates is inhibited by pNiXa, but to a limited extent, even at high pNiXa concentrations. An octadecapeptide that models the His-rich domain (-HRHRHEQQGHHDSAKHGH-) of pNiXa forms six-coordinate, octahedral Ni(II)-complexes when the N-terminus is acetylated, and a square-planar Ni(II)-complex when the N-terminus is unblocked. Spectroscopy reveals two distinct types of octahedral Ni(II)-coordination to the N-acetylated octadecapeptide, involving, respectively, 3-4 and 5-6 imidazole nitrogens; the octadecapeptide undergoes partial, reversible precipitation in pH- and Ni(II)-dependent fashion, suggesting an insoluble, Ni(II)-coupled (Hx)n-dimer. Such (Hx)n-peptide interaction is confirmed by an enzyme-linked biotinavidin assay with N-biotin-KHRHRHE-amide and N-acetyl-KHRHRHE-resin beads, which become coupled after adding Ni(II) or Zn(II). H2O2 oxidation of 2'-deoxyguanosine to mutagenic 8-hydroxy-2'-deoxyguanosine is enhanced by the octahedral Ni(II)-octadecapeptide complex, although the effect is more intense with the square-planar Ni(II)-octadecapeptide complex. Immunoperoxidase staining of whole mounts with pNiXa antibody shows that pNiXa is distributed throughout gastrula-stage embryos and is localized during organogenesis in the brain, eye, spinal cord, myotomes, craniofacial tissues, and other sites of Ni(II)-induced anomalies. Patterns of pNiXa staining are similar in controls and Ni(II)-exposed embryos. Binding of Ni(II) to pNiXa may cause embryotoxicity by enhancing oxidative reactions that produce tissue injury and genotoxicity. Although the natural target proteinases for pNiXa inhibition have not been established, pNiXa may be an important regulator of proteolysis during embryonic development. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Plný text