CHARACTERIZATION OF PNIXA, A SERPIN OF XENOPUS-LAEVIS OOCYTES AND EMBRYOS, AND ITS HISTIDINE-RICH, NI(II)-BINDING DOMAIN

A Ni(II)-binding serpin, pNiXa, is abundant in Xenopus oocytes and emb ryos. Kinetic assays show that purified pNiXa strongly inhibits bovine alpha-chymotrypsin (K-i = 3 mM), weakly inhibits porcine elastase (K- i = 0.5 mu M), and does not inhibit bovine trypsin. The reversible, sl ow-binding inhibition of alpha-chymotrypsin by pNiXa is unaffected by Ni(II). Ovochymase in egg exudates is inhibited by pNiXa, but to a lim ited extent, even at high pNiXa concentrations. An octadecapeptide tha t models the His-rich domain (-HRHRHEQQGHHDSAKHGH-) of pNiXa forms six -coordinate, octahedral Ni(II)-complexes when the N-terminus is acetyl ated, and a square-planar Ni(II)-complex when the N-terminus is unbloc ked. Spectroscopy reveals two distinct types of octahedral Ni(II)-coor dination to the N-acetylated octadecapeptide, involving, respectively, 3-4 and 5-6 imidazole nitrogens; the octadecapeptide undergoes partia l, reversible precipitation in pH- and Ni(II)-dependent fashion, sugge sting an insoluble, Ni(II)-coupled (Hx)(n)-dimer. Such (Hx)(n)-peptide interaction is confirmed by an enzyme-linked biotin-avidin assay with N-biotin-KHRHRHE-amide and N-acetylKHRHRHE-resin beads, which become coupled after adding Ni(II) or Zn(II). H2O2 oxidation of 2'-deoxyguano sine to mutagenic 8-hydroxy-2'-deoxyguanosine is enhanced by the octah edral Ni(II)-octadecapeptide complex, although the effect is more inte nse with the square-planar Ni(II)-octadecapeptide complex. Immunoperox idase 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 rea ctions that produce tissue injury and genotoxicity. Although the natur al target proteinases for pNiXa inhibition have not been established, pNiXa may be an important regulator of proteolysis during embryonic de velopment. (C) 1996 Wiley-Liss, Inc.