Structural influence of hydrophobic core residues on metal binding and specificity in carbonic anhydrase II.

Aromatic residues in the hydrophobic core of human carbonic anhydrase II (CAII) influence metal ion binding in the active site. Residues F93, F95, and W97 are contained in a beta-strand that also contains two zinc ligands, H94 and H96. The aromatic amino acids contribute to the high zinc affinity and slow zinc dissociation rate constant of CAII [Hunt, J. A., and Fierke, C. A. (1997) J. Biol. Chem. 272, 20364-20372]. Substitution of these aromatic amino acids with smaller side chains enhances Cu(2+) affinity while decreasing Co(2+) and Zn(2+) affinity [Hunt, J. A., Mahiuddin, A., & Fierke, C. A. (1999) Biochemistry 38, 9054-9062]. Here, X-ray crystal structures of zinc-bound F93I/F95M/W97V and F93S/F95L/W97M CAIIs reveal the introduction of new cavities in the hydrophobic core, compensatory movements of surrounding side chains, and the incorporation of buried water molecules; nevertheless, the enzyme maintains tetrahedral zinc coordination geometry. However, a conformational change of direct metal ligand H94 as well as indirect (i.e., "second-shell") ligand Q92 accompanies metal release in both F93I/F95M/W97V and F93S/F95L/W97M CAIIs, thereby eliminating preorientation of the histidine ligands with tetrahedral geometry in the apoenzyme. Only one cobalt-bound variant, F93I/F95M/W97V CAII, maintains tetrahedral metal coordination geometry; F93S/F95L/W97M CAII binds Co(2+) with trigonal bipyramidal coordination geometry due to the addition of azide anion to the metal coordination polyhedron. The copper-bound variants exhibit either square pyramidal or trigonal bipyramidal metal coordination geometry due to the addition of a second solvent molecule to the metal coordination polyhedron. The key finding of this work is that aromatic core residues serve as anchors that help to preorient direct and second-shell ligands to optimize zinc binding geometry and destabilize alternative geometries. These geometrical constraints are likely a main determinant of the enhanced zinc/copper specificity of CAII as compared to small molecule chelators.

Immunohistochemical evidence for IgE involvement in Graves' orbitopathy.

Orbital muscle and adipose tissues from seven Graves' orbitopathy patients were studied with in situ assays for IgE. The cases varied in disease severity and site biopsied. Two monoclonal and one polyclonal anti-IgE reagents produced similar results. Identically prepared monoclonal anti-IgM and anti-IgG antibodies and tissues obtained from five patients treated for unrelated orbital disorders were used as controls. Graves' tissues exhibited extravasated leukocytes and leukocyte-rich vessels. These leukocytes were mostly lymphoid. Some basophils and mast cells were identified and polymorphonuclear cells were abundant within vessels of adipose tissue. IgE-positive material was found in association with the majority of leukocytes and with muscle fibers. Parallel sections reacted with anti-IgM antibody were negative, whereas anti-IgG produced diffuse staining with no specific structures highlighted. Control, non-Graves' tissues had no evidence of immune cell activity and were either negative or displayed reactions with anti-IgE reagents that were in most cases different from those of Graves' tissue. Serum IgE was measured in six of the seven patients and was elevated in the two patients with fulminating disease.

The protective action of zinc against the deleterious effects of cadmium in the regenerating forelimb of the adult newt, Notophthalmus viridescens.

Forelimbs of adult male newts (Notophthalmus viridescens) were amputated and immediately dipped in cadmium nitrate for 2 minutes; in addition, some of the newts were injected with zinc chloride 24 hours prior to, or 24 hours after amputation. Dipping the amputated forelimb of a newt in a solution of 0.4 M cadmium nitrate completely inhibited or retarded regeneration throughout the 65 days of observation. Other effects of cadmium administration included erythema of the limb, an extensive protrusion of the humerus, and in some cases atypical differentiation of regenerates. When zinc chloride was injected (0.04 mg/g of body weight) intraperitoneally into the newt 24 hours prior to limb amputation and cadmium dipping, the deleterious effects of cadmium treatment were prevented and normal regeneration occurred. When zinc chloride was administered 24 hours after amputation and cadmium dipping, it gave no protection against the cadmium. It is suggested that cadmium might inhibit regeneration through the inactivation of zinc metalloenzymes as a result of an exchange of cadmium for zinc. Zinc chloride administered to newts prior to cadmium treatment may prevent the replacement of zinc by cadmium.