Evidence for Cu(I) Clusters and Zn(II) Clusters in Neuronal Growth‐Inhibitory Factor Isolated from Bovine Brain

Abstract

Neuronal growth-inhibitory factor (GIF), a central-nervous-system-specific metallothionein-like protein, has been isolated by means of an improved isolation procedure from bovine brain. The native protein contains 4-5 Cu+ and 2-2.5 Zn2+, which results in an overall stoichiometry of 6-7 mol metal ions/mol protein. Native Cu, ZN-GIF and the Zn2+ -substituted and Cd2+-substituted metalloforms have been characterized by means of electronic-absorption, CD, magnetic-circular-dichroism (MCD) and low-temperature (77 K) Cu(I)-luminescence spectroscopy. Analysis of the metal-induced-charge-transfer transitions below 300 nm in the electronic-absorption and CD spectra of Cu, ZN-GIF revealed spectral features characteristic of metal-thiolate coordination. The presence of formally spin-forbidden 3d --> 4s Cu(I)-cluster-centered transitions, above 300 nm in the corresponding CD and MCD spectra indicate the existence of a Cu(I) cluster. The 77-K luminescence spectrum of Cu, ZN-GIF revealed two emissive bands at approximately 420 nm and 570 nm, which were reported also for CU4 clusters in mammalian Cu8-metallothionein. By analogy with Cu8-metallothionein, we propose the presence of a Cu4 cluster with similar electronic structure in native GIF. However, the determined Cys/Cu+ ratio of approximately 2:1 in Cu, Zn-GIF is higher than the ratio found in mammalian Cu(I)-metallothionein forms (approximately 1.6:1 ), which implies that the coordination geometry of CU+-binding sites is different in the CU4 Cluster. The spectroscopic characterization of Zn2+-substituted and Cd2+-substituted GIF (6-7 metal ions/protein) showed CD and MCD features at positions identical to those reported for the well-characterized mammalian Zn7-metallothionein and Cd7-metallothionein. Therefore, it is inferred that the cluster organization in GIF with divalent metal ions is comparable to that found in mammalian metallothioneins. The effect of metal ions on the protein structure with regard to the biological function of GIF is discussed.