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The first human tumor derived protein with in vivo angiogenic activity to be obtained in pure form has been isolated from serum-free supernatants of an established human adenocarcinoma cell line (HT-29) and named angiogenin. It was purified by cation-exchange and reversed-phase high-performance liquid chromatography; the yield was approximately 0.5 microgram/L of medium. Biological activity of angiogenin was monitored throughout purification by using the chick embryo chorioallantoic membrane assay. Statistical evaluation demonstrates that it displays activity in this system with as little as 35 fmol per egg. Moreover, only 3.5 pmol is required to induce extensive blood vessel growth in the rabbit cornea. The amino acid composition of this basic (isoelectric point greater than 9.5), single-chain protein of molecular weight approximately 14 400 has been determined. The amino terminus is blocked, and the carboxyl-terminal residue is proline.

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tions for the nitration reaction and its quantitation have been established by comparison of spectrophotometric results with those obtained by amino acid analyses.

We postulate a novel and general mechanism in which the redox-active sulfur donor group of cyst(e)ine confers oxidoreductive characteristics on stable zinc sites in proteins. Thus, the present, an earlier, and accompanying manuscripts Metallothionein (MT) is a unique metalloprotein in which cysteine constitutes one-third of its amino acids and histidine and aromatic amino acids all are completely absent. All 20 cysteines bind seven zinc atoms such that each metal atom has a complement of four cysteine ligands. It is important to emphasize that the by now well known multinuclear cluster network, identified by both x-ray diffraction and NMR spectroscopy only less than a decade ago (1,2), shows the exceptional structural arrangement that was hitherto unknown in zinc/sulfur chemistry and has thus far been encountered solely in biological material such as MT. The zinc/cysteinyl interactions in the two clusters are of two different types: they are either bridging or terminal cysteine thiolates. In the ␤-domain cluster, three bridging and six terminal cysteine thiolates provide a coordination environment that is formally identical for each of the three zinc atoms. In the ␣-domain cluster, there are two different zinc sites; two of them have one terminal ligand and three bridging ligands, respectively, while the other two have two terminal and two bridging ligands.We have performed and advocated experiments to relate the structure of MT to its possible function(s) on the basis of the nature of zinc coordination in MT (3-5). We have suggested that the characteristics of the cluster motif might be the key to the mode of cellular zinc distribution (6). MT binds zinc with high thermodynamic stability [K d ϭ 1.4 ϫ 10 Ϫ13 M for human MT at pH 7.0 (7)] while simultaneously providing a mechanism for kinetic lability whereby zinc can be released at rates that are orders of magnitude greater than those observed for zinc metalloenzymes. Zinc, as well as cadmium, is known to undergo rapid inter-and intracluster exchange (8, 9).MT apparently binds zinc with higher affinity than do many other proteins. For MT to serve as a source for the distribution of zinc, mechanisms would be required that could regulate the binding and release of the metal. It has been shown that an interaction of MT with glutathione disulfide (GSSG) or other biological disulfides releases zinc (10, 11) and that the combination of reduced glutathione (GSH) and GSSG enhances transfer of zinc from MT to an apoenzyme (12). This has led us to infer that the reactivity and redox behavior of the sulfur ligands in the MT clusters are crucial for the dynamic state of zinc. We proposed that the zinc-sulfur cluster chemistry might be sensitive to changes of the cellular redox state and that oxidizing conditions induce the transfer of zinc from its binding sites in MT to those of lower affinity in other proteins.We here provide additional support for this concept and show that a number of compounds, including some of potential biological importance, can oxidize the ...

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