Zinc binding ligands and cellular zinc trafficking: Apo-metallothionein, glutathione, TPEN, proteomic zinc, and Zn-Sp1

Many cell types contain metal-ion unsaturated metallothionein (MT). Considering the Zn2+ binding affinity of metallothionein, the existence of this species in the intracellular environment constitutes a substantial “thermodynamic sink”. Indeed, the mM concentration of glutathione may be thought of in the same way. In order to understand how apo-MT and the rest of the Zn-proteome manage to co-exist, experiments examined the in vitro reactivity of Zn-proteome with apo-MT, glutathione (GSH), and a series of common Zn2+ chelating agents including N,N,N′,N′-(2-pyridylethyl)ethylenediammine (TPEN), EDTA, and [(2,2′-oxyproplylene-dinitrilo]tetraacetic acid (EGTA). Less than 10% of Zn-proteome from U87 mg cells reacted with apo-MT or GSH. In contrast, each of the synthetic chelators was 2–3 times more reactive. TPEN, a cell permeant reagent, also reacted rapidly with both Zn-proteome and Zn-MT in LLC-PK1 cells. Taking a specific zinc finger protein for further study, apo-MT, GSH, and TPEN inhibited the binding of Zn3-Sp1 with its cognate DNA site (GC-1) in the sodium-glucose co-transporter promoter of mouse kidney. In contrast, preformation of Zn3-Sp1-(GC-1) prevented reaction with apo-MT and GSH; TPEN remained active but at a higher concentration. Whereas, Zn3-Sp1 is active in cells containing apo-MT and GSH, exposure of LLC-PK1 cells to TPEN for 24 h largely inactivated its DNA binding activity. The results help to rationalize the steady state presence of cellular apo-MT in the midst of the many, diverse members of the Zn-proteome. They also show that TPEN is a robust intracellular chelator of proteomic Zn2+.