Measurement of zinc in hepatocytes by using a fluorescent probe, zinquin: relationship to metallothionein and intracellular zinc.
Zinquin [ethyl (2-methyl-8-p-toluenesulphonamido-6-quinolyloxy)acetate], a new intracellular zinc fluorophore, was used to reveal and to measure Zn in cultured rat hepatocytes before and after metallothionein (MT) induction. Hepatocytes labelled with an intense extranuclear fluorescence. Culture with combinations of Zn, dexamethasone and interleukin-6, increased intracellular MT by 24-fold, Zn 3-fold, and Zinquin fluorescence by approx. 2-fold above control values. Zinquin fluorescence correlated in descending order with the total cellular Zn (r = 0.747), exchangeable Zn (r = 0.735), soluble cytosolic Zn (r = 0.669) and MT (r = 0.666). When Zinquin was incubated with a cytosolic fraction of liver proteins before Sephadex G-75 column chromatography, it fluoresced with free, MT-incorporated and protein-bound Zn. Although only a slight attenuation of fluorescence was seen with high-molecular-mass protein-bound Zn, MT was degraded by 60% in the presence of Zinquin. The undegraded Zn-MT fluoresced at about 20% of the expected intensity. Although Zinquin fluoresces with all cytosolic Zn, caution is required when comparisons are made between samples with different concentrations of MT. This limitation was demonstrated by staining liver slices from adjuvant-treated rats where MT was increased 24-fold, intracellular Zn by 77%, but Zinquin fluorescence by only 19% above controls. Nevertheless, Zinquin should prove to be a useful tool for studying the distribution of Zn in living cells.
Division of Clinical Biochemistry, Institute of Medical and Veterinary Science, Adelaide, Australia.
P
P
Coyle
P D
PD
Zalewski
J C
JC
Philcox
I J
IJ
Forbes
A D
AD
Ward
S F
SF
Lincoln
I
I
Mahadevan
A M
AM
Rofe
D000818:Animals; D002478:Cells, Cultured; D005456:Fluorescent Dyes; D008099:Liver / Q000737:chemistry* / Q000166:cytology; D008297:Male; D008668:Metallothionein / Q000096:biosynthesis*; D015363:Quinolones; D051381:Rats; D014105:Tosyl Compounds; D015032:Zinc / Q000032:analysis*
D005456:Fluorescent Dyes; D015363:Quinolones; D014105:Tosyl Compounds; D008668:Metallothionein; D015032:Zinc; C084731:zinquin