Determination of zinc-65, copper-64 and sulphur-35 labelled rat hepatoma tissue culture metallothioneins by high-performance liquid chromatography with on-line radioactivity detection.
Molecular size exclusion (MSE), reversed-phase (RP), and anion-exchange (AE) high-performance liquid chromatography (HPLC) techniques were employed in combination with on-line radioactivity detection, in a study on the kinetic behaviour of 65Zn-, 64Cu- and [35S]cysteine-labelled metallothionein (MT) in rat hepatoma tissue culture (HTC) cells. MSE-HPLC of [35S]cysteine-labelled HTC cell cytosol resulted in co-eluting MT-I and MT-II isoforms (tR 19.80 min; Ve/Vo: 1.85). AE-HPLC of 65Zn-treated HTC cell cytosol yielded separated 65Zn MT-I (tR 11.5 min; I = 64 mM) and 65Zn MT-II (tR 14.5 min; I = 104 mM). RP-HPLC of 64Cu-treated HTC cytosol resulted in separated 64Cu MT-I (tR 26.4 min) and 64Cu MT-II (tR 23.4 min). Determination of the amino acid composition, apparent molecular mass and cysteine content of HTC MT-I and MT-II isoforms showed the characteristics of class I metallothioneins. The rate of dissociation of Zn2+ from Zn-MT could be determined from the losses of 65Zn from MT during a single AE-HPLC run, showing a Zn-MT dissociation half-life of 0.66 h. RP-HPLC showed a delay in incorporation of newly accumulated 64Cu into MT, possibly owing to the appearance of reduced glutathione as an intracellular copper-transfer compound. Application of compartmental analysis in [35S]-cysteine accumulation experiments permitted the determination of the actual rate of MT degradation; when 200 microM of Zn were applied, the MT degradation half-life was 2.0 +/- 0.8 h. These results indicate the potential of combined HPLC techniques and application of radionuclides in studies on the synthesis and degradation of MT and metal-MT complexes.
Steinebach
O M
OM
Department of Radiochemistry, Interfaculty Reactor Institute, Delft University of Technology, Netherlands.
Wolterbeek
H T
HT
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002478:Cells, Cultured; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003301:Copper Radioisotopes / Q000032:analysis; D003600:Cytosol / Q000737:chemistry; D007536:Isomerism; D008114:Liver Neoplasms, Experimental / Q000737:chemistry*; D008668:Metallothionein / Q000032:analysis*; D051381:Rats; D013056:Spectrophotometry, Ultraviolet; D013462:Sulfur Radioisotopes / Q000032:analysis; D015037:Zinc Radioisotopes / Q000032:analysis
D000596:Amino Acids; D003301:Copper Radioisotopes; D013462:Sulfur Radioisotopes; D015037:Zinc Radioisotopes; D008668:Metallothionein