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The concentrations of metallothionein-I (MT-I) and related immunoreactive products in bile from adult female rats were measured by radioimmunoassay. Concentrations in normal animals were 20-30 ng/ml, but increased to 600 and 75 ng/ml after injection of Cu2+ and Zn2+ respectively (3 mg of metal/kg body wt.). However, only 1-2% of the biliary Cu was bound to MT, and less than 1% of the total liver MT in control or Cu2+-injected rats appeared to be secreted in intact form into bile. Other major immunoreactive components in bile from Cu2+-injected rats included an aggregated form of MT-I and a possible degradation product of the isoprotein.

A sensitive radioimmunoassay for rat liver metallothionein-I has been developed using avid and high-titre antibodies obtained from sheep that were immunized with a conjugate of metallothionein and rabbit immunoglobulin G. The assay was specific for metallothionein-I, and did not depend on the particular metal bound to the protein. There was no significant cross-reaction with rat liver metallothionein-II. The use of the assay to measure metallothionein concentrations in rat plasma and kidneys is described.

The mixed copper- and zinc-binding proteins, with mol.wt. of approx. 12000, induced in rat liver after injection of copper were isolated and characterized as metallothioneins. Three separate forms were obtained, with 7-11% total metal in the protein, equivalent to 2.6-1.6 cysteine residues/metal atom.

The isolation of two forms of hepatic zinc-thioneins after either zinc injection into rats or partial restriction of their food intake is described. The proteins differed slightly in their amino acid composition and electrophoretic mobilities. Increases in liver zinc content after both treatments were synchronous with, and associated almost completely with, increased zinc-binding to these proteins. The time-course for the appearance and disappearance of the zinc proteins is shown. It is suggested that metallothionein is involved in the normal metabolism of zinc, perhaps in some temporary storage or detoxication capacity.

Zn-metallothionein 1 from rabbit liver was investigated by means of Zn K-edge extended X-ray-absorption fine structure (e.x.a.f.s.). Also, the Cu and Zn K-edge e.x.a.f.s. were measured for two samples of mixed Cu Zn-metallothionein 2, with Cu/Zn ratios of 5:2 and 6:3, from pig liver. Detailed simulation of the Cu sites shows a primary co-ordination with three sulphur atoms, presumably from cysteine residues at 0.225 nm +/- 0.001 nm (2.25 +/- 0.01 A). The data for the Zn sites are best reproduced by four Zn-S separations at 0.233 +/- 0.001 nm (2.33 +/- 0.01 A). The Zn K-edge e.x.a.f.s. recorded for rabbit metallothionein 1 at 77 K shows, in addition to the primary co-ordination shell, evidence for two Zn-Zn separations at approx. 0.50 nm (5.0 A). This latter result provides the first information concerning the internal arrangement of zinc atoms in Zn7-metallothionein.

The appearance and excretion of metallothionein-I (MT-I) was studied in rats given a diet containing 1000 mg of Cu/kg for several weeks. No significant increase in MT-I concentrations in liver, plasma or bile was detected in rats with liver copper concentrations less than 600 micrograms of Cu/g fresh wt. Above this concentration, liver MT-I concentrations increased in proportion to the increase in hepatic copper content. Plasma and bile MT-I concentrations were directly related to those in the liver and were about 10 times those in normal rats. Urinary MT-I concentration also increased 10-fold within 1 week. Fractionation of bile and urine on Sephadex G-50 revealed the presence of monomeric MT-I and a range of possible degradation products of the isoprotein.

The degradation of purified 35S-labelled rat liver isometallothioneins (MT) by lysosomal extracts was studied. Zn-MT-I was more readily hydrolysed than Zn-MT-II, but no significant degradation of the Cu-containing metallothioneins could be detected, even after 24 h incubation. The susceptibility of MT to degradation in vitro may be related to the strength of the metal-thiolate bonds. However, the turnover rates of cytosolic MT in vivo, as established by pulse-labelling techniques, are apparently subject to different controls. The half-lives of MT-I and -II in the liver cytosol of Cu2+-injected rats were only 15.4 +/- 1.5 and 18.2 +/- 1.1 h respectively. Approx. 25% of the total liver MT was present in particulate fractions (probably in lysosomes) of the liver and had a half-life of 25.1 +/- 4.1 h.

Large amounts of Cu-metallothionein were obtained by 2-mercaptoethanol and sodium dodecyl sulphate extractions of the particulate fractions of the liver of pigs given high-Cu2+ diets or rats injected with Cu2+. Three isoproteins were purified from pig liver and characterized on the basis of their physicochemical properties, metal content and amino acid composition. No such pool of Cu-metallothionein was present in the liver of Cu2+-loaded sheep or of rats given Cu2+-supplemented diets.

The concentrations of metallothionein-I in the plasma and liver of neonatal rats were measured by radioimmunoassay. Plasma concentrations of the protein in male and female 4-day-old rats were 350 and 740 ng/ml respectively, and declined rapidly to only 3.5 ng/ml at 32 days of age. Concentrations in liver were also high in the newborn rats (200 micrograms/g), and declined from 12 days of age onwards.

Injection of Zn2+-adequate and Zn2+-deficient rats with Cu2+ stimulated the incorporation of l-[35S]cysteine into a low-molecular-weight Cu2+-binding protein in both liver and kidney. No significant incorporation of l-[4,5-3H]leucine into this protein occurred, confirming the previous claim that it was metallothionein and not some other leucine-rich protein. The half-life of the protein was found to be 16.9 +/- 1.0 (S.E.)h in the liver of Zn2+-adequate rats but only 12.3 +/- 0.5h in Zn2+-deficient animals. The degradation rate of the metallothionein was similar to the rate of disappearance of Cu2+ and Zn2+ from the protein, indicating that the release of mental from the protein and its catabolism occurred simultaneously. There was no significant difference in the half-lives of the hepatic or renal copper-thioneins in Zn2+-adequate rats.