Comparison of the catalytic oxidation of cysteine and o-dianisidine by cupric ion and ceruloplasmin.

Several features of the catalytic oxidation of cysteine by ceruloplasmin and nonenzymic Cu(II) at pH 7 have been compared. The oxidation of cysteine by ceruloplasmin has several properties in common with the Cu(II) catalyzed oxidation of cysteine: pH maxima, thiol specificity, lack of inhibition by anions, and high sensitivity to inhibition by copper complexing reagents. These two catalysts differed in their molecular activity, in their ability to oxidize penicillamine and thioglycolate, and in that H2O2 was produced as a primary product only during Cu(II) oxidation. The oxidation of cysteine by ceruloplasmin was compared also with the ceruloplasmin catalyzed oxidation of o-dianisidine, a classical pH 5.5 substrate. The mechanism of the oxidation of cysteine by ceruloplasmin at pH 7 differed from that of o-dianisidine oxidation because the latter substrate was inhibited by anions but not by copper complexing agents. Spectral and other data suggest that during the ceruloplasmin reaction with cysteine there is a one electron transfer from cysteine to ceruloplasmin resulting in the specific reduction of type 1b Cu(II).

Degradation of rat liver metallothioneins in vitro.

The degradation of zinc and cadmium-induced hepatic metallothionineins was investigated in vitro. Both zinc-thionein and cadmium-thionein were labeled in vivo with [35S]cystine. The labeled proteins were isolated and purified by gel filtration and DEAE-ion exchange chromatography. Purified zinc[35S]thionein and cadmium-[35S]thionein were incubated with trypsin, chymotrypsin and pronase for varying times up to 24 h. The rate of degradation of zinc-thionein was twice that of cadmium-thionein when the proteins were incubated with trypsin. Virtually no digestion occurred when the proteins were incubated with chymotrypsin, whereas the rates of degradation were about equal when they were incubated with pronase. In contrast, degradation of zinc-thionein was twice that observed with cadmium-thionein when the proteins were incubated at pH 5.0 with a purified lysosomal extract. Degradation of these proteins by the lysosomal proteases was 77 and 46% within 3 h for zinc-thionein and cadmium-thionein, respectively. Thionein, the metal-free form of metallothionein, was degraded extremely rapidly by both neutral and lysosomal proteases. Chromatography of the digestion products on Sephadex G-25 demonstrated that all three forms of metallothionein were degraded to species of approximately 100-300 daltons. These data indicate that metals stabilize thionein polypeptides and suggest that the degradation of metallothionein in vivo is regulated in part by the species of metal bound.

Degradation of cadmium-thionein in rat liver and kidney.

3H-cystine and 115mCd were incorporated into hepatic and renal cadmium-thionein in response to a subcutaneous administration of 4.4 micronmol of Cd2+ containing 115mCd. Cadmium-thionein bound 115mCd reached a plateau by 24 hrs. and 72 hrs. after the Cd2+ injection in liver and kidney, respectively. The half-life (t 1/2) of 3-H-labeled hepatic cadmium-thionein was 3.5 days, whereas the average t 1/2 of the soluble proteins was 3.7 days. The t 1/2 of the soluble renal proteins was 3.8 days. In marked contrast, the 115mCd content of both hepatic and renal cadmium-thionein was virtually unchanged even 9 days after administration of this radionuclide. These data indicate that the protein moiety of metallothionein is degraded, although there appears to be a concomitant rebinding of Cd2+ to nascent thionein polypeptide chains. Thus the lack of metallothionein degradation, per se, does not account for the long-term retention of Cd2+ in liver and kidney during chronic exposure.

Degradation of cadmium-thionein in rat liver and kidney.

[3 H] Cystine and 115mCd were incorporated into hepatic and renal Cd-thionein in response to sc administration of 4.4 mumol of Cd2+ containing 115mCd. Cd-thionein-bound 115mCd reached a plateau by 24 and 72 h after the Cd2+ injection in liver and kidney, respectively. The half-life (t1/2) of 3 H-labeled hepatic Cd-thionein was 3.5 d, whereas the average t1/2 of the soluble proteins was 3.7 d. The t1/2 of 3 H-labeled renal Cd-thionein was 3.7 d, whereas the average t1/2 of the soluble renal proteins was 3.8 d. In marked contrast, the 115mCd content of both hepatic and renal Cd-thionein was virtually unchanged, even 9 d after administration of this radionuclide. These data indicate that the protein moiety of metallothionein is degraded, although there appears to be a concomitant rebinding of Cd2+ to nascent thionein polypeptide chains. Thus the lack of metallothionein degradation per se does not account for the long-term retention of Cd2+ in liver and kidney during chronic exposure.

Control of zinc-thionein synthesis in rat liver.

The rate of [35S]cystine incorporation into hepatic zinc-thionein (a metallothionein) was stimulated, with a maximum of 5-6h, after parenteral administration of 2mg of Zn2+ containing 65Zn. The binding of 65Zn to zinc-thionein was measurable by 2-1/2h and reached a plateau by 18h after the injection. A net increase in the hepatic 65Zn content was observed subsequent to the decrease in the rate of zinc-thionein synthesis. The incorporation of both 65Zn and [35S]cystine into zinc-thionein was inhibited by prior administration of either actinomycin D or cordycepin. A second injection of Zn2+, 20h after the initial injection, yielded a 4.9-fold greater increase in zinc-thionein synthesis compared with that after only one injection; however, this synthesis was also inhibitable by actinomycin D. These data support the concept that hepatic zinc-thionein synthesis responds quickly to changes in Zn2+ status and that Zn2+ is bound subsequent to synthesis of nascent thionein chains. The mechanism of control of zinc-thionein synthesis by Zn2+ appears to involve changes in the amounts of a short-lived, poly(A)-containing RNA whose translation can be derepressed by additional exposure to Zn2+.

Biomedical responses of rats to chronic exposure to dietary cadmium fed in ad libitum and equalized regimes.

Forty 100 g male rats were fed, in groups of eight, either 0, 5, or 25 ppm cadmium in a purified diet for 14 wk. Three groups were fed each of the levels of cadmium on an ad libitum basis. Two other groups were fed either 0 or 5 ppm cadmium in amounts that were equalized to that consumed by the 25 ppm group fed ad libitum. Cadmium ingestion decreased daily diet consumption, weight gain, and terminal body weight. These parameters were not significantly different in rats whose diet consumption was equalized. Packed cell volume and serum iron as well as serum zinc were decreased in the rats fed 25 ppm cadmium. These effects were not related to diet intake. No major differences were observed in serum ceruloplasmin, glucose, protein, leucine aminopeptidase activity, or copper in any of the groups. Blood urea nitrogen and renal leucine aminopeptidase activity were decreased by cadmium ingestion in the rats fed ad libitum only. In contrast, serum alkaline phosphatase activity was elevated by cadmium in the equalized-intake groups only. Cadmium and zinc concentrations were elevated and the iron concentration was decreased in the kidney, liver, and intestinal mucosa of the cadmium-fed rats irrespective of level of diet consumption. The increased uptake of cadmium in these tissues was coincident with the increased content of the cadmium-binding protein, metallothionein, in the cytosol fraction. The results indicate that some parameters of chronic cadmium toxicity are associated with diet consumption whereas others are not.

Degradation of hepatic zinc-thionein after parenteral zinc administration.

A low-molecular-weight protein, zinc-thionein, a metallothionein, was implicated as having a regulatory function in zinc metabolism. The half-life (t 1/2) of hepatic zinc-thionein was determined by pulse-labelling with either L-[35S] cystine and/or 65Zn. In two experiments with L-[35S]cystine, the t 1/2 of zinc-thionein was 18h and 19h. Most of the soluble 35S-labelled hepatic proteins had a t 1/2 of 4 days. The t 1/2 of zinc-thionein calculated by using 65Zn was 20h. The close similarity between the calculated and measured t 1/2 values for zinc-thionein suggests that release of Zn2+ from zinc-thionein probably occurs simultaneously with degradation of the protein moiety.