Induction of a light-inducible gene in Arthrobacter sp. by exposure of cells to chelating agents and pH 5.

Transcription of a light-inducible gene in the prokaryote Arthrobacter sp. is induced in the dark when cells are incubated with chelating agents or in medium at pH 5. However, repletion of metal ions such as Ca2+, Mn2+ or Zn2+ or an increase in pH is required for accumulation of the gene product, an Mr 21,000 polypeptide. But such changes in condition restore repression of the gene, and the decay in the rate of synthesis of the polypeptide follows the same time-course as when photodynamically induced cells are transferred to the dark. These results are consistent with regulation of expression of this gene at transcriptional and posttranscriptional steps by mechanisms that involve metal-protein complexes.

Effects of divalent metals on the specificity of inhibitors of the cyclic nucleotide phosphodiesterases from bovine heart.

Divalent metals used to support phosphodiesterase (EC 3.1.4.-) activity have been found to influence the substrate and enzyme specificity of many phosphodiesterase inhibitors in studies of the hydrolysis of cyclic AMP and cyclic GMP by the calmodulin-dependent and cyclic AMP-specific phosphodiesterases from bovine heart. Many compounds displayed marked differences in substrate specificity and inhibitory potency in the presence of Mg2+, as compared with Mn2+, when studied with the unactivated form of calmodulin-dependent phosphodiesterase, while few compounds displayed differences in the presence of calmodulin. With a single divalent metal, marked differences in inhibitory potency and substrate specificity were also observed in the absence or presence of calmodulin suggesting that alterations in calmodulin and/or Ca2+ levels may greatly affect the response to phosphodiesterase inhibitors. Divalent metals did not alter the effects of inhibitors on the hydrolysis of cyclic AMP by the cyclic AMP-specific phosphodiesterase, however divalent metals would probably indirectly influence the relative cellular level of cyclic AMP hydrolyzed by this enzyme, and therefore the effects of inhibitors, through metal effects on the calmodulin-dependent phosphodiesterase. No correlation was found between the inhibitory activity of the compounds, many of which were cyclic nucleotide analogs, and their ability to activate cyclic AMP-dependent or cyclic GMP-dependent protein kinases or to affect cyclic AMP-dependent protein kinase activity by displacing bound cyclic AMP.

Effect of calcium on proteolytic activity and conformation of hemorrhagic toxin I from five pace snake (Agkistrodon acutus) venom.

AaHI, a proteolytic hemorrhagic toxin from A. acutus venom, contains one g-atom Zn per mole protein and 2 g-atoms Ca per mole protein. AaHI is activated by calcium and slightly inhibited by zinc. When treated with EDTA, AaHI is completely inactivated. When dialyzed against 1,10-O-phenanthroline, 50-80% of the metal content and activities are lost, while 90% of the hemorrhagic and proteolytic activities are retained when dialyzed against 1,10-O-phenanthroline containing 5mM Ca2+. The results suggest that calcium is essential for the hemorrhagic and proteolytic activities. The circular dichroism spectra show that calcium may play an important role in maintaining a proper structure for AaHI. The function of zinc is not clear.

In vitro leukemic cell differentiation in metal-depleted media.

Lymphocytes of children with T cell and pre-B cell acute lymphocytic leukemia (ALL) were cultured in media with different concentrations of Fe, Cu and Zn to detect a possible effect of these ions on leukemic cells. Thymidine uptake was elevated in non-mitogen stimulated ALL cells and increased after mitogen stimulation, whereas intracellular ferritin, Fe and Cu, elevated before culture, decreased thereafter; pre-B ALL cells, positive only for cytoplasmic mu chains, became positive for surface immunoglobulins and released a detectable amount of them; low T4/T8 ratio in cells from T cell ALL returned to normal values after culture. These findings were present only in media with minimal concentrations of Fe, Cu and Zn and suggest that regulation of intracellular levels of these metals may induce some differentiation of leukemic cells.

The receptor concept: prejudice, prediction, and paradox.

Major progress has been achieved in understanding the chemical nature of receptors for certain hormones and neurotransmitters. Some of these units have been obtained as homogenous proteins; concept and technology are adequate to permit the detailed chemical and structural analysis of these macromolecular units. The critical problem which now arises is to define how a receptor once "occupied" and "activated" serves to initiate action. Elucidation of details of receptor structure, in of itself, will not solve the "coupling problem" in hormone action. New concepts, as well as new technics, will probably be necessary. In this discussion I have raised the possibility that metals coordinated to hormone receptor complexes may be the "trigger" element involved in initiating action, serving to alter the state of functional units in binary fashion. In effect, the metal determines whether the "state" of the system is active or inactive. As stated previously in connection with the action of steroid hormone receptor complexes, the specific suggestions made relating metals to the hormone coulping process have been advanced primarily to illustrate the conceptual gap which exists with respect to "coupling." The present suggestions may prove to be correct or untenable, in whole or in part. If it turns out that metals play a central role in the coupling process of hormone action, perhaps via completely different mechanisms than those suggested here, one of the central ideas of receptor action developed by the pioneers who created the receptor concept will have been resurrected in principle, if not in detail. In science, as in life generally, conceptual progress once achieved sometimes turns out to be the rediscovery of the past.

The role of metal ions in ischemia/reperfusion injury in skin flaps.

The role of hydroxyl radical generation by the metal-catalyzed Haber-Weiss reaction in producing injury to postischemic skin flaps in rats was evaluated. The venous drainage from groin island flaps was occluded for 7 hr and then reperfused. The flaps were infused with either deferoxamine, CaNa2EDTA, histidine, salicylate, or vehicle (saline) at the time of reperfusion. In another experimental group, the role of hydrogen peroxide was evaluated by the infusion of catalase at the time of reperfusion. Treatment with a single dose of deferoxamine (40 mg/kg), CaNa2EDTA (50 mg/kg), or histidine (50 mg/kg), significantly increased the flap survival rate from 24 to 63, 75, and 63%, respectively. A large dose of salicylate (80 mg/kg) improved the flap survival rate (to 63%): a smaller dose (40 mg/kg) offered no improvement. A large dose of catalase ameliorated the survival rate (to 88%). The results suggest that the presence of metal ions is required for the expression of free radical-induced tissue damage. Hydrogen peroxide appears to be essential for the production of this injury.

[Role of zinc and other metals in the biological activity of the serum thymic factor (thymulin)]. / Role du zinc et d'autres métaux dans l'activité biologique du FTS (thymuline).

The serum thymic factor (FTS) used in synthetic or natural form, loses its biological activity after passage on a chelating agent, Chelex 100. Such activity is recovered after addition of zinc and, to a lesser degree, of certain other metals. FTS activation is secondary to zinc binding to the peptide. These results indicate the existence of two FTS forms: the first one, deprived of zinc and biologically inactive, the second one containing zinc and biologically active, for which we purpose to coin the name of thymulin.

Trace elements and rheumatoid arthritis (RA)--pathogenetic and therapeutic aspects.

Rheumatoid arthritis is characterized by increased activity of macrophages which produce toxic forms of oxygen. Such oxygen has been suggested as mediator also of rheumatoid inflammation. Gold accumulates in lysosomes of the macrophages and stabilizes lysosomal and other cell membranes leading to reduced liberation of toxic oxygen. Intracellular production of metallothionein can be induced. Zinc in high doses parenterally can immobilize macrophages and also induce metallothionein-like proteins. Copper and zinc are components of SOD which detoxifies oxygen, and copper-thiolate complexes are reported to be anti-inflammatory. The therapeutic effect of penicillamine and other thiols like aurothiomalate may also be related to an anti-oxidative action. Therapeutic induction of increased intracellular levels of glutathione or administration of selenium in such a form that it incorporates into glutathione-peroxidase and increases the efficacy of the enzyme may lead to accelerated metabolism of toxic oxygen.

Metals and activity of bovine heart cytochrome c oxidase are independent of polypeptide subunits III, VII, a, and b.

Bovine heart cytochrome c oxidase, depleted of polypeptide subunits by alkaline detergent treatment, was characterized with respect to metal content, optical spectral properties, and oxidase activity. Treatment with 1.0% Triton X-100 at pH 9.5 followed by anion-exchange chromatography caused removal of subunit III, subunit VII, and polypeptides a and b. The metal atom stoichiometries of the control and the polypeptide-depleted enzyme were in both cases 2.5Cu/2Fe/1Zn/1Mg with metal-to-protein ratios significantly greater in the latter. The treated enzyme exhibited a red shifted oxidized Soret maximum and bound carbon monoxide upon reduction. Activity was markedly decreased by the treatment but was restored to control levels by incubation with 0.3% Tween 80 at pH 6.0. Therefore, subunit III, subunit VII, polypeptide a, and polypeptide b do not contain Cu, Fe, Zn, or Mg and are not essential for reduction of O2 by ferrocytochrome c.

Insulin-receptor interactions. Possible involvement of metals.

Addition of Zn2+ or Cu2+ ions to plasma membrane preparations or to purified insulin receptors from rat liver resulted in an increase of specific insulin binding; no effect was observed with the addition of Fe3+, Ca2+ or Na+. Dialysis of membrane preparations, or of purified receptors, against chelating agents such as zincon (2-carboxy-2'-hydroxy-5'-sulfoformazyl-benzene) or 1,10-phenantroline resulted in a decrease in specific binding of insulin. With the readdition of Zn2+ or Cu2+ to the medium an increase in specific binding was observed, and values much higher than those of the original preparations were obtained; the addition of Ca2+, Fe3+ or Na+ to dialyzed preparations did not cause any effect on the specific binding. Dialysis of purified receptors against chelating agents resulted in a decrease in the content of Zn2+ and Cu2+. Zincon has been found to be a competitive inhibitor of insulin interfering with the specific binding to the receptor, and noncompetitive with the nonspecific binding. These results suggest the possible involvement of a metal ion present in the receptor in the formation of the insulin-receptor complex.

Growth inhibition and metallothionein induction in cadmium-resistant cells by essential and non-essential metals.

Essential and non-essential metal ions were compared on the basis of their growth-inhibitory potency and their mediation of metallothionein induction in a Chinese hamster ovary cell line resistant to cadmium. Cadmium-resistant cells were found to be 20-fold and 6-fold more resistant than wild-type Chinese hamster ovary cells to the non-essential metals CdCl2 and HgCl2, respectively. In contrast, cadmium-resistant cells showed 2-fold or less resistance to growth inhibition due to the metals with known or possible biological essentiality, ZnCl2, CuSO4, CoCl2, and NiCl2. Resistance to either cadmium or mercury was not due to decreased uptake as measured isotopically or by X-ray fluorescence. At concentrations near the threshold of growth inhibition, CdCl2 and ZnCl2 induced metallothionein 8- to 10-fold above background levels in cadmium-resistant cells within 8-10 hr. A 2- to 3-fold induction of this protein was produced in resistant cells by levels of HgCl2, CuSO4, and CoCl2 near the threshold of growth inhibition whereas NiCl2 produced no measurable elevations of metallothionein at concentrations below, near, and above those that inhibit cell growth. Induction of metallothionein was measured by a modified 203Hg binding assay and by [35S]cysteine incorporation. No measurable induction of metallothionein was evident in wild-type cells with any metal treatment using a reasonable quantity of cells consistent with our assay. These results in cadmium-resistant cells demonstrate selective induction of metallothionein by various metals and suggest that induction of this protein alone is not solely responsible for differences in the growth-inhibitory potential of these elements.