A study of copper treatment and tissue copper levels in the murine congenital copper deficiency, mottled.

The injection of copper chloride overcomes the lethality and pigment deficiency in the brindled ($\text{Mo}$^br) mouse mutant but copper levels remain depressed in the liver and brain, and a further accumulation occurs in the kidney. The copper-dependent synthesis of brain noradrenaline returns to normal but the activity of brain cytochrome c oxidase, although increased, remains depressed. Significant changes in tissue copper content of female brindled heterozygotes are reported and in each case, the changes exceed those expected on the basis of X-inactivation. The significance of these results to the development of a satisfactory treatment regime for this disease is discussed.     

The enzyme-bound copper of dopamine beta-monooxygenase. Reaction with copper chelators, preparation of the apoprotein, and kinetics of the reconstitution by added copper.

The enzyme-bound copper of dopamine beta-monooxygenase reacted rapidly with the chelator bathocuproine disulfonate; the reaction in the presence of ascorbate was completed in 2 min at 25 degrees C with 1mM chelator. This reaction and also the reaction with EDTA could be used to prepare the apoenzyme, which in both cases was completely reactivated in less than 10 s. The reactivation data gave apparent Michaelis constants for copper 0.03 -- 0.2 micron. Trace amounts of copper in buffers and assay mixtures gave significant reactivation without added copper, unless they had been treated with a chelating resin. Titrations using the different chelation rates of free and enzyme-bound copper indicated that four copper atoms are bound per enzyme molecule of four subunits. The native enzyme was more stable against thermal inactivation than the apoenzyme, but this stability was only partially restored by addition of copper to the apoenzyme.     

The effect of copper supplementation on the concentration of copper in the brain of the brindled mouse.

The brindled mutant mouse is a useful model to study Menkes kinky-hair syndrome. The metabolic dysfunctions in both human and rodent are related to insufficient levels of bioavailable copper. Recently, copper supplementation therapy has been able both to prevent the appearance of various neuropathological changes and to prolong the life of these mutant mice. The optimum conditions for supplementation have been shown to be two intraperitoneal injections on postnatal days 7 and 10. The present study reports on the brain copper concentrations before, during and after the intraperitoneal copper therapy. The results demonstrate that postnatal days 7 and 10 correspond to two important epochs in copper homoeostasis. The supplementation therapy seems to provide sufficient bioavailable copper to respond to the needs of the animal at these crucial time points. The results are discussed in terms of their importance to the human copper disorder.     

Studies upon the copper fungicides: VIII. The penetration of copper into germinating peas

Three varieties of pea seed showed less damage when treated with solutions of simple cupric salts than with solutions of equivalent strength in which the copper could occur in complex form. On adding supplementary pea exudate to copper sulphate solution, thereby converting some cupric to complex copper, greater damage was caused. In spite of the greater phytotoxicity of complex forms of copper, the copper intake by the seeds was less. Sealing of the micropyle had little effect on the water and copper intake of peas.
Colorimetric tissue tests on treated peas indicated a high concentration of copper in the testa and the embryonic radical with only small amounts in the cotyledonary tissues. These observations were largely confirmed by chemical analyses-on the test as, cotyledons and embryos of copper treated peas.
The results suggest that when copper dissolves from dressings on pea seeds much becomes fixed on the testa, so providing fungicidal protection. The soluble complex forms of copper produced by pea exudate are markedly phytotoxic and injury to the embryonic radical is regarded as the main cause of damage to peas.     

Purification of low molecular weight copper proteins from copper loaded liver.

Purification of low molecular weight copper binding proteins from the livers of copper loaded male rats was achieved by sequential ultracentrifugation (186,000g, 2h), ultrafiltration (Amicon PM 30), gel filtration (Sephadex G-75) and anion exchange chromatography (DEAE - Biogel A) of soluble tissue extracts. The three major copper-associated polypeptides obtained which had molecular weights of about 7000, 9,000, and 12,000 daltons contained approximately 2.5g atoms of copper per mole. Amino acid analyses indicated a similarity between these proteins and the copper protein ‘L-6D’ isolated earlier from livers of Wilson's disease patients and distinguished them from metallothioneins which have been isolated from animals administered other trace metal ions.     

A conditional mutation affecting localization of the Menkes disease copper ATPase. Suppression by copper supplementation

Copper is an essential co-factor for several key metabolic processes. This requirement in humans is underscored by Menkes disease, an X-linked copper deficiency disorder caused by mutations in the copper transporting P-type ATPase, MNK. MNK is located in the trans-Golgi network where it transports copper to secreted cuproenzymes. Increases in copper concentration stimulate the trafficking of MNK to the plasma membrane where it effluxes copper. In this study, a Menkes disease mutation, G1019D, located in the large cytoplasmic loop of MNK, was characterized in transfected cultured cells. In copper-limiting conditions the G1019D mutant protein was retained in the endoplasmic reticulum. However, this mislocalization was corrected by the addition of copper to cells via a process that was dependent upon the copper binding sites at the N-terminal region of MNK. Reduced growth temperature and the chemical chaperone, glycerol, were found to correct the mislocalization of the G1019D mutant, suggesting this mutation interferes with protein folding in the secretory pathway. These findings identify G1019D as the first conditional mutation associated with Menkes disease and demonstrate correction of the mislocalized protein by copper supplementation. Our findings provide a molecular framework for understanding how mutations that affect the proper folding of the MNK transporter in Menkes patients may be responsive to parenteral copper therapy.     

Molecular mechanisms of copper homeostasis.

Copper is an essential trace element which plays a pivotal role in cell physiology as it constitutes a core part of important cuproenzymes. Novel components of copper homeostasis in humans have been identified recently which have been characterised at the molecular level. These include copper-transporting P-type ATPases, Menkes and Wilson proteins, and copper chaperones. These findings have paved the way towards better understanding of the role of copper deficiency or copper toxicity in physiological and pathological conditions.     

Studies upon the copper fungicides: VII. The solution of copper from dressings on the pea seed

During the process of swelling pea seeds yielded substances to water which were capable of dissolving the insoluble copper dressings normally applied to the seed to prevent 'pre-emergence damping off'. This occurred under conditions in which soil factors were eliminated and irrespective of whether the dressing was on the surface of the seed or treated separately. Copper was readily taken into solution from dried 4:4:50 Bordeaux Mixture by water in which peas had been soaked, a circumstance which proved that the formation of soluble complex forms of copper was involved.
A chemical investigation on the solution obtained by soaking peas in water showed that volatile acids or their salts were present together with a protein-like material in colloidal solution. The occurrence of amino derivatives and other nitrogenous materials as well as carbohydrates was indicated and evidence of the presence of asparagine and citrate was also obtained.
The suggestion is made that both the fungicidal and phytocidal actions of insoluble copper dressings on pea seed depend largely on the effect of the pea seed exudates, notably colloidal protein-like material and certain amino derivatives, in bringing copper into solution.     

Production of copper coproporphyrin III by Bacillus cereus. I. Purification and identification of copper coproporphyrin III.

Bacillus cereus strains 2 and T did not form spores and accumulated a large amount of purple pigment inside the cells, when cultured in a yeast extract-ammonium salt medium with excess glucose. The pigment was extracted and crystallized as the ethyl ester. It was identified as copper coproporphyrin III.