Nuclear magnetic relaxation of 19F as a novel assay method of superoxide dismutase.

The 19F longitudinal nuclear magnetic relaxation rate (T-1(1p)) of F- solutions is greatly enhanced by copper, zinc or manganese superoxide dismutase. The measured T-1(1p) values are at least an order of magnitude, and in most cases 2 orders of magnitude, higher than those of other metal-containing proteins and low molecular weight complexes. This property is suitable for a direct, specific, and sensitive assay of superoxide dismutase, free of interference by other molecules. For mixtures of the copper, zinc and manganese enzymes, addition of CN- permits quantification of the relative amounts of each enzyme. In the case of copper, zinc enzyme, the method proved exquisitely sensitive to the native state of the active site. Results are reported to show that the 19F relaxation method can be used to assay biological fluids and crude homogenates for copper, zinc and manganese superoxide dismutases, under conditions that offer considerable advantages in comparison to other assay methods.     

Copper Transport in Mammalian Cells: Special Care for a Metal with Special Needs

Copper plays an essential role in human physiology. It is required for respiration, radical defense, neuronal myelination, angiogenesis, and many other processes. Copper has distinct physicochemical properties that pose uncommon challenges for its transport across biological membranes. Only small amounts of copper are present in biological fluids, and essentially none of it exists in a free ion form. These properties and the low redox potential of copper dictate special structural and mechanistic features in copper transporters. This minireview discusses molecular mechanisms through which copper enters and exits human cells.     

Copper-histidine ternary complex equilibria with glutamine,asparagine and serine. The implications for computer-simulated distributions of copper(II) in blood plasma

The use of computer simulation techniques for assessing the percentage distribution of metal ions in biological fluids has recently been questioned on the grounds of observed discrepancies between such simulations and the results obtained experimentally by Neumann and Sass-Kortsak in their pioneering work on low-molecular-weight copper in reconstituted human serum. Recent work by our group has resolved the most serious of these discrepancies, but some worrying uncertainties remain regarding the ternary equilibria of copper(II) and histidine with glutamine, serine and asparagine.This paper describes an experimental investigation of these equilibria and the effect that the newly-determined data have on the computer-simulated distribution of copper(II) in blood plasma. Simulations have also been performed using the actual low-molecular-weight compositions of the biofluids studied by Neumann and Sass-Kortsak. The results show good agreement between calculated and experimentally observed distributions and confirm the usefulness of the simulation techniques.     

Chemical reactivity of metallic copper in a model system containing biological metabolites

Chemical reactivity of metallic copper in a model system containing biological metabolites is described. Methionine, methional, and propanal produced ethylene when exposed to metallic copper in the presence of oxygen. It may be that metallic copper in this system serves as the '1 electron reducing agent' in the proposed chemical model system (Kumamoto et al). The requirement for oxygen was verified by removing this electron acceptor and observing the reduced ethylene production. Preliminary studies have shown that other reaction products of the reaction of copper metal with methionine include dimethyl sulfide and dimethyl disulfide or methyl mercaptan or both. These data further suggest that these chemicals are liberated from methionine when copper comes in contact with methionine-containing biological fluids.     

Candida albicans dimorphism and virulence: Role of copper

Previously reported observations that Candida albicans grows in the yeast phase at 30° C and the mycelial phase at 37° C and that the former phase is more virulent than the latter were confirmed. A novel factor, copper, was discovered to suppress filamentation. Injection of copper into mice permitted the filamentous phase to be as virulent as the yeast phase. In subsequent studies on candidosis, copper assays should be performed on relevant body fluids to determine if there might be a correlation between elevated copper and heightened susceptibility to the fungus.     

An inhibitor of opiate receptor binding from human erythrocytes identified as a glutathione-copper complex.

A “peptide-like” inhibitor of opiate receptor binding and of N-methyltransferase previously purified by us from rabbit brain was also found in human red blood cells. Boiled extracts of erythrocytes were fractionated on Sephadex followed by chromatography of the active fractions on silica gel layers. Both, a migrating ninhydrin-positive spot and a naturally blue substance which did not migrate from the origin coincided with the active fractions. The blue substance was identified as copper and the ninhydrin-positive material was identified as oxidized glutathione. While glutathione $\text{per se}$ has no effect, copper and other transition metals are potent inhibitors of opiate receptor binding. A mixture of glutathione and copper plus serum albumin in proportions simulating erythrocyte extracts gave results identical to the latter. Several other laboratories have extracted, from various tissues and body fluids, “opiate-like peptides” which are distinct from the β-LPH derived endorphins. In view of our findings it is possible that metal bound to glutathione or to other peptide ligand may be a complicating factor in some of these studies.     

Zinc, copper, and iron metabolism during porcine fetal development

Zinc, copper, and iron levels in maternal and fetal pig tissues and fluids were measured starting on d 30 of gestation and continuing to term (d 114) at 10-d intervals. Fetal hematocrit increased from a low of 19% on d 30 to 32% by d 50, after which it remained above 30% to term. Amniotic fluid zinc, copper, and iron all reached maximal levels by d 60 of gestation. Maternal serum zinc levels fluctuated little during gestation, but fetal serum zinc concentration was significantly elevated above maternal levels during the second trimester. Fetal serum copper levels were significantly lower than maternal values throughout gestation and this was also the case for ceruloplasmin oxidase activity. Maternal serum iron reached its lowest level by d 80 of gestation when rate of transfer of iron to the developing fetuses was high. Fetal serum iron declined throughout gestation, reaching its lowest level on d 100. In general, fetal liver concentrations of zinc, copper, and iron were higher than the corresponding maternal values throughout gestation. Distinct increases were noted for fetal hepatic zinc and copper concentrations during the second trimester of pregnancy and these were accompanied by increases in cytosolic and metallothionein-bound zinc and copper levels. Maternal hepatic iron declined during the second trimester, reaching its lowest point on d 80, indicative of the shunting of maternal iron reserves to fetal tissues. Fetal kidney metal levels did not demonstrate any distinctive developmental patterns with respect to zinc, copper, or iron concentrations, but a general accumulation of each metal was observed as gestation progressed. The results of this study highlight some of the distinct changes occurring in the metabolism of zinc, copper, and iron in both maternal and fetal tissues and fluids during gestation in the pig. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other suitable products.     

The behaviour of caeruloplasmin in stored human extracellular fluids in relation to ferroxidase II activity, lipid peroxidation and phenanthroline-detectable copper.

No Cu(II) ion is measurable in human serum or synovial fluid by the phenanthroline assay. On storage of human serum or synovial fluid at 4 degrees C, phenanthroline-detectable copper appears, lipid peroxidation occurs, ferroxidase I activity declines and ferroxidase II activity rises, yet there is no fall in immunologically detectable caeruloplasmin. Storage of body fluids at -20 degrees C or -70 degrees C slows, but does not prevent, these deteriorative changes. It is suggested that the presence of low-molecular-mass Cu(II) ion complexes, ferroxidase II activity, "cytotoxic factors' and "immunosuppressive factors' in body fluids may be, in part or in whole, an artifact of the storage and handling of the fluids. A report [Blake, Blann, Bacon, Farr, Gutteridge & Halliwell (1983) Clin. Sci. 64, 551-553] that the caeruloplasmin present in rheumatoid synovial fluid is deficient in ferroxidase activity is shown to be such an artifact. It is strongly recommended that all such experiments be performed upon freshly taken fluid samples.     

Is the Western diet adequate in copper?

Copper has been known to be essential for health for more than three quarters of a century. Myriad experiments with animals reveal that the cardiovascular, musculoskeletal and nervous systems are most sensitive to deficiency. Copper in the Western diet has been decreasing at least since the 1930s; half of the adult population consumes less than the amount recommended in the European Communities and the United Kingdom. At least one fourth of adults consume less than the estimated average requirement published for the United States and Canada. Hundreds of people have been reported in journals about medicine and neurology rather than nutrition to have impaired copper nutriture based on the criteria of low copper concentrations and low activities of enzymes dependent on copper in various fluids and tissues. In contrast, only 46 people have participated in depletion/repletion experiments needed to define requirements. Almost 1000 people have benefited from supplements containing copper in controlled trials. People deficient in copper are being identified increasingly; it is unknown if unusually high requirements or unusually low diets are causal. Alzheimer's disease, ischemic heart disease and osteoporosis are the most likely human illnesses from low copper intakes.     

Antioxidant properties of the proteins caeruloplasmin, albumin and transferrin. A study of their activity in serum and synovial fluid from patients with rheumatoid arthritis

Patients with rheumatoid arthritis have altered protein patterns in their serum and synovial fluid which influences the antioxidant activity of these fluids. Rheumatoid serum has a higher antioxidant activity than control serum when ferrous and ferric ions stimulate membrane damage. The raised levels of caeruloplasmin and the lower iron saturation of transferrin contribute to these differences. When membrane damage is stimulated by a copper salt, rheumatoid serum does not show an increased antioxidant protection and has probably a lower protective activity than control serum. Attempts to damage caeruloplasmin and transferrin with oxygen radicals were unsuccessful. However, prolonged incubations with trypsin reduced the iron-binding capacity of transferrin and decreased the ferroxidase and antioxidant properties of caeruloplasmin. Copper was released from caeruloplasmin under these conditions.     

Hyaluronidase activity of human endometrial tissues & uterine fluids.

Hyaluronidase activity of human endometrial tissues and uterine fluids was investigated. Endometrial tissue and uterine fluid specimens were obtained from normal human subjects, and different cases of uterine dysfunction induced by steroidal contraceptives, copper IUD, lactational amenorrhea, and in early pregnancy. Hyaluronidase activity was found to increase from Cycle Days 8 to 10 and reach the maximum value during the secretory phase. Hyaluronidase activity was reduced in both endometrial tissue and uterine fluid during lactational amenorrhea and early pregnancy, and was drastically reduced in copper-IUD and steroidal contraceptive users. The low hyaluronidase activity in the early phase of the cycle may be due to rapid growth of endometrial tissue. In the secretory phase, the corresponding activities were found to increase because of high secretory activity and enhanced catabolic processes. In early pregnancy, the low lysosomal enzyme activity may also be explained on the basis of increased endometrium tissue growth. Low hyaluronidase activity of amenorrhic subjects may be due to the absence of ovarian steroids.     

Optimization of a culture medium for ligninolytic enzyme production and synthetic dye decolorization using response surface methodology

A Box-Wilson central composite design was applied to optimize copper, veratryl alcohol and l-asparagine concentrations for Trametes trogii (BAFC 212) ligninolytic enzyme production in submerged fermentation. Decolorization of different dyes (xylidine, malachite green, and anthraquinone blue) by the ligninolytic fluids from the cultures was compared. The addition of copper stimulated laccase and glyoxal oxidase production, but this response was influenced by the medium N-concentration, with improvement higher at low N-levels. The medium that supported the highest ligninolytic production (22.75 U/ml laccase, 0.34 U/ml manganese peroxidase, and 0.20 U/ml glyoxal oxidase) also showed the greatest ability to decolorize the dyes. Only glyoxal oxidase activity limited biodecoloration efficiency, suggesting the involvement of peroxidases in the process. The addition of 1-hydroxybenzotriazole (a known laccase mediator) to the ligninolytic fluids increased both their range and rate of decolorization. The cell-free supernatant did not decolorize xylidine, poly R-478, azure B, and malachite green as efficiently as the whole broth, but results were similar in the case of indigo carmine and remazol brilliant blue R. This indicates that the mycelial biomass may supply other intracellular or mycelial-bound enzymes, or factors necessary for the catalytic cycle of the enzymes. It also implies that this fungus implements different strategies to degrade dyes with diverse chemical structures.     

Role of diffusion in nonaqueous enzymology. 1. Theory.

Using a set of standard equations, we have calculated the role of internal and external mass transfer in limiting the rate of enzyme-catalysed reactions in anhydrous organic solvents and supercritical fluids. We have shown that enzyme particles suspended in anhydrous organic solvents will be subject to increasing diffusional limitation as the enzyme activity and particle size increase. Using particle dimensions, as measured by scanning electron microscopy, we have prepared a series of graphs that will enable investigators to determine whether their combination of particle size and activity will result in internal or external diffusional limitations. We have shown that supercritical fluids can be expected to enhance the activity of enzymes in nonaqueous environments as a result of the high diffusivity of the bulk solvent. The plots also clearly indicate that enzyme particles in supercritical fluids require nearly two orders of magnitude less agitation than those suspended in conventional solvents in order to overcome any external mass transfer limitations.     

Comparison of pathways of copper metabolism in aorta and liver. A functional test of metallothionein.

Soluble fractions from chick liver and aorta were examined for copper-binding proteins. In liver a zinc-binding thionein appeared to be the major binding protein for copper. Aortic tissue contained only traces of this thionein protein. Unlike liver, moderate amounts of soluble copper in aorta showed no association with macromolecules. Chicks fed on copper-deficient diets for 8 days had one-third the liver copper concentrations of controls. Aortic copper concentration was decreased only slightly, but the activity of lysyl oxidase, a copper-dependent enzyme in aorta, was decreased significantly. Treating the deficient chicks with CuSO4 (1 mg/kg) restored liver copper rapidly. The increase correlated with the binding of copper to a 10 000-mol.wt. component in the soluble fraction. Aortic copper concentrations responded much less to the CuSO4 treatment, but lysyl oxidase activity was again measurable in the tissue. Radioactive isotopes of copper bound almost exclusively to the 10 000-mol.wt. component in liver and to components of mol.wt. 30 000 or above in aorta. Hardly any of the administered radioactivity appeared with the 10 000-mol.wt. components in aorta, and none was found with unbound copper. The 30 000-mol.wt. components in aorta showed superoxide dismutase activity that was sensitive to NaCN. They also showed the highest specific activity of copper of any other aorta component. A clear distinction was seen between the metabolism of copper in liver and aortic tissues. Whereas a copper thionein, metallothionein, was a major component in the liver pathway, it is doubtful that this protein plays a major role in the intracellular metabolism of copper in aortic tissue.     

Induction of metallothionein in rat tissues following subchronic exposure to mercury shown by radioimmunoassay

Although the analysis of metallothionein (MT) by radioimmunoassay (RIA) is not a common technique, its use is preferred over other methods since it offers the advantages of sensitivity and specificity. In this paper we present data on the basal levels of MT in rat tissues and physiological fluids of female Sprague-Dawley rats. The mean basal MT concentrations of the following organs and fluids were determined by RIA to be: liver (9.8 μg/g), kidney (68 μ/g), brain (0.8 μg/g), spleen (1.0 μg/g), heart (5.4 μg/g), plasma (11 ng/ml), and urine (200–300 μg/g creatinine). Following subcutaneous exposure to inorganic mercury (0.2 μmol/kg/d, 5 d a week for up to 4 wk), the metal accumulated primarily in the kidney. There was also a simultaneous accumulation of zinc in the liver and of zinc and copper in the kidney. Induction of MT did take place in liver, kidney, brain, and spleen. No increases in the MT contents of blood and urine were noted. The excess zinc and copper in the kidney of exposed animals were found to be associated predominantly with MT. No overt signs of mercury toxicity were noted in these animals and the incidence of proteinurea was nil. The data are discussed with reference to methods of MT determination in animal tissues and in relation to mercury metabolism and toxicity.     

Chemotaxis of rat lymphocytes.

Rat lymphocytes obtained from spleens, lymph nodes, and thymus glands showed migratory responses to a variety of factors including fluids from mixed lymphocyte culture fluids from concanavalin A-stimulated cells, fluids from phagocytizing macrophages, and to anti-rat IgG. Migratory responses to the last factor were bimodal over a dose range of anti-Ig; at high concentrations of anti-Ig, the response appeared to be nonspecific, whereas, at low concentrations, the responses seemed to be chemotactic in character. When lymphocytes from spleens, lymph nodes, and thymic glands were compared, qualitative and quantitative differences on the responses were evident with use of the three attractants. When spleen lymphocytes were separated into T cell- and B cell-enriched fractions, T cells responded to the culture fluids from mixed lymphocyte cultures, whereas B cells seemed to respond poorly, if at all. Only B cells responded to anti-Ig. These findings may explain, at least in part, the accumulation of lymphoid cells at sites of inflammatory stimuli.     

Studies upon the copper fungicides. IX. Investigations with the exudate from fungus spores

Determinations have been made of the copper dissolved from pure copper compounds by water, by a standardized solution of the exudate of Neurospora sitophila spores, by standard solutions of malic and succinic acids and their sodium salts, and by glycine and mannitol.
The results compared with the known fungicidal performances of the copper compounds show that the amounts of copper yielded to water and to the standardized spore extract or glycine solution provide an indication of fungicidal properties.
Although soluble copper in excess of that dissolved by water can only appear from alkaline Bordeaux deposit by complex ion formation, results show that this is not necessarily the only means by which copper can dissolve from other copper compounds.
In a chemical investigation of the exudate of N. sitophila spores, mannitol, succinate and fumarate have been isolated. None of these materials can dissolve an appreciable amount of copper from alkaline Bordeaux deposit. Malate, found by other workers to be a constituent of the exudate of N. sitophila spores cultured on potato dextrose agar, has not been detected in such a solution from spores grown on malt agar. The quantity of mannitol in spore exudate has been shown to depend on the nutrient medium on which the fungus is grown.     

An e.p.r. study of the non-equivalence of the copper sites of caeruloplasmin.

The two Type 1 (blue) copper-binding sites of caeruloplasmin were spectroscopically differentiated by the kinetic analysis of the e.p.r. spectra during the redox cycle. One blue copper, with a hyperfine splitting constant (A parallel) of 6.8 mT, which was rapidly reduced, was not reoxidized by oxygen, whereas it was reoxidized by H2O2. The other blue copper (A parallel = 5.8 mT), which was reduced slowly, was rapidly reoxidized by either oxygen or H2O2. A conformational change of the Type 2 copper was concomitant with the fast reduction of Type 1 copper, whereas its reduction occurred during the slow phase. This sequence of events was reversed in the reoxidation step, that is, the Type 2 copper reappeared rapidly as the species with altered conformation and reverted to the symmetry typical of the native state in the slow phase. The specific reaction of a blue-copper site with the H2O2 can tentatively be related to the established ability of caeruloplasmin to prevent 'oxidative' attack of proteins and lipids.     

Complexes of sulfur-containing ligands. I. Factors influencing complex formation between D-penicillamine and copper (II) ion.

Complex formation and redox reactions between copper (II) ion and D-penicillamine were studied in detail as functions of the metal/-ligand ratio and the concentration of halide ions. It was established that a copper (I)- D-penicillamine polymeric complex of amphoteric character is formed when excess D-penicillamine is present. When the D-penicillamine/copper (II) ratio = 1.45 in the starting reaction mixture, a mixed valence complex with an intense red-violet color is formed. The formation of this compound, which contains 44% copper (II) ion, is greatly influenced by the experimental conditions, primarily by the concentration of halide ions. The main chemical and physical characteristics of the mixed valence complex were determined via magnetic and spectroscopic measurements. It was further established that a very intense blue complex is formed when the D-penicillamine/copper (II) ratio = 2 and halide ions are present. On the basis of the nature of the products formed under various conditions it was concluded that the copper (II)-D-penicillamine system may serve as a good model for studying the binding sites of copper-containing proteins.     

Xylem Fluids of White Lupin (Lupinus albus) Cultivars Susceptible and Resistant to Fusarium oxysporum f. sp. Lupini Race 3

Xylem fluids from white lupin cv. Neuland, susceptible to wilt causes by Fusarium oxysporum f. sp. lupini , promoted germination of conidia and chlamydospores of he pathogen, and the production of micro-conidia, to a greater extent than did xylem fluids from the resistant cv. Primorskij No sugars, organic acids or phenolic compounds were detected in the xylem fluids, but a number of amino acids were found, and the concentration of some differed in xylem fluids from the two cultivars; on the whole, concentrations were greater in the susceptible cv. Neuland. Synthetic amino acids were supplied to the fungus in vitro and both stimulatory and inhibitory effects (according to the compound and its concentration) were noted upon the germination of conidia and chlamydospores and on mycelial growth and the production of conidia. The role of amino acids and other compounds in the susceptibility of white lupin to wilt is discussed.