Biomedical subjects

S S Percival

Publications and source records attributed to S S Percival.

26 records · Page 2Linked to original sources

PubMed1992

HL-60 cells can be made copper deficient by incubating with tetraethylenepentamine.

A system for studying copper deficiency was developed in a cell culture model. HL-60 cells were incubated with three chelators known to bind copper. One chelator, tetraethylenepentamine (TEPA), reduced cellular copper levels and the activities of two copper-requiring enzymes, Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and cytochrome c oxidase. The specificity of the chelator was assessed by incubating cells with both copper and TEPA and, in other experiments, with zinc and TEPA. Copper levels, Cu/Zn-SOD activity and cytochrome c oxidase activity were restored to control values when copper and TEPA were added to cultures simultaneously, indicating the TEPA was responsible for reducing these aspects of copper metabolism. Incubating with both zinc and TEPA reduced copper levels relative to the control, but did not reduce Cu/Zn-SOD activity to the same extent as TEPA alone. The chelation of copper was a time-dependent process that was stable for at least 4 d. Cell growth and viability were not affected by TEPA. Respiratory burst activity, an indicator of differentiation, was not affected by TEPA, demonstrating that the reduction of Cu/Zn-SOD activity was due to copper chelation and not due to changes in Cu/Zn-SOD protein levels that occur during differentiation. Loss of copper, as well as a reduction of the activity of two copper-requiring enzymes, provides evidence that TEPA is a useful compound for creating a functional copper deficiency in cell culture.

Copper↗

PubMed1992

Iron metabolism is modified by the copper status of a human erythroleukemic (K562) cell line.

Copper deficiency is known to result in a microcytic, hypochromic anemia. Red cells of copper-deficient animals have less hemoglobin than their copper-adequate counterparts. The objective of this work was to determine what role copper plays in maintaining hemoglobin levels. It was hypothesized that the primary defect lies in intracellular iron metabolism. The influence of copper supplementation on iron uptake and storage was examined in a cell line capable of hemoglobin synthesis. The results demonstrated that copper supplementation of human K562 cells was associated with higher cytosolic iron levels and ferritin levels. Copper supplementation of the cell culture altered the initial rate of iron uptake from transferrin and enhanced iron uptake in noninduced cells; however, in hemin-induced K562 cells, which express fewer transferrin receptors on the cell surface, copper appeared to reduce iron uptake. Subsequent studies showed that the cells were able to take up the same amount of iron from transferrin when incubated over a longer period of time (24 hr). In the noninduced (non-hemoglobin synthesizing) cells, proportionally more iron was associated with the ferritin. We concluded from these studies that copper affects both uptake and storage of iron and that copper supplementation reduces cellular iron turnover.

Cell Line↗

PubMed1991

Regulation of Cu,Zn superoxide dismutase with copper. Caeruloplasmin maintains levels of functional enzyme activity during differentiation of K562 cells.

K562 cells, a human erythroleukaemic cell line blocked for differentiation, commit towards erythrocytes when exposed to haemin (20 microM). The cells synthesize fetal haemoglobins and show site-specific binding of caeruloplasmin, a plasma copper protein. These events are set into motion by haemin. On the assumption that the binding of caeruloplasmin could reflect a greater need for copper, we sought to determine whether the transfer of 67Cu from caeruloplasmin was accelerated in haemin-induced compared with non-induced K562 cells. Cu,Zn superoxide dismutase (CuZnSOD) was the recipient. Haemin induction caused the K562 cells to lose CuZnSOD activity. By 96 h, the level of SOD activity was less than 60% of that of non-induced cells. The loss was confined entirely to the CuZn form, MnSOD activity staying essentially unchanged. Although CuZnSOD activity declined with the haemin induction, the incorporation of [4,5-3H]lysine into immunoprecipitable CuZnSOD protein was unaffected. There was also no change in CuZnSOD mRNA concentration in haemin-induced cells. Thus a loss of enzyme did not correlate with a decline in the synthesis de novo of CuZnSOD protein. When 48 h-induced cells were transferred to a medium supplemented with 0.2 microM-caeruloplasmin, CuZnSOD activity was restored to control levels in 24 h. Caeruloplasmin also stimulated the incorporation of [3H]lysine into immunoprecipitable CuZnSOD protein. Caeruloplasmin addition may have affected a post-translational regulatory site for CuZnSOD biosynthesis, possibly by providing copper for the newly synthesized enzyme.

Biological Transport↗

PubMed1991

A role for ascorbic acid in copper transport.

Scurvy-like symptoms have been seen in experimental copper deficiency. This forecasts a role for the vitamin in copper metabolism. Ascorbate has been known to antagonize the intestinal absorption of copper. More recent studies have characterized a postabsorption role for ascorbate in the transfer of copper ions into cells. The vitamin reacts directly or indirectly with ceruloplasmin, a serum copper protein, specifically labilizing the bound copper atoms and facilitating their cross-membrane transport. Ascorbate at physiological levels and above impedes the intracellular binding of copper to Cu,Zn superoxide dismutase. The mechanism is unclear but nonetheless suggests both positive and negative regulatory functions for ascorbate in copper metabolism.

Absorption↗

PubMed1990

Copper transport from ceruloplasmin: characterization of the cellular uptake mechanism.

Copper uptake from 67Cu-labeled ceruloplasmin (67CuCp) was studied in K-562 cells, a human erythroleukemic cell line. 67CuCp was prepared by an ascorbate-catalyzed exchange of recrystallized ceruloplasmin with 67CuCl2. The labeled protein was treated with Chelex-100 and gel filtration to ensure that 67Cu was tightly bound to the structure. 67CuCp bound specifically to the K-562 cells at 4 degrees C. The binding was linear with protein in the range of 200-800 nM and in the presence of 3% albumin. In this concentration range, 67CuCl2 showed no binding that could be interpreted as specific; 80-90% of the cell-bound 67Cu was removed by washing the cells with acid buffer. When binding was attempted at 37 degrees C, a significant fraction of the 67Cu resisted acid washing and with time accumulated in the cells. Fractionating the cytosolic components on Percoll gradients located the 67Cu in buoyant fractions of densities 1.030-1.05, with a peak at 1.035. Repeating the experiment with 125I-labeled ceruloplasmin failed to localize any 125I label in Percoll fractions; very little 125I was detected in the cytosol. Double-labeled 67Cu-125I-ceruloplasmin confirmed that copper and not the protein moiety of ceruloplasmin was taken up by the cells. The uptake reaction was inhibited by 1 mM bathocuproine sulfonate and by 1 mM sodium iproniazid. Ascorbate (100 microM) strongly stimulated uptake. These studies provide evidence that K-562 cells are able to extract copper atoms from ceruloplasmin and transport the copper to the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport↗

PubMed1989

Copper transport: insights into a ceruloplasmin-based delivery system.

Ceruloplasmin binds to the membranes of K562 cells. The binding has been shown to result in a temperature-dependent transfer of ceruloplasmin-bound copper into the cytosol. Ascorbic acid (100 microM) stimulates the transmembrane transfer nearly 10-fold, depending on the initial concentration of 67Cu-ceruloplasmin. The protein moiety of ceruloplasmin does not enter the cells. Bathocuproine disulfonate, a chelator specific for cuprous copper, inhibits the uptake, suggesting copper atoms are reduced concomitant with their removal from ceruloplasmin. Cytosolic 67Cu from ceruloplasmin was found mainly bound to Cu, Zn superoxide dismutase, the major cytosolic copper protein in these cells. Evidence supporting the various phases in the ceruloplasmin-mediated transport mechanism are presented.

Ascorbic Acid↗

PubMed1989

Ascorbate enhances copper transport from ceruloplasmin into human K562 cells.

Copper uptake from human ceruloplasmin (Cp) into cells of a human erythroleukemic cell line, K562, was investigated. The interaction between ascorbic acid and the copper atoms in ceruloplasmin was a focal point of the study. Nondenatured 67Cu-labeled ceruloplasmin (67Cu-Cp) was prepared by an ascorbate-catalyzed exchange of Cp with 67CuCl2 in vitro. The complex was stable, even in the presence of 1.0 mM ascorbate. Adding K562 cells and incubating at 37 degrees C resulted in an immediate transfer of 67Cu from ceruloplasmin to the cells. At 37 degrees C the copper accumulated by the K562 cells resisted dissociation by mild acid washing. The rate of transfer of 67Cu was proportional to the Cp concentration in the medium. Ascorbate (100 microM) enhanced the uptake of 67Cu at least fourfold. D-Isoascorbate worked as well as L-ascorbate, suggesting that the reducing potential of the vitamin (or its isomer) was important in the uptake of copper. Approximately 20% of the 67Cu absorbed into the cytosol was precipitable with antibodies to Cu-Zn superoxide dismutase (Cu-Zn SOD). Ascorbate, however, did not enhance the incorporation of radioactivity into Cu-Zn SOD, suggesting that copper may not be the only rate-limiting factor in the synthesis of this enzyme in K562 cells. The possible relevance of these observations to vitamin C deficiency is discussed.

Ascorbic Acid↗

PubMed1979

Long term pancreatic response to feeding heat damaged casein in rats.

Rats were fed a heat damaged casein (autoclaved 24 hours, 121 degrees, 2 atm) diet to determine the effect of poorly digested protein on pancreatic enzyme levels and response to a meal. After 10 days of feeding, the pancreas showed no signs of atrophy, however, chymotrypsin and amylase activities were lower in proportion to body weight. An estimation of secretion during the meal was similar or slightly lower in rats fed heated casein (HC) as compared to the casein diet (C), but a greater levels of enzyme activity was found in the intestinal contents of rats fed HC relative to control rats. These results suggest that the turnover of enzymes in the gut is reduced when a less digestible protein is fed, and that the endogenous pancreatic secretions and the dietary protein are not digested and absorbed as well.

Amylases↗