Selenocysteine-containing proteins and glutathione peroxidase.
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Animals↗
Biomedical subjects
M A Motsenbocker
Publications and source records attributed to M A Motsenbocker.
Effect of dietary selenium on plasma selenoprotein P, selenoprotein P1 and glutathione peroxidase in the rat.
The purpose of this study was to determine the effect of dietary selenium on the abundance of selenium in plasma selenoprotein P, selenoprotein P1 and glutathione peroxidase. Weanling rats were provided water that contained 1.0, 0.1 or 0.01 ppm selenium and 75Se for 21 days. Gel filtration of denatured subunits was used to identify 75Se in the selenoproteins. Rats provided 1.0 ppm selenium accumulated 1.5 times more 75Se in liver cytosolic selenoprotein P1, but not in the two other selenoproteins, than did rats provided 0.1 ppm selenium. Most of the liver and blood selenium in rats provided 1.0 ppm selenium was insoluble and in an unknown chemical form. The tissue accumulation of unrecoverable selenium was apparently a response to the high dietary level of selenium. The proportion of selenium in plasma selenoprotein P, a putative selenium-transport protein, reflected the long-term selenium status of rats and varied from approximately 11-58% depending on the level of selenium supplementation. Turnover of selenium from this protein was affected by the dietary selenium of the rats. The results indicate that selenium incorporation into plasma selenoprotein P and selenoprotein P1 is affected by diet in ways that may reflect their importance to the rat.
Animals↗
Selenium and selenoproteins in the rat kidney.
Kidney tissue contains a high concentration of selenium that is not accounted for by the known selenoprotein glutathione peroxidase (glutathione: hydrogen-peroxide oxidoreductase, EC 1.11.1.9). In order to investigate the nonglutathione peroxidase selenium, rats were isotopically labeled with [75Se]selenite over a 10-day period. After this time half of the 75Se in kidney homogenate was found in the particulate subcellular fractions. The kidney lysosomes contained unusually high levels of 75Se, yet they did not contain correspondingly high levels of glutathione peroxidase activity. Two selenoproteins having molecular weights less than 40 000 were resolved by gel filtration from a kidney supernatant fraction. A third selenoprotein exhibited a molecular weight of 75 000. This protein contained one 75 000 molecular-weight subunit, and its selenium was in the amino acid selenocysteine. The 75 000 molecular-weight protein was chromatographically distinct from glutathione peroxidase. In order to determine if these selenoproteins protect against cadmium toxicity, 109CdCl2 was administered to rats that were isotopically prelabeled with 75Se. At 3, 25 and 72 h after 109Cd administration, no 109Cd was associated with selenium-containing proteins. Two of the nonglutathione peroxidase selenoproteins were apparently unique to the kidney.
Animals↗
A selenocysteine-containing selenium-transport protein in rat plasma.
A selenocysteine-containing rat plasma protein (selenoprotein P) was examined for a possible role in the transport of selenium in the rat. A time-course study of the localization of injected 75Se from [75Se]selenite indicated that one-half of the selenium was sequestered by liver tissue 1 h after injection and that one-fourth of the 75Se in the plasma was attached to selenoprotein P 3 h after injections. By 25 h there was little 75Se in plasma, and much of the 75Se had accumulated in nonhepatic tissues. 75Se was incorporated into selenoprotein P by liver slices in a process that was sensitive to the protein synthesis inhibitor cycloheximide. The fate of 75Se from intracardially injected 75Se-labeled selenoprotein P was followed in rats maintained on selenium-deficient and selenium-sufficient diets. Substantially more of the injected 75Se was present per gram wet weight in the testes and kidneys than the livers of the selenium-deprived rats after 5 h. The results indicate that selenoprotein P is synthesized in rat liver and that it transfers selenium from the liver to extrahepatic tissues.
Animals↗
Selenocysteine-containing proteins from rat and monkey plasma.
This investigation was carried out to determine whether a selenium-containing plasma protein in rat and monkey (Macaca mulata) plasma might be involved in selenium transport. Injection of [75Se]selenite or [75Se]selenomethionine was used to label a plasma protein. The native molecular weight of the protein from rat and monkey plasma was determined by gel filtration to be about 80 000. The molecular weight of a selenium-containing polypeptide prepared from the protein was about 45 000, as determined by gel filtration in the presence of sodium dodecyl sulfate. Selenium was attached to both the rat and monkey plasma protein in the form of the amino acid selenocysteine. The proportion of plasma selenium normally bound to the rat protein in vivo was less than 5%, and the half-life of selenium bound to the protein was a few hours. These findings are consistent with a selenium-transport function for this protein.
Animals↗
Sensitivity limitations encountered in enhanced horseradish peroxidase catalysed chemiluminescence.
Conditions for the enhanced horseradish peroxidase (HRP) catalysed reaction between luminol and hydrogen peroxide were optimized to determine detection limits for HRP conjugated to antibody fragment (HRP-Fab) in solution phase. Light output was linear with respect to HRP-Fab concentration but became nonlinear at low HRP-Fab concentrations when an accelerator (enhancer) of the reaction was used. para-Phenylphenol was a more effective enhancer than p-iodophenol at HRP-Fab concentrations below 20 pmol/l. The detection limit for HRP-Fab was 1.2 femtomoles in the absence of p-phenylphenol and 0.08 femtomole in the presence of p-phenylphenol. The acceleration of peroxidase activity at the lowest HRP-Fab concentrations occurred after an incubation time period of up to five minutes. This lag time limited the sensitivity and the mechanism for it was sought. Preincubation experiment results indicated that the lag time phenomenon may involve a reversible alteration in HRP catalytic activity and that enhancer, peroxide, luminol and HRP-Fab had to be incubated together some time before maximum activation could occur.
Horseradish Peroxidase↗
Enhancers of the non-peroxidative metal porphyrin chemiluminescence reaction.
Metal porphyrins catalyse luminol chemiluminescence at pH13 without added peroxide. The effects of 22 different surface active compounds on this reaction were studied using six metal porphyrins and one metal porphyrin conjugate. The most active catalyst was Mn-meso-tetra(4-sulphonatophenyl)porphine. Tween-20 enhanced the activity of this catalyst best at a Tween-20 to luminol ratio of 74:1. However, lauryl sulphate enhanced best at an optimum lauryl sulphate to luminol ratio of over 1000:1 and both detergents enhanced the reaction when present below their critical micelle concentrations. Negatively charged aliphatic compounds such as fatty acids enhanced the reaction but positive-charged aliphatic compounds inhibited it. Small differences in enhancer structure resulted in differing enhancement. For example, linoleic acid enhanced Mn-meso-tetraphenyl porphine more than 10-fold, yet linolenic acid inhibited this catalyst. Conjugation of a metal porphyrin to antibody did not influence its enhancement by detergents. The results indicate that the enhancement mechanism does not require formation of pure detergent micelles but that direct association between enhancer and catalyst may be important.
Hydrogen-Ion Concentration↗
Improvements to enhanced horseradish peroxidase detection sensitivity.
At very low horseradish peroxidase (HRP) concentrations, the enhanced chemiluminescence reaction is often characterized by a lag time between initiation of the reaction and beginning of light output. In this study, four treatments of luminol solution were examined in an effort to remove the lag time and to improve chemiluminescence light output. Addition of ammonium persulphate stimulated light output more than tenfold. Ultraviolet irradiation and photoactive dye pretreatment of luminol solution both increased light output fourfold. Luminol purity was the most important factor affecting detection sensitivity. Recrystallization of luminol from base improved the detection limit 13-fold although there was an improvement in the detection limit from 13 attomoles per millilitre to 5 attomoles per millilitre with highly purified luminol when photoactive dye pretreatment was utilized. The results are consistent with a simple interference mechanism whereby enhancer radicals produced by the enzyme are preferentially quenched by contaminants present in the luminol, in the enhancer and in the solvent used to dissolve the enhancer. Consumption of these interferences prior to light emission results in a lag time and a less favourable HRP detection limit.
Ammonium Sulfate↗