Calcium uptake and ATPase activity of sarcoplasmic reticulum vesicles isolated from control and selenium deficient lambs.

The calcium uptake and ATPase activity were studied using fragmented sarcoplasmic reticulum (FSR) vesicles from normal and selenium (vitamin E)--deficient lambs. The latter group was suffering from white muscle disease (WMD). The calcium uptake of FSR vesicles from muscle of WMD lambs was reduced 10-fold as compared to those from normal lambs. An inverse relationship was found with the calcium uptake ability of the FSR vesicles and the severity of WMD. ATPase activity was nonsignificantly lower in vesicles from WMD lambs. The most active FSR vesicles from both normal and WMD lambs banded at 27% when purified on linear sucrose density gradients. The number of protein bands appearing in acrylamide gels of the purified vesicles appeared to be directly proportional to the severity of WMD. The 75Se cosedimented with the calcium uptake and ATPase activity when FSR vesicles from a lamb injected with 75Se-selenite were subjected to linear sucrose density gradient centrifugation, suggesting that selenium is incorporated into these vesicles. Injection of selenium into WMD lambs resulted in significantly greater calcium uptake activity in vesicles 18 and 38 days later as compared with untreated WMD lambs. Injection of selenium in WMD lambs resulted in a marked decrease in plasma CPK activity and a significant increase of glutathione peroxidase activity in the blood.

Association of selenium with tissue membranes of ovine and rat tissues.

Rats and lambs were injected with(75)Se-selenite and the microsomal fraction obtained by differential centrifugation from homogenates of muscle, heart, liver, brain, kidney, and testes. These fractions were subsequently centrifuged on sucrose density gradients from 25 to 45% and the distribution of selenium-75 between the labeled membranes of different densities monitored by scintillation counting. The majority of the selenium-75 was associated with membranes of similar density in muscle, heart, liver, and testes from both rats and lambs. The selenium-75 was distributed between many different density membranes in brain from both lambs and rats. The selenium-75 was associated predominantly with one density membrane from ovine kidney but with several density membranes from rat kidney.

Incorporation of selenium-75 into semen and reproductive tissues of bulls and rams.

After intramuscular injections of 500 muCi 75Se, semen was collected periodically over a 63-day period from a selenium-deficient and a selenium-injected ram which were then killed for collection of the reproductive organs for the gel filtration studies. Testes, accessory glands and semen were also obtained from a bull injected intravenously with 75Se. Gel filtration (Sephadex G 150) of ram testis cytosol resulted in 4 75Se peaks (Ve/Vo ratios of 1 X 1, 1 X 5, 2 X 3, 2 X 9). In the selenium-injected ram the glutathione peroxidase (GSH-Px) peak (Ve/Vo 1 X 5) predominated, but in the selenium-deficient ram, radioactivity of the GSH-Px peak was less than that of the higher molecular weight peak (Ve/Vo 1 X 1). Gel filtration chromatograms of bull testis cytosol yielded 5 75Se peaks (Ve/Vo 1 X 1, 1 X 5, 1 X 9, 2 X 4, 2 X 8). In chromatograms of ram seminal plasma on Sephacryl S-200 there were 2 major (Ve/Vo 1 X 4, 1 X 1) and 2 minor peaks (Ve/Vo 1 X 7, 2 X 4). 75Se increased with time up to 49 days after injection in all peaks. 75Se-labelled bull seminal plasma yielded 2 75Se peaks (Ve/Vo 1 X 1, 1 X 4) which corresponded to the major peaks of ram seminal plasma. Bull and ram seminal plasma GSH-Px activities per mg protein were comparable (28 and 29 nmol NADPHox/min, respectively), but when expressed per ml seminal plasma, activity of the bull was more than 7 times the highest activity of ram seminal plasma (2908 and 385 nmol NADPHox/min, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of physical exercise on plasma creatine kinase activity in healthy and dystrophic turkeys and sheep.

Plasma creatine kinase (P-CK) activities were significantly increased after physical exercise in healthy turkeys and in turkeys with genetic muscular dystrophy. Activities as high as 100,500 mU/ml of plasma were observed. Activities remained high for at least 29 hours after exercise. Moderate exercise did not significantly affect P-CK activity in lambs. Increases in P-CK activity during expression of nutritional muscular dystrophy were readily distinguished from exercise effects; activity exceeded 160,000 mU/ml in lambs during expression of that condition.

Blood selenium and glutathione peroxidase activity in pregnant women: comparative assays in primates and other animals.

The influence of pregnancy on blood selenium levels and glutathione peroxidase (GSH-Px) activity was studied in women. Whole blood and plasma selenium levels decreased whereas erythrocyte and plasma GSH-Px activities increased with the progress of pregnancy. The ratio of erythrocyte GSH-Px activity to whole blood selenium levels was 4- to 5-fold higher in rats and sheep than in primates (humans and monkeys), suggesting that more selenium is associated with GSH-Px in erythrocytes from rats and sheep than from primates. In assays of blood with low GSH-Px activity such as that from humans or selenium-deficient animals, a component of the erythrocyte other than GSH-Px was found to contribute more to the peroxidase activity. Evidence was obtained to indicate that t-butyl hydroperoxide is a better substrate than hydrogen peroxide for the assay of low GSH-Px erythrocyte activity. The length of time that the blood was stored before assay was found to have an effect on erythrocyte GSH-Px activity, and the storage patterns may be dependent on the species of animal from which the blood is drawn.

Evidence for selenocysteine in ovine tissue organelles.

Subcellular fractions were prepared by differential centrifugation from heart and liver of a lamb labeled with 75Se-selenite. Crude fractions of nuclei, mitochondria, and microsomes from both tissues were solubilized with sodium dodecyl sulfate and chromatographed on columns of sephacryl S-200. A low molecular weight (MW) 75Se labeled cardiac cytosol protein (approximately 10,000 daltons) was partially purified by gel filtration chromatography. The major 75Se peaks from the sephacryl columns and the low MW cardiac protein were hydrolyzed in HCl under an inert atmosphere. When chromatographed on an amino acid analysis column, 75Se from each hydrolysate chromatographed in the identical position of 2,7-diamino-4-thia-5-selenaoctanedioic acid, the mixed oxidized dimer of cysteine and selenocysteine. The low MW cardiac protein was reacted with chloroacetate after reduction with borohydride. 75Se from a hydrolysate of this derivatized protein eluted in the same position as Se-carboxymethylselenocysteine on the amino acid analysis column. Thus, selenocysteine appears to be the predominant form of selenium in ovine heart and liver.

Influence of silver, mercury, lead, cadmium, and selenium on glutathione peroxidase and transferase activities in rats.

At the levels used in the experiments, mercury and silver significantly depressed the activity of glutathione peroxidase (assayed with either H2O2 or cumene-OOH) in rat tissues, whereas cadmium or lead had no effect on this activity. The most pronounced effects of mercury and silver on glutathione peroxidase were found in the liver and kidneys, with much less effect in the testes and erythrocytes. Similar trends for the effects of these metals were noted for tissue selenium levels. Silver and mercury significantly depressed the selenium concentrations, but cadmium and lead had no effect upon the selenium levels. Mercury and silver had no effect upon the activity of glutathione transferase in liver and testes, but mercury caused a significant initial increase of its activity in the kidneys. At no time did silver have any significant effect on its activity in this organ.

Selenium proteins in ovine tissues: III. Distribution of selenium and glutathione peroxidases in tissue cytosols.

Three 6 week-old lambs were injected with carrier-free selenium-75 as sodium selenite initially and again after 6 days. One lamb received no further injections whereas the other two received injections of either vitamin E or unlabeled Na2SeO3 when the first selenium-75 injection was given. Selected tissues were removed at autopsy 10 days after the first injection. The cytosol from homogenates of these tissues was subjected to gel chromatography, and the elution profiles determined for radioactivity, protein content, and glutathione peroxidase activity using either hydrogen peroxide or cumene hydroperoxide as substrates. The selenium-75 was found to be distributed mainly between 2 different MW peaks. The larger MW seleno-peak (90,000) possessed both glutathione:hydrogen peroxide oxidoreductase, and glutathione:cumene hydroperoxide oxidoreductase activities, but the smaller MW seleno-peak (about 10,000) possessed no glutathione peroxidase activity. A peak of about 60,000 daltons containing only glutathione:cumene hydroperoxide oxidoreductase activity and no selenium-75 was found primarily in the liver and kidney. Vitamin E had no effect on the elution profiles. Selenium status of the animal had only a minor effect on the selenium-75 distribution in the cytosol, but had a marked effect on the absolute amount of the label taken up by tissues.

Effects of selenium and vitamin E deficiencies in lambs on hepatic microsomal hemoproteins and mitochondrial respiration.

Microsomal hemoprotein levels and delta-amino levulinic acid dehydrase activity were determined in livers and rate of oxygen uptake, ADP:O ratios, and respiration control index were determined on mitochondria from muscle, liver, and heart of normal and white muscle diseased (WMD) lambs. WMD lambs were produced by feeding their dams either low selenium purified or alfalfa hay diets. Vitamin E and/or selenium was injected in a 2 x 2 factorial treatment in the ewes fed purified diets. Hepatic microsomal cytochrome P 450 levels and total heme content were significantly lower in WMD lambs. Cytochrome b5 content was significantly lower in lambs on the -E-Se or -E + Se treatments than those on the +E--Se treatment, but the cytochrome b5 content was not different between WMD and normal lambs from ewes fed the hay diet. No differences were found in hepatic delta-amino levulinic acid dehydrase activity, or in the rate of oxygen uptake, ADP:O ratios or respiratory control index between mitochondria from normal and WMD lamb tissue on any of the treatments.