Effect of molybdenum treatments on the distribution of Cu and metallothionein in tissue extracts from rats and sheep.

An examination was made of the effects of tetrathiomolybdate (TTM), ammonium molybdate (AM), sodium sulphide, and two molybdo amino acids (cysteine-Mo, cysteine-Mo-S) on the distribution of Cu and Zn among proteins in extracts of the livers and kidneys of rats and sheep. Tetrathiomolybdate caused a shift in the chromatographic distribution of Cu from low molecular weight proteins such as metallothionein (MT) to proteins of higher molecular weight (greater than 100,000 daltons). This was not due to polymerization or cross-linking of metallothionein with the latter, but to the formation of protein-TTM complexes that had a strong affinity for Cu. There was a concomitant redistribution of Zn towards proteins of low molecular weight. Pretreatment of high molecular weight proteins from rat liver with TTM greatly increased the capacity of the proteins to remove Cu from MT. When AM or sodium sulphide were added together to extracts of rat liver, changes similar to those induced by TTM were observed in the chromatographic distribution of Cu and Zn. Individually, these compounds had no significant effect on the distribution of the metals. Of the two molybdo amino acids, only cysteine-Mo-S altered the chromatographic distribution of Cu in extracts of rat liver. The redistribution was in the same direction as that induced by TTM, but was not as pronounced.

Involvement of organic molybdenum compounds in the interaction between copper, molybdenum, and sulfur.

The effect on Cu metabolism of two organic Mo compounds, cysteine-Mo (CM) and cysteine-Mo-S (CMS), and of an inorganic compound, tetrathiomolybdate (TTM), was investigated in sheep. Intravenous administration of CMS or TTM increased total plasma Cu concentrations and promoted the appearance of trichloroacetic acid (TCA)-insoluble Cu in plasma. Plasma Mo was increased by both compounds. Column chromatography of plasma showed that CMS caused the accumulation of TCA-insoluble Cu in high-MW fractions, whereas TTM increased the Cu content of low-MW fractions, mainly albumin. CMS and TTM increased the concentration of Cu and Mo in kidney. In liver, Mo concentrations were elevated by both CMS and TTM, and Cu concentrations were reduced by TTM when it was given at a low dose rate. The subcellular distribution of Cu and Mo in liver and kidney was investigated. The findings are discussed in relation to the proposal that Mo-containing, organic compounds are intermediaries in the interaction between Cu, Mo, and S in ruminants.

Distribution of Cu, Zn, and Fe in the soluble fraction of the kidney in normal, copper-poisoned, and thiomolybdate-treated sheep.

Twenty-seven sheep were used in two experiments to study the distribution of copper (Cu), zinc (Zn), and iron (Fe) in the kidney cytosol of control, Cu-loaded, and thiomolybdate (TM)-treated sheep. A comparison was made of the patterns of distribution on Sephadex G-75 of Cu, Zn, and Fe from the cytosol of fresh and frozen kidneys and after thawing of frozen cytosol. In both Cu-dosed and TM-treated sheep, the absolute level of Cu increased in the cytosol, but the percent of Cu decreased. The percent recovery of Cu from the frozen kidney was comparable to that from fresh kidney, but the extraction of Cu from the supernatant of frozen cytosol was approximately 10% less. This was due to a loss of Cu in a precipitate that formed when the frozen cytosol was thawed. Most of the Cu in the cytosol from the kidney of Cu-loaded sheep was in a metallothionein (MT)-like protein fraction and was trichloroacetic acid (TCA) soluble. In contrast, that from the cytosol of TM-treated sheep was mostly in a high molecular weight fraction that was TCA insoluble. The chromatograms obtained from cytosol derived from frozen kidneys, or cytosol that had itself been frozen, contained a similar distribution of Cu, Zn, and Fe, but the peak heights were lower in the latter samples.

Subcellular distribution of copper in the kidneys of normal, copper-poisoned, and thiomolybdate-treated sheep.

Twenty-seven sheep given either copper (Cu) and/or tetrathiomolybdate (TM) were used to study the subcellular distribution of Cu within the kidney and to monitor the location of lysosomes within the subcellular fractions using acid phosphatase (AP) as a marker enzyme. Copper dosing alone increased the Cu content in the liver and the kidneys. The administration of intravenous TM prevented the development of chronic copper poisoning (CCP) in sheep, reduced the rate of accumulation of Cu in the liver of Cu-dosed animals, but increased the Cu content of kidneys in both the control and Cu-dosed sheep. The total amount of Cu that accumulated in the kidneys of sheep given TM appears to depend on several factors: a) liver Cu concentration, b) Cu intake, and c) dosage of TM. Thus, the highest Cu concentration was found in the kidneys of sheep that continued to receive Cu orally at the same time as they were given TM. The intracellular distribution of Cu and AP in the kidneys showed that in the control sheep given neither Cu or TM, the highest proportion of Cu was in the cytosol fraction, and the highest specific activity of AP was in the light mitochondrial (lysosomal) fraction. Dosing with Cu markedly increased the Cu concentration and greatly elevated the total activity of AP in the heavier fractions, i.e., the nuclear (N) and heavy mitochondrial (MH). Thus, the increase in Cu observed in the N and MH fractions was not caused by an accumulation of Cu by nuclei and mitochondria, but was due to an accumulation of Cu by lysosomes that sedimented with the heavier fractions. The intracellular distribution of Cu in the kidneys of TM-treated sheep was similar to that seen in Cu-loaded sheep. Although Cu accumulated readily in the kidneys of animals receiving TM, kidney function tests showed neither glomerular nor tubular functional impairment.

Effect of thiomolybdate and ammonium molybdate in pregnant guinea pigs and their offspring.

Groups of eight guinea pigs and their offspring were given drinking water containing molybdenum as ammonium molybdate (AM) or thiomolybdate (TM) throughout and subsequent to pregnancy. All adult females had oestrous cycles and conception rates were unaffected. Fetal death was common in groups given the high dose of TM. The concentration of copper in liver was reduced in all groups at all ages except for pups killed at birth from animals given AM. The concentration of molybdenum was elevated in liver and kidney of all groups and was statistically significant in the majority. The concentration in plasma of copper, molybdenum and copper insoluble in trichloroacetic acid was elevated in all groups. Superoxide dismutase activity was significantly reduced in dams and six-week-old pups in which TM administration commenced before mating. Histological damage occurred in the pancreas of animals given AM or TM. The effects on the fetus and pancreas were considered to result from copper deficiency rather than molybdenum toxicity.

Polioencephalomalacia (cerebrocortical necrosis) induced by experimental thiamine deficiency in lambs.

Lesions of polioencephalomalacia (PEM, cerebrocortical necrosis) have been demonstrated in pre-ruminant lambs fed on synthetic, thiamine-free milk. Thiamine deficiency in these lambs was confirmed by transketolase assay.

Induced thiamin deficiency in lambs.

By following a feeding regimen which consistently induced polioencephalomalacia in pre-ruminant lambs it was possible to study certain characteristics just before the terminal stage in polioencephalomalacia. There was always a marked deficit in erythrocyte precursors in bone marrow and this preceded any pathological changes in the brain. Erythrocyte transketolase activity decreased in control lambs, and decreased to an even greater extent in thiamin-deficient lambs. Glucose was the only substrate of those measured which was used by the brain, and its rate of use was not affected by thiamin deficiency. After a single intravenous injection of 35S-thiamin, the decrease of 35S in the plasma was consistent with its dispersal into two pools: the half-time of disappearance of 35S into the slowly equilibrating pool was less in thiamin-deficient lambs than in their controls. Characteristics which were not altered in thiamin deficiency were concentrations of calcium and magnesium in various regions of brain and concentrations of calcium, magnesium, sodium and potassium in plasma.

Involvement of the solid phase of rumen digesta in the interaction between copper, molybdenum and sulphur in sheep.

1. Merino sheep fed on a diet of chopped wheaten hay, chopped lucerne (Medicago sativa) hay and oat grain were the source of rumen contents for the study. The diet contained (mg/kg dry weight) 3.3 copper, 0.24 molybdenum and 2.8 sulphur. The effects of adding between 5 and 25 mg Mo/kg as ammonium molybdate (AM) or tetrathiomolybdate (TTM) on the distribution and forms of Cu and Mo in rumen contents were investigated in vivo and in vitro. 2. Approximately 88% of the Cu and 94% of the Mo in rumen contents were associated with the solid phase. When AM or TTM was added to rumen contents in vivo or in vitro the proportion of these elements in the solid phase was increased at the expense of the fluid phase. 3. The addition of AM and TTM to rumen contents also decreased the proportion of Cu that was soluble in trichloroacetic acid (50 g/l; TCA) and increased the proportion of Cu that was not extractable by sequential treatment with TCA and neutral detergent. 4. Column chromatography of neutral-detergent extracts of rumen contents revealed that TTM treatment caused Cu to be strongly bound to proteins of high molecular weight. 5. Addition of sulphide to rumen contents did not result in significant changes in the distribution of Cu between the fluid and solid phases, or in the solubility of Cu in TCA. 6. It is postulated that constant removal of TTM from the fluid phase via reaction with proteins and other macromolecules in the solid phase results in greater formation of TTM in vivo than would be expected from solution chemistry. The molybdo-proteins so formed are strong chelators of Cu and may be the agents responsible for the decrease in Cu absorption in animals that consume diets containing high concentrations of Mo.

Apple software for analysis of the size of restriction fragments.

A software package that has been specifically written for an Apple II microcomputer is described. The program, DNAFRAG, is used for the determination of the sizes of the DNA restriction fragments that are observed in electrophoresis experiments and is based on a program previously reported by Duggleby et al., 1981. DNAFRAG allows the operator to use standard sets of fragment sizes that are stored on a floppy disk and to add new sets of standards to that disk. In addition, the program has an improved capacity to manipulate data once it has been entered into the program.

The effect of molybdenum on the conversion of sulphate to sulphide and microbial-protein-sulphur in the rumen of sheep.

1. [35S]sulphate was used to measure the apparent turnover of sulphate, sulphide and microbial-protein-S in the rumen contents of four sheep that were intraruminally infused with 10 g sodium sulphate/d alone, or together with 126 mg sodium molybdate (50 mg molybdenum). 2. Infusion of molybdate increased the concentration of sulphate in rumen fluid from 2.2 to 7.2 mug S/ml and decreased the rate of reduction of sulphate to sulphide by 50%. Although the rate of sulphide production was slower, the concentration of sulphide in the rumen is suggested to explain these changes. 3. In animals that were not infused with molybdate, only one-third of the S (3.0 g/d) that passed through the sulphate pool in rumen fluid was converted to sulphide, decreasing to one-sixth when molybdate was infused. 4. The turnover of S amino acids in microbial protein was not significantly affected by molybdate. Only 52-57% of the S amino acid content of microbial protein was synthesized de novo by way of the sulphide pool.

Folic acid metabolism in vitamin B12-deficient sheep. Effects of injected methionine on methotrexate transport and the activity of enzymes associated with folate metabolism in liver.

1. A study was made of the effects of injected l-methionine on the activity of several enzymes of folate metabolism, and on the transport of methotrexate in liver preparations from vitamin B(12)-deficient ewes and their pair-fed controls receiving vitamin B(12). 2. The activities of dihydrofolate reductase (EC 1.5.1.3) and 5-methyltetrahydrofolate-homocysteine transmethylase were significantly decreased in the liver of vitamin B(12)-deficient animals, but were unaffected by l-methionine. 3. The concentration of S-adenosyl-l-methionine in the liver of deficient animals was about one-half of that in normal animals, and was restored to normal by either vitamin B(12) or l-methionine. 4. Methylenetetrahydrofolate reductase (EC 1.1.1.68) from sheep liver was inhibited by S-adenosyl-l-methionine in vitro, but not by concentrations of S-adenosyl-l-methionine found in the liver of vitamin B(12)-deficient animals after injection of physiological amounts of l-methionine. 5. Pteroylpolyglutamate synthetase activity was significantly increased in the liver of vitamin B(12)-deficient animals, and was decreased by intravenous injections of l-methionine. 6. l-Methionine injections increased the initial rate of uptake of methotrexate in liver slices from deficient animals and acted synergistically with vitamin B(12) to increase the quantity taken up in 40min. The failure of folate metabolism in vitamin B(12) deficiency can be satisfactorily explained if l-methionine similarly affects the membrane transport of naturally occurring folates. 7. Further details of the results have been deposited as Supplementary Publication SUP 50028 (4 pages) at the British Library (Lending Division), (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.

The synthesis of pteroylpolyglutamates by sheep liver enzymes in vitro.

1. Sephadex G-15 was used to separate pteroylmonoglutamates from corresponding polyglutamate derivatives. 2. Pteroylpolyglutamates were formed when 5-formyltetrahydro[2-(14)C]pteroylglutamic acid, 5-[methyl-(14)C]tetrahydropteroylglutamic acid or tetrahydro[2-(14)C]pteroylglutamic acid was incubated at pH8.4 with ATP, MgCl(2), KCl, l-glutamic acid and sheep liver cytosol. The gamma-glutamyl side chain appeared to be lengthened by the stepwise addition of single glutamate moieties. 3. The subcellular distribution of pteroylpolyglutamates paralleled that of pteroylpolyglutamate synthetase activity, and followed the order cytosol>;nuclear' fraction>microsomal fraction>mitochondria.

Factors affecting the activity of cellulases isolated from the rumen digesta of sheep.

Sodium phosphate buffer was used to extract cellulases from the plant solids fraction of rumen contents. The mixed cellulase preparation had maximal activity at pH 6.9 and 49 degrees C. The V(max) and the apparent K(m) for wheaten hay cellulose were 19.8 glucose units/min and 6.35 mg/ml, respectively, and for microcrystalline cellulose (Sigmacell) at the same enzyme concentration, they were 33 glucose units/min and 27.5 mg/ml, respectively. For these assays a glucose unit was defined as nanomoles of glucose plus twice the nanomoles of cellobiose. Consideration of thermodynamic and kinetic data suggested that the hydrolysis of a relatively labile arabino-xylan comprising 3% of the wheaten hay cellulose was dependent on prior removal of the protecting beta-1,4-glucose chains at the outer surface of the cellulose preparation. Sequential removal of structural polysaccharides from the plant cell wall rendered the latter more susceptible to cellulase activity. Cellulase activity was stimulated by increasing the concentration of phosphate from 5 to 50 mM. The stimulation was magnified in the presence of cell-free rumen fluid. Cellulase activity was not stimulated by calcium, magnesium, iron, zinc, manganese, copper, or cobalt ions and was unaffected by the chelators ethylenediaminetetraacetic acid and ethyleneglycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid. O-phenanthroline inhibited activity by 30 to 50%, but this may have been due to nonchelate properties. Anaerobic conditions or thiol protective agents were not essential for either the activity or stability of the cellulases during assay. An ultrafiltrable inhibitor of cellulase activity was detected in cell-free rumen fluid.

Thiamin deficiency in the lamb: changes in thiamin phosphate esters in the brain.

Concentration of thiamin (unphosphorylated), thiamin monophosphate (TMP), thiamin diphosphate (TDP), and thiamin triphosphate (TTP) were measured in three regions of the brain of seven pairs of lambs. The lambs were maintained on a thiamin-free synthetic diet for 2, 3, or 4 weeks. Controls were pair-fed and supplemented with thiamin. The three brain regions were: (1) dorso-lateral aspect of the cortex [common site for lesions of polioencephalomalacia (PEM)]; (2) pyriform lobe of the cortex (no PEM lesions are found here); (3) white matter of the internal capsule (no PEM lesions found here). The concentration of TTP ina ll three sections of brain was maintained at control values for up to 4 weeks on the thiamin-deficient diet. TDP concentration decreased to 22% of control values in both regions of grey matter after 4 weeks on the diet. Unphosphorylated thiamin and TMP decreased to a smaller extent than TDP.

Skeletal-muscle alpha-glucosidases in bovine generalized glycogenosis type II.

The skeletal muscle of cattle suffering from generalized glycogenosis type II was shown to lack acid alpha-glucosidase (EC 3.2.1.3) activity. Furthermore, there was no evidence of enzymically inactive proteins that cross-reacted with antibodies raised against acid alpha-glucosidase from the muscle of normal animals.

Intravenous administration of thiomolybdate for the prevention and treatment of chronic copper poisoning in sheep.

1. Twenty-six sheep were used in experiments designed to test the effectiveness of ammonium tetrathiomolybdate in the prevention and treatment of chronic copper poisoning. 2. Intravenous injections of 100 mg ammonium tetrathiomolybdate twice weekly (a) prevented the occurrence of haemolytic crisis in sheep repeatedly dosed with copper sulphate and (b) minimized tissue damage and prevented further haemolytic crisis when given to sheep already in haemolysis. 3. Thiomolybdate prevented excessive deposition of Cu in the liver of sheep receiving orally large amounts of Cu and decreased liver Cu levels in sheep that were not given additional Cu. In the latter sheep, 50 mg ammonium tetrathiomolybdate given twice weekly did not produce histologically-detectable tissue damage even though liver and kidneys contained high levels of molybdenum, and kidneys contained elevated levels of Cu. 4. It is concluded that chronic Cu poisoning can be successfully prevented or treated by intravenous injection of appropriate doses of ammonium tetrathiomolybdate.

An investigation of the effects of intravenous administration of thiomolybdate on copper metabolism in chronic Cu-poisoned sheep.

1. Fourteen sheep were dosed repeatedly with a solution of copper sulphate (2 g/l) in order to induce chronic copper poisoning and four similar undosed animals acted as controls 2. Thiomolybdate (TM) was intravenously administered to all control sheep and to all except two of the test sheep. A variety of biochemical factors were studied before and after injections of TM. 3. The direct-reacting Cu, whole-blood Cu and plasma Cu concentrations were elevated in animals given TM injections and at the "haemolytic crisis" of untreated chronic Cu-poisoned animals. But most of the increased Cu observed on injecting TM was insoluble in trichloroacetic acid (TCA) and did not enter erythrocytes. The results indicate that uptake of Cu by erythrocytes is essential for haemolysis to occur and that for this to happen the Cu must be in a direct reacting, TCA-soluble form. 4. Increased amounts of Cu were excreted in the urine at haemolysis and at commencement of TM injections. High levels of direct-reacting Cu were found in plasma at these times. 5. Marked changes were not found in caeruloplasmin activity, packed cell volume or the osmotic fragility of erythrocytes except at haemolysis. TM injection s did no alter these factors in any of the sheep studied.

Folic acid metabolism in vitamin B12-deficient sheep. Effects of injected methionine on liver constituents associated with folate metabolism.

1. The effects of injected l-methionine (2g every second day for 28 days) on liver folates and other constituents of liver associated with folate metabolism were studied in vitamin B(12)-deficient ewes and their pair-fed controls receiving vitamin B(12). The dose rate of methionine used was sufficient to restore almost to normal the elevated excretion in the urine of formiminoglutamate in the deficient animals. 2. Liver folates active for Lactobacillus casei, Streptococcus faecalis R and Pediococcus cerevisiae were severely depressed in deficient livers and were partly restored by methionine. Analysis of the folates after ion-exchange chromatography showed that the major effect of methionine was to increase the concentrations of tetrahydrofolates and formyltetrahydrofolates. Methyltetrahydrofolates were also increased, but there was no effect of methionine on the small amounts of incompletely reduced folates present in deficient livers. The folates present were predominantly penta-, hexa- and hepta-glutamates whether or not animals received vitamin B(12) or methionine. 3. Concentrations of ATP, NAD(+), NADH and NADPH were lower in freeze-clamped liver from vitamin B(12)-deficient sheep than in liver from pair-fed, vitamin B(12)-treated sheep. These changes were not affected by methionine which was also without effect on the elevated K(+)/Na(+) ratios found in deficient livers. 4. The livers of vitamin B(12)-deficient animals contained lower concentrations of choline and higher concentrations of lipid than their pair-fed controls. These effects were reversed by methionine.

Infantile and late onset form of generalised glycogenosis type II in cattle.

A herd of cattle which produces calves with generalised glycogenosis type II has been established. Seven affected animals have been born and their disease status as indicated by a decreased acid alpha-glucosidase activity and excessive glycogen deposition in muscle, can be detected on the day of birth. Two animals have died of heart failure aged 3 and 5 months and have shown cardiomegally. Five animals were clinically normal until 9 months of age when they failed to maintain weight gain, showed muscle weakness and four were killed aged between 12 and 16 months after showing difficulty in rising. All affected animals had abnormal ECG tracings and had elevated levels of CK, LDH and HBDH in serum. Excessive amounts of glycogen were deposited in voluntary, cardiac and smooth muscle, and in cells of the nervous system. The muscles showed a vacuolar myopathy. Both the infantile and late onset forms of generalised glycogenosis type II are present in this herd of cattle. The condition appears to be controlled by a recessive allele at a single autosomal locus.