William W. Wells

Improved rapidity and precision in the determination of brain 2′,3′-cyclic nucleotide 3′-phosphohydrolase☆

Abstract A rapid and precise method for the determination of brain 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) activity has been developed. Total brain homogenates were treated with deoxycholate, and CNP activity was measured as inorganic phosphate (phosphomolybdic acid, 410 nm) released from the product, 2′-AMP, by alkaline phosphatase. Measurements were carried out under optimal conditions of temperature (30°C) and pH (6.2) using the whole brain of the rat, chicken, and quaking mouse. The entire assay was applicable to multiple samples and could be completed in less than 1 hr.

COPPER DEFICIENCY IN THE DEVELOPING RAT BRAIN: A POSSIBLE MODEL FOR MENKES’ STEELY‐HAIR DISEASE

Abstract— In comparison to controls, copper‐deficient suckling rats showed an appreciable decrease in body growth, a slight decrease in whole brain and cerebellar growth, and a highly significant decrease in myelination based on the activity of cerebellar 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase—a myelin enriched protein. Specific effects of a fivefold reduction in the copper content of brain were seen in a drastic decrease in cerebellar cytochrome c oxidase and smaller but significant drops in cerebellar super‐oxide dismutase and brain norepinephrine concn. These observations are discussed with respect to the neuropathology and biochemistry of Menkes’ steely‐hair disease, a sex‐linked recessive disorder in humans characterized by copper deficiency.

Biosynthesis of myo-inositol in rat mammary gland. Isolation and properties of the enzymes☆

Abstract myo-Inositol 1-phosphate synthase (EC 5.5.1.4) and 1 l -myo-inositol 1-phosphatase (EC 3.1.3.25) were isolated and partially purified from lactating rat mammary gland. The synthase had an apparent molecular weight of 290,000 as determined by gel filtration; its pH optimum was 7.2, and the Km for glucose 6-phosphate was 0.5 m m . No other compound could act as a substrate, but the synthase was inhibited 100% by d -gluconic acid 6-phosphate, 54% by d -fructose 6-phosphate, 31.8% by d -galactose 6-phosphate, and 29.6% by d -mannose 6-phosphate each at 5m m . Activity was stimulated 2-fold by the addition of 1 m m NAD+ and 40% by 14 m m ammonium ions, whereas it was inhibited by 30% in the presence of 1 m m NADH and by 93.6% when incubated with 1 m m p-mercuribenzoate. Reagents which interfere with Schiff-base formation, pyridoxal 5′-phosphate and trinitrobenzenesulfonate, inhibited the enzyme, but EDTA was without effect. The 1 l -myo-inositol 1-phosphatase from rat mammary tissue appears to exist in a native tetrameric form of 210,000 as determined by gel filtration which, upon heating at 70 °C for 15 min, is converted into a stable monomer of approximately 52,000. Mg2+ (1.5 m m ) was an absolute requirement for activity though Mn2+ gave 17% of the activity provided by Mg2+. Sodium, potassium, or ammonium ions were stimulatory, but lithium ions were strongly inhibitory. 1 l -myo-Inositol 1-phosphatase specifically cleaved 1 l -myo-inositol 1-phosphate and was 60% as active toward l -α-glycerol phosphate with only minor activity toward other phosphorylated compounds. The pH optimum was 8.0 and the Km for 1 l -myo-inositol 1-phosphate was 0.8 m m .

COPPER DEFICIENCY IN THE DEVELOPING RAT BRAIN: EVIDENCE FOR ABNORMAL MITOCHONDRIA

Abstract— Copper deficiency was produced in developing rats by feeding a low copper diet to rats during gestation and lactation and providing the offspring the same diet. The progeny developed similar to those of an earlier model based on preconception depletion followed by marginal supplementation during gestation. Copper levels were greatly reduced in the brain, iron levels were slightly depressed, and no differences in zinc content were found. Electron microscopic examination of brain tissue revealed the presence of enlarge mitochondria from copper‐deficient animals. Isolated mitochondria from copper‐deficient rats showed a 30% reduction in the rate of both succinate and glutamate oxidation, and for glutamate, the respiratory control ratio (RCR) was decreased by 60%. Difference spectra displayed a four‐fold reduction in cytochrome a+a3 and slight increases in cytochrome b, c1 and c. Enzyme analysis of isolated mitochondria revealed a five‐fold decrease in cytochrome oxidase, slight increases in succinic dehydrogenase and fumarase, and small decreases in hexokinase and monoamine oxidase. No difference in peroxidation of brain lipids was evident. Determination of metabolites from fast frozen tissue suggested that the copper‐deficient brain was in a more reduced state based on a doubling of both the lactate/pyruvate and α‐glycerol‐P/dihydroxyacetone‐P ratios. Creatine‐P, ATP, and ADP levels were not different.

ALPHA -LIPOIC ACID DEPENDENT REGENERATION OF ASCORBIC ACID FROM DEHYDROASCORBIC ACID IN RAT LIVER MITOCHONDRIA

Rat liver mitochondria were examined for their ability to reduce dehydroascorbic acid to ascorbic acid in an α-lipoic acid dependent or independent manner. The a-lipoic acid dependent reduction was stimulated by factors that increased the NADH dependent reduction of α-lipoic acid to dihydrolipoic acid in coupled reactions. Optimal conditions for dehydroascorbic acid reduction to ascorbic acid were achieved in the presence of pyruvate, α-lipoic acid, and ATP. Electron transport inhibitors, rotenone and antimycin A, further enhanced the dehydroascorbic acid reduction. The reactions were strongly inhibited by 1 mM iodoacetamide or sodium arsenite. Mitoplasts were qualitatively similar to intact mitochondria in dehydroascorbate reduction activity. Pyruvate dehydrogenase and α-ketoglutarate dehydrogenase reduced dehydroascorbic acid to ascorbic acid in an α-lipoic acid, coenzyme A, and pyruvate or α-ketoglutarate dependent fashion. Dehydroascorbic acid was also catalytically reduced to ascorbic acid by purified lipoamide dehydrogenase in an α-lipoic acid (K0.5=1.4±0.8 mM) and lipoamide (K0.5=0.9±0.3 mM) dependent manner.

[7] Gas chromatography of carbohydrates

Publisher Summary This chapter discusses the preparation of gas chromatography of carbohydrates. Analyses of carbohydrates by gas–liquid partition chromatography are carried out with volatile derivatives of the substances under investigation. Extensive studies are made of the chromatographic behavior of derivatives such as O -methyl ethers, O -acetyl esters, O -trimethylsilyl (TMS) ethers, and various acetals and ketals. The separations can be obtained with any of these modified forms of carbohydrates, the volatilities of which are generally sufficient for gas chromatography under a wide variety of operating conditions. The reaction yielding a derivative must be rapid and quantitative with any carbohydrate; preferably it should be carried out at room temperature, and must be suitable for use over a wide range of concentration of the starting material. Nonpolar derivatives are usually less reactive than polar ones and are preferred for quantitative determinations. Packed columns or open tubular columns may be operated isothermally or with temperature programming. A short packed column containing a low percentage of dimethylsilicone gum (SE-30) is the most useful, all-purpose column for the determination of carbohydrates by gas chromatography.

[4] Glutathione : Dehydroascorbate oxidoreductases

Publisher Summary This chapter focuses on the dehydroascorbate oxidoreductases of glutathione. Dehydro- L -ascorbic acid (DHA), the first chemically stable oxidation product of L -ascorbic acid (AAH 2 ), is generated in plant or animal cells from L -ascorbic acid by one- or two-electron oxidation reactions in which semidehydroascorbic acid (AAH . ) may or may not be a required intermediate. Superoxide is produced by respiration and by a number of enzymes, for example, xanthine oxidase, aldehyde oxidase, and others. Oxidation of many compounds including hydroquinone, flavins, thiols, catecholamines, dialuric acid, herbicides, paraquat, and CCl 4 produces O 2 - . Two mammalian DHA reductases have been identified. The cytoplasmic reductase, also known as thioltransferase or glutaredoxin, has been purified from numerous sources, including rat liver, pig liver, calf thymus, rabbit bone marrow, human placenta, and human erythrocytes. The DHA reductase properties of thioltransferase and protein disulfideisomerase have been reported. By using site-directed mutagenesis techniques, the essential amino acids at the catalytic center of porcine thioltransferase (dehydroascorbate reductase) are determined.

Ascorbic acid and cell survival of adriamycin resistant and sensitive MCF-7 breast tumor cells.

The ability of human cells to regenerate ascorbic acid from dehydroascorbate is partially dependent on the glutathione redox status of the cell and the relative activity of dehydroascorbate reductases. Mammalian dehydroascorbate reductase activity is associated with two proteins known as thioltransferase (glutaredoxin) and protein disulfide isomerase. We compared the specific activity of thioltransferase, protein disulfide isomerase, and other GSH-related enzymes in Adriamycin-resistant human breast tumor cells, MCF-7 ADRR, and Adriamycin-sensitive, MCF-7 WT, tumor cells. MCF-7 ADRR cells had higher activities of glutathione peroxidase (34.7 fold), nonseleno-glutathione peroxidase (glutathione S-transferases; 5.3 fold), thioredoxin (2.3 fold), and thioltransferase (4.0 fold) compared with the WT Adriamycin-sensitive cell line. Thioltransferase was detected in Western blots in extracts of ADRR MCF-7 cells but not in WT MCF-7 cells. alpha-Tocopherol in the membrane and cytosolic fractions was 2.8 and 3.0 fold higher, respectively, in Adriamycin-resistant compared with Adriamycin-sensitive cells. Supplementation of MCF-7 cells with L-ascorbic acid 2-phosphate (2 and 10 mM) had no effect on WT cell viability after 5 days incubation with up to 0.33 microM Adriamycin. In contrast, supplementation of ADRR MCF-7 cells with L-ascorbic acid 2-phosphate resulted in enhanced resistance up to 3.4 microM Adriamycin over a 5-day incubation. Both lines of MCF-7 cells demonstrated the ability to utilize ascorbic acid as the 2-phosphate derivative. After 48 h incubation with 8.6 microM Adriamycin, the resistant cells maintained normal viability and ascorbate-dehydroascorbate levels, whereas drug-sensitive cells had significantly lower ascorbate with a higher percent dehydroascorbate and increased cell death as judged by cell protein levels (52% of controls).

Studies on the developmental pattern of the enzymes converting glucose 6-phosphate to myo-inositol in the rat

Abstract The myo -inositol level of plasma was determined during pre- and postnatal development of the rat. Fetal concentrations exceeded those of maternal rats by nearly 10-fold. Immediately after birth, the myo -inositol level decreased but was maintained at values 3–4 times that of the lactating dams. The cyclitol content of rat milk was high and rose during lactation to a maximum of 1.6 m M . The biosynthesis of myo -inositol from glucose 6-phosphate is catalyzed by glucose 6-phosphate: l - myo -inositol-1-phosphate cyclase and l - myo -inositol-1-phosphate phosphatase. The activity of both enzymes was monitored in fetal and neonatal liver, maternal liver, placenta, and mammary gland. Results indicated that the fetal liver accounted for over 48% of the total carcass cyclase and 26% of the total carcass phosphatase activity. Developmental changes correlated well with the pattern of myo -inositol in fetal rat plasma. Similarly, the enzymes of the myo -inositol biosynthetic pathway increased in rat mammary gland in close agreement with the myo -inositol content of milk and diminished to prelactation activities within 24 hr after the onset of involution. The myo -inositol level of colostrum and milk of five human subjects was highest (2.8 m M ) before birth and decreased to 40% of that level 5 days postpartum, where it remained for at least 3 weeks. Even after 7 months of lactation, the milk of one subject contained 3–4-fold more myo -inositol than all commercial infant formulas analyzed.

Studies on the interaction of chick brain microsomal (Na+ + K+)-ATPase with copper.

Abstract Chick brain microsomal ATPase was strongly inhibited by Cu 2+ . (Na + + K + )-ATPase was more susceptible to low levels of Cu 2+ than Mg 2+ -ATPase. The inhibition of (Na + + K + )-ATPase could be partially protected from Cu 2+ in the presence of ATP in the preincubation period. When Cu 2+ (6 μM) was preincubated with the enzyme in the absence of ATP, only sulfhydryl-containing amino acids ( d -penicillamine and l -cysteine) could reverse the inhibition. At lower concentrations of Cu 2+ ( μ M), in the absence of ATP during preincubation, the inhibition could be completely reversed by the addition of 5 mM l -phenylalanine and l -histidine as well as d -penicillamine and l -cysteine. Kinetic analysis of action of Cu 2+ (1.0 μM) on (Na + + K + )-ATPase revealed that the inhibition was uncompetitive with respect to ATP. At a low concentration of K + (5 mM), V with Na + was markedly decreased in the presence of Cu 2+ and K m was about twice that of the control. However, at high K + concentration (20 mM), the K m for Na + was not affected. At both low (25 mM) and high (100 mM) Na + , Cu 2+ displayed non-competitive inhibition of the enzyme with respect to K + . On the basis of these data, we suggest that Cu 2+ at higher concentrations (> 6 μ M) inactivates the enzyme irreversibly, but that at lower concentrations ( μ M), Cu 2+ interacts reversibly with the enzyme.