Self-inactivation of an erythrocyte NAD glycohydrolase

Summary NAD glycohydrolase activity was studied using bovine erythrocytes, erythrocyte ghosts and partially purified enzyme preparations. During catalysis the enzyme becomes irreversibly inactivated in a process related to substrate turnover. Self-inactivation was observed with intact cells, ghosts and solubilized enzyme and could be demonstrated with NAD, NADP and nicotinamide 1,N⁶ ethenoadenine dinucleotide as substrates. Thionicotinamide adenine dinucleotide and NADH, which are not substrates for the enzyme, do not inactivate but are reversible substrate-competitive inhibitors. Added thiols had no effect on enzyme self-inactivation. Of the reaction products, added nicotinamide partially protected the enzyme while added ADPR had no effect. Thermodynamic parameters calculated from Arrhenius plots for rate constants of self-inactivation indicate a large negative S for transition state formation suggesting a process other than extensive denaturation. Erythrocyte ghost NADases from several other mammalian sources have been demonstrated to undergo a self-inactivation similar to that observed with the bovine enzyme.This work was supported by Research Grant PCM 76-05839 from the National Science Foundation.     

Superoxide Dismutases of Escherichia coli: Intracellular Localization and Functions

Escherichia coli B contains two superoxide dismutases which differ with respect to their localization within the cell, the nature of their prosthetic metals, their responses to changes in (p)O(2), and their functions. One of these enzymes, which was liberated from the cells by osmotic shock and which was therefore presumed to be localized in the periplasmic space, is an iron-containing superoxide dismutase. The amount of this iron enzyme did not vary in response to changes in (p)O(2) during growth. In contrast, the other superoxide dismutase was not solubilized by osmotic shock, was a mangano-protein, and was found in greater amounts in cells which had been grown at high (p)O(2). E. coli, which had low levels of the iron-enzyme and high levels of the mangano-enzyme, as a consequence of growth in iron-deficient aerated medium, was killed by exposure to an exogenous flux of O(2) (-) which was generated either photochemically or enzymatically. The addition of bovine superoxide dismutase to the suspending medium protected these cells against this stress. On the other hand, E. coli, which had high levels of the iron-enzyme and low levels of the mangano-enzyme, as a consequence of growth in iron-rich anaerobic medium, was resistant to exogeneous O(2) (-). On the basis of these and of previously reported results (4a, Yost, F. J. and I. Fridovich, J. Biol. Chem., 1973, in press), it appears that the iron superoxide dismutase, of the periplasmic space, serves as a defense against exogenous O(2) (-), whereas the mangano-superoxide dismutase, in the matrix of these cells, serves to counter the toxicity of endogenous O(2) (-).     

<Emphasis Type="Italic">Miscanthus × Giganteus</Emphasis> Growth and Nutrient Export on 22 Producer Fields

On-farm assessments of Miscanthus × giganteus growth and nutrient export across a wide range of management and environmental conditions are needed to determine and model how this crop performs and where it should be placed on the landscape. Therefore, Miscanthus growth and nutrient concentration and nutrient export at harvest were monitored during 2014 and 2015 at several landscape positions within 22 commercial production fields in central and southwestern Missouri and northeast Arkansas. Miscanthus shoot density and/or yield were best when it was grown: (i) following pasture converted to annual row crops or following row crops, (ii) on soils with colluvium parent material, (iii) on north-facing backslopes or footslopes, (iv) on soils with medium to fine texture, and (v) on well-drained/high runoff/low available water soils. Factors influencing nutrient concentrations varied by nutrient, but all concentrations consistently decreased as stands matured and most were more influenced by weather than were yield or nutrient export. Most effects on nutrient export were similar to effects on yield, but some nutrient exports were also influenced by manure history and weather conditions. Overall, cropping history prior to Miscanthus, landscape position, and soil properties such as parent material, soil textural class, and drainage class had the largest influence on Miscanthus growth and nutrient concentrations and exports. Weather conditions and inferior soils did not strongly influence Miscanthus production, but excessive soil moisture caused by various soil and weather factors often limited its growth. Thus, Miscanthus may be especially well-suited following annual crops on erosion-prone soils that drain well and have slope. These results will assist with the strategic cultivation of Miscanthus on Midwest landscapes.     

Calmodulin-binding proteins: visualization by 125I-calmodulin overlay on blots quenched with Tween 20 or bovine serum albumin and poly(ethylene oxide)

To streamline detection of calmodulin-binding proteins, blotting techniques for the electrophoretic transfer of proteins onto nitrocellulose filters, followed by overlay with 125I-calmodulin, have been adapted. Autoradiography of the 125I-calmodulin-labeled blots allows the identification and quantitation of proteins that possess affinity for calmodulin. Five protocols for suppressing nonspecific binding and for enhancing specific interactions of 125I-calmodulin with electrophoretically separated proteins were investigated. Tween 20 and bovine serum albumin alone, as well as combinations of bovine serum albumin and poly(ethylene oxide) or hemoglobin and gelatin, were evaluated as quenching and enhancing agents. Tween 20 proved highly effective for quenching nonspecific binding and for enhancing specific 125I-calmodulin binding of a 61,000-Mr rat brain protein, which was only faintly observed on blots quenched with proteins alone. However, Tween 20 dissociated 50% of 68,000-Mr proteins and 80% of 21,000-Mr 125I-labeled protein standards from the nitrocellulose filter. An alternative, the combination of bovine serum albumin followed by incubation with 15,000- to 20,000-Mr poly(ethylene oxide), proved satisfactory for the recovery of 61,000-Mr calmodulin-binding activity and for the detection of calmodulin-binding peptides (50,000 to 14,000 Mr) produced by limited proteolysis of rat brain 51,000-Mr calmodulin-binding protein. These blotting procedures for detection of calmodulin-binding proteins are compatible with a variety of one-dimensional and two-dimensional electrophoresis systems, including a two-dimensional electrophoresis system utilizing urea and sodium dodecyl sulfate in the first dimension and nonurea sodium dodecyl sulfate electrophoresis in the second, a system which proved useful for resolving calmodulin-binding proteins displaying anomalous electrophoretic migration in the presence of urea.     

Growth inhibition of Streptococcus mutans and Leuconostoc mesenteroides by sodium fluoride and ionic tin.

Sodium fluoride caused inhibition of growth rate and growth levels of Streptococcus mutans with glucose as the primary energy and carbon source. Stannous fluoride increased growth lag nad caused a much greater inhibition of growth rate than did sodium fluoride. Neither compound was found to be bactericidal when culture viability was measured after 6 days of incubation. Leuconostoc mesenteroides, which lacks a phosphotransferase system for sugar transport, showed less inhibition of growth rate with both inhibitors than did S. mutans, which possesses a phosphotransferase system. Metabolism of glucose or lactose which requires enolase activity shoed sodium fluoride inhibition, whereas metabolism of arginine or pyruvate does not involve enolase activity and showed no inhibition of growth.     

A flavonoid mutant of barley (Hordeum vulgare L.) exhibits increased sensitivity to UV-B radiation in the primary leaf

The aim of the present investigation was to define the role of soluble flavonoids as UV-B protectants in the primary leaf of barley (Hordeum vulgare L.). For this purpose we used a mutant line (Ant 287) from the Carlsberg collection of proanthocyanidin-free barley containing only 7% of total extractable flavonoids in the primary leaf as compared to the mother variety (Hiege 550/75). Seven-day-old leaves from plants grown under high visible light with or without supplementary UV-B radiation were used for the determination of UV-B sensitivity. UV-B-induced changes were assessed from parameters of chlorophyll fluorescence of photosystem II, including initial and maximum fluorescence, apparent quantum yield, and photochemical and non-photochemical quenching. A quartz fibre-optic microprobe was used to evaluate the amount of potentially harmful UV-B (310 nm radiation) penetrating into the leaf as a direct consequence of flavonoid deficiency. Our data indicate an essential role of flavonoids in UV-B protection of barley primary leaves. In leaves of the mutant line grown under supplementary UV-B, an increase in 310nm radiation in the mesophyll and a strong decrease in the quantum yield of photosynthesis were observed as compared to the corresponding mother variety. Primary leaves of liege responded to supplementary UV-B radiation with a 30% increase in the major flavonoid saponarin and a 500% increase in the minor compound lutonarin. This is assumed to be an efficient protective response since no changes in variable chlorophyll fluorescence were apparent. In addition, a further reduction in UV-B penetration into the mesophyll was recorded in these leaves.