A new biosensor for specific determination of glucose or fructose using an oxidoreductase of Zymomonas mobilis

Cells of Zymomonas mobilis were permeabilized with toluene in order to utilize the enzymes, glucose-fructose oxidoreductase and gluconolactonase, inside the intact cells. Permeabilized cells were immobilized in a gelatin membrane, and a whole cell enzyme electrode was constructed by fixing the membrane on pH electrode. The biosensor developed was used for specific determination of glucose or fructose by detecting the production rate of hydrogen ion. Optimum conditions for biosensor response were pH 6.2 and temperature of 39 degrees C. The biosensor was highly specific and reproducible, and calibration curves for glucose and fructose were excellent, being linear up to 5 and 50 g/L, respectively.     

Cleavage of spermidine as the first step in deoxyhypusine synthesis. The role of NAD

The biosynthesis of deoxyhypusine (N-(4-aminobutyl)lysine) occurs by the transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in a precursor of eukaryotic translation initiation factor 4D (eIF-4D). Deoxyhypusine synthase, the enzyme that catalyzes this reaction, was purified approximately 700-fold from rat testis. The Km values for the substrates, spermidine, the eIF-4-D precursor protein, and NAD+, were estimated as approximately 1, 0.08, and 30 microM, respectively. After incubation of partially purified enzyme with [1,8-3H]spermidine, NAD+, and the eIF-4D precursor, equal amounts of radioactivity were found in free 1,3-diaminopropane and in protein-bound deoxyhypusine. However, when the protein substrate (eIF-4D precursor) was omitted, radioactivity was found in 1,3-diaminopropane and in delta 1-pyrroline in nearly equal quantities, providing evidence that the cleavage of spermidine occurs, albeit at a slower rate, in the absence of the eIF-4D precursor. That NAD+, which is required for this reaction, functions as the hydrogen acceptor was demonstrated by the fact that radioactivity from spermidine labeled with 3H at position 5 is found in NADH as well as in delta 1-pyrroline. Transfer of this hydrogen from spermidine to the re face of the nicotinamide ring of NAD+, as determined by the use of dehydrogenases of known stereospecificity, defines the first step of deoxyhypusine synthesis as a pro-R, or A, stereospecific dehydrogenation. Based on these findings, an enzyme mechanism involving imine intermediate formation is proposed.     

Prevention of Dahl salt-induced hypertension by chronic dietary tryptophan

Four-week-old inbred Dahl salt-sensitive (DS/JR) and Dahl salt-resistant (DR/JR) rats were placed on an 8% salt diet with or without a supplemental 2.5% tryptophan (Trp). Blood pressures were monitored for the next 5 weeks. Urine volumes and ion concentrations were measured during the 6th week. Blood pressures of DS/JR rats on control diets elevated rapidly and markedly, whereas pressures of DS/JR rats on the Trp-supplemented diet were not significantly elevated over those of DR/JR rats. Pressures of DR/JR rats were unaffected by Trp supplementation. Urinary sodium was significantly greater in DR/JR rats compared with DS/JR rats and was unaffected by Trp supplementation. This suggests that the antihypertensive effect of Trp was not at the level of the kidney. We conclude that dietary Trp blocks the development of hypertension in DS/JR rats maintained on a high salt diet.     

Specificity in the inactivation of enzymes by periodate-oxidized nucleotides

Periodate-oxidized ATP (oATP) inactivates the partial reaction of aminoacyl-tRNA synthetases in which amino acid is transferred to tRNA without altering the other partial reaction in which ATP is a substrate or a product. The inactivation has been shown to be nonspecific with regard to substituents on the dialdehyde and with regard to the enzymes susceptible to inactivation; oxidized GTP and oxidized uridine react as well as oATP with aminoacyl-tRNA synthetases and all three dialdehydes also inactivate rabbit muscle aldolase.     

Selective stimulation of the synthesis of an 80,000-dalton protein by calcium ionophores

Brief exposure of cultured chicken pectoralis muscle cells to ionomycin or A23187 selectively increases the rate of incorporation of [35S]methionine into an 80,000-dalton protein was also observed upon cell-free translation of poly(A)-enriched RNA isolated from ionomycin-treated, as compared with control, cultures. These observations suggest that ionomycin selectively increases the cellular concentration of mRNA, which codes for the 80,000-dalton protein. The effect is probably mediated through an increase in cytoplasmic [Ca2+] caused by the ionophore. A similar effect of ionomycin was observed in cultured fibroblasts, HeLa cells, mouse LSP cells, and monkey kidney CV1 cells.     

Light microscopic localization of labile calcium in hypertrophied chondrocytes of long bone with alizarin red S.

A method which localizes labile 5% ethylene glycol-bis-(beta-amino-ethyl ether)N-N'-tetraacetic acid-removable calcium in spherules within hypertrophied chondrocytes and in pericellular matrix using alizarin red S (ARS) is described. Fresh blocks of epiphyseal cartilage approximately 1 mm thick were immersed into 0.5-2% ARS solution containing 7% mounted on glass slides in 7% sucrose or in glycerol-gelatin. The stained tissue blocks were also dehydrated in acetone, cleared in xylene and mounted in Preservaslide. The ARS precipitated ionic calcium as red Ca-ARS salt which was birefringent in polarizing microscope, stable in water at pH 4-9 and in nonpolar organic solvent but soluble in polar solvents, especially in dimethyl sulfoxide. In contrast, ARS-stained insoluble calcium phosphate was stable even in dimethyl sulfoxide. Calcium in the hypertrophied chondrocytes, therefore, was thought to be present in a readily ionizable state instead of as insoluble calcium phosphate. Since addition of 7% sucrose retained as well as improved ARS localization of cellular calcium, the calcium was believed to be present in an osmotically sensitive, membrane-bound cytoplasmic compartment. The ARS-positive labile calcium in spherules which develop in the hypertrophied chondrocytes as well as in the pericellular matrix at the zone of provisional calcification suggested a preparatory stage in the process of cartilage calcification.     

Organic synthesis by quench reactions

The effects of chemical quench reactions on the formation of organic compounds at a water surface under simulated primordial earth conditions were investigated for the study of chemical evolution. A mixture of gaseous methane and ammonia over a water surface was exposed to an arc discharge between an electrode and the water surface. This discharge served as a source of dissociated, ionized and excited atomic and molecular species. Various organic molecules were formed in the gaseous, aqueous, and solid states by a subsequent quenching of these reactive species on the water surface. The effects of these water-surface quench reactions were assessed by comparing the amounts of synthesized molecules to the amounts which formed during the discharge of an arc above the water level. The results showed that: (1) the water-surface quench reaction permitted faster rates of formation of an insoluble solid and (2) the quench discharge yielded twice as much amino acids and 17 times more insoluble solids by weight than the other discharge. The highest yield of amino acids with the quench reaction was 9×10⁷ molecules per erg of input energy. These observations indicate that quench reactions on the oceans, rain, and clouds that would have followed excitation by lightning and shock waves may have played an important role in the prebiotic milieu. Furthermore, the possibility exists that quench reactions can be exploited for the synthesis of organic compounds on a larger scale from simple startng materials.