Direct evidence for separate binding sites for L-Glu and amino acid feedback inhibitors on unadenylylated glutamine synthetase from E. coli.

Glutamine synthetase from $\text{E. coli}$ is modulated by adenylylation of a tyrosine residue on each subunit of the dodecamer, as well as by feedback inhibition. With the stopped-flow fluorometric method, the binding constants for L-Glu, L-Ala, D-Val, and Gly to E_1.0—Mg, E⁷, in the absence or presence of ATP or ADP, and NH₃ were evaluated at pH 7.0, 15°. Strong synergistic effects between the amino acids and the nucleotide were observed. The fluorescence amplitude observed due to either simultaneous or sequential addition of 2 different amino acids to E or E·ATP indicate that L-Glu can bind to the enzyme simultaneously with L-Ala, Gly and D-Val; L-Ala can coexist with D-Val, Gly or D-Ala. NMR method also shows that L-Glu and L-Ala can bind simultaneously. Therefore, within our experimental conditions, the unadenylylated enzyme possesses allosteric site(s) for the amino acid inhibitors.     

A high-affinity calcium-stimulated ATPase activity in the hen oviduct shell gland

The hen oviduct shell gland is a highly active calcium-transporting epithelial tissue which is responsible for the mineralization of the egg shell. We have identified a calcium-stimulated ATPase present at high specific activity in membrane preparations from shell gland mucosal shavings. In the presence of optimal MgCl₂ (5 mm) and a Ca²⁺ buffer, ATP hydrolysis was stimulated by addition of low concentrations of free Ca²⁺ (K_0.5 ~0.4 μm); but not by similar concentrations of Mn²⁺, Zn²⁺, Co²⁺, or La²⁺. This stimulation was specific for ATP; there was little or no effect of Ca²⁺ on hydrolysis of ADP, AMP, GTP, ITP, or p-nitrophenyl phosphate. Calcium-stimulated ATPase activity was inhibited by chlorpromazine, trifluoperazine, and quercetin, as well as by sulfhydryl-blocking agents, but not by oligomycin or ouabain. No significant effect of calmodulin was observed. Finally, low concentrations of free Ca²⁺ (10 to 100 μm) in the presence or absence of Mg²⁺ stimulated transfer of ³²P from [γ-³²P]ATP to a 105,000 molecular weight shell gland membrane protein. This phosphoprotein was sensitive to hydrolysis by heating or by hydroxylamine treatment at acidic pH, and its formation was not inhibited by addition of K⁺. The specific activity of Ca²⁺-ATPase in total membrane preparations from laying hen shell gland ranged from 80 to 150 nmol/min/ mg protein, similar to or greater than levels found in purified plasma membrane fractions from a variety of tissues. No significant activity was found in membrane preparations from the magnum or isthmus regions of the oviduct, which are not involved in egg shell calcification. The characteristics of the Ca²⁺-ATPase, its high specific activity, and its preferential localization in the shell gland region of the oviduct suggest a role for an ATP-dependent calcium transport system in egg shell mineralization.     

Purification and properties of glutamine synthetase from the plant cytosol fraction of lupin nodules

Glutamine synthetase from the plant cytosol fraction of lupin nodules was purified 89-fold to apparent homogeneity. The enzyme molecule is composed of eight subunits of M_r 44,700 ± 10%. Kinetic analysis indicates that the reaction mechanism is sequential and there is some evidence that Mg-ATP is the first substrate to bind to the enzyme. Michaelis constants for each substrate using the ammonium-dependent biosynthetic reaction are as follows: ATP, 0.24 mm; l-glutamate, 4.0–4.2 mm; ammonium, 0.16 mm. Using an hydroxamate-forming biosynthetic reaction the K_m ATP is 1.1 mm but the K_m for l-glutamate is not altered. The effect of pH on the K_m for ammonium indicates that NH₃ rather than NH₄⁺ may be the true substrate. At 10 mm Mg²⁺, the pH optimum of the enzyme is between 7.5 and 8, but increasing Mg²⁺ concentrations produce progressively more acidic optima while lower Mg²⁺ concentrations raise the pH optimum. The rate-response curve for Mg²⁺ is sigmoidal becoming bell-shaped in alkaline conditions. The enzyme is inhibited by l-Asp (K_i, 1.4 mm) and less markedly by l-Gln and l-Asn. Inhibition by ADP and AMP is strong, both nucleotides exhibiting K_i values around 0.3 mM. Investigations of the probable physiological conditions within the nodule plant cytosol indicate that in situ glutamine synthetase has an activity greater than that required to support the efflux of amino acid nitrogen from the nodule. A possible role for glutamine synthetase in the control of nodule ammonium assimilation is suggested.     

Separation and quantification of the B6 vitamers in plasma and 4-pyridoxic acid in urine of adolescent girls by reversed-phase high-performance liquid chromatography

The vitamin B₆ status of seemingly healthy adolescent girls was determined using several accepted and proposed parameters in an effort to establish guidelines for status evaluation. High-performance liquid chromatography-derived plasma B₆ vitamers (pyridoxal phosphate, PLP; pyridoxine phosphate, PNP; pyridoxamine phosphate, PMP; pyridoxal, PL; pyridoxine, PN; and pyridoxamine, PM) and 4-pyridoxic acid (4-PA) concentrations and urinary 4-PA levels of 28 white adolescent females, 12–15 years, having radiomonitored plasma PLP concentrations and coenzyme stimulation of erythrocyte alanine aminotransferase activities indicative of adequate status were determined. Mean vitamin B₆ and protein intakes were 1.48 mg and 78.3 g. Ranges for plasma B₆ vitamer and 4-PA concentrations (nmol/1) were: PLP, 40.9–122.2; PNP, non-detectable (ND)—16.1; PMP, ND—8.1; PL, ND—15; PN, ND—21.9; PM, ND—17.8; and 4-PA, ND—55.7. PLP was the only vitamer found in plasma of all subjects. Urinary 4-PA concentrations ranged from 0.11 to 2.50 μmol/mmol of creatinine. B₆ vitamer values of these girls should be of use in the establishment of normal ranges for vitamin B₆ status parameters.     

Cultivation characteristics of immobilized Aspergillus oryzae for kojic acid production

Aspergillus oryzae in situ grown from spores entrapped in calcium alginate gel beads was used for the production of kojic acid. The immobilized cells in flask cultures produced kojic acid in a linear proportion while maintaining the stable metabolic activity for a prolonged production period. Kojic acid was accumulated up to a high concentration of 83 g/L, at which the kojic acid began to crystallize, and, thus, the culture had to be replaced with fresh media for the next batch culture. The overall productivities of two consecutive cultivations were higher than that of free mycelial fermentation. However, the production rate of kojic acid by the immobilized cells was suddenly decreased with the appearance of central cavernae inside the immobilized gel beads after 12 days of the third batch cultivation.     

Continuous hydrolysis of olive oil by immobilized lipase in organic solvent

Lipase (EC 3.1.1.3) from Candida rugosa was immobilized with DEAE-Sephadex A50, Sephadex G50, Sephadex LH-20, Amberlite IRA94, and Amberlite XAD-7. The enzye immobilized with DEAE-Sephadex A50 was found to be most effective for continuous hydrolysis of olive oil in isooctane. For the continuous reaction, 0.2 g of dry immobilized enzyme was swollen with predetermined amount of water, and packed in a glass column reactor. When the organic solvent (Isooctane) containing olive oil substrate was cocurrently fed with aqueous buffer, the two phases were evenly distributed throughout the packed bed without surfactant supplement or prior mixing of the two phases. A small amount of the surfactant (AOT) was used only in packing procedure, and no additional surfactant was necessary thereafter. Effects of initial water content of the swollen gel, buffer types, and strength were examined in the continuous reaction. Our results suggest that the operational half-life was affected by desorption of the bound enzyme. Under the conditions of 20% olive oil in isooctane and 25 mM triethanolamine buffer (pH 7.0), operational half life was 220 h at 30 degrees C. The reactor was also operable with n-hexane, but the operational stability of the immobilized enzyme in n-hexane was only half of that in isooctane. Our results indicate that various enzyme carrier having hydrophilic or amphiphilic properties could be used for two-phase continuous reaction in packed-bed column, reactor without any surfactant supply or prior dispersion of the two immiscible phases. (c) 1992 John Wiley & Sons, Inc.     

Nitrogen and carbon fixation byAnabaena sp. isolated from a rice paddy and grown under P and light limitations

Anabaena sp., isolated from a rice paddy, was investigated for its nitrogen fixation as measured by acetylene reduction activity (ARA) in P-limited continuous and light-limited semi-continuous cultures. Growth rate (μ) under P limitation was a function of cell P content (q _p). Both the photosynthetic capacity (P_max) and photosynthetic efficiency (α) increased with μ when expressed per cell, but not per unit chla. The ARA of steady-state cells under P limitation increased with μ and was linearly related to C-fixation rate. This was apparently a consequence of the control of C-fixation by P limitation. In light-limited cells, steady state ARA, both at the culture light intensity and in the dark, increased asymptotically with μ, but the activity in the dark was only about 51% of that in the light. When the light level of steady-state cells grown at a high in intensity was switched to a low level, ARA decreased exponentially with time. Dark ARA activity also showed a similar decline, but at much lower levels. Thus, ARA depended not only on light history, but also immediate photosynthesis. Steady-state ARA at the ambient intensity or in the dark showed a strong correlation with¹⁴C-fixation rate. ARA of light-limited cells showed the same light-saturation characteristics as their¹⁴C-fixation, with the same initial saturation intensity,I _k. The ratios of P_max to the maximum ARA (ARA_max), and α to the slope of ARA (α_ara) were identical. A comparison of gross to net photosynthesis and N₂ fixation suggested that there was little leakage or excretion of fixed C or N.     

Sorption of hydrophobic organic contaminants and trace metals on phytoplankton and implications for toxicity assessment

The partitioning of trace metals and hydrophobic organic contaminants to phytoplankton determines their toxicity as well as their fate and transport in aquatic ecosystems. Accurate impact assessments, therefore, depend on a good understanding of the factors regulating the sorption of these compounds to biotic particles. The accumulation of chlorinated organic compounds in phytoplankton is generally considered as being due solely to physical sorption, described by reversible equilibrium models based on Langmuir or Freundlich isotherms. On the other hand, the uptake of trace metals is a two phase process: a fast sorption component viewed as an ionexchange or a covalent bonding process with cell surface ligands, followed by an intracellular transport phase that is dependent on cellular metabolic activity. The uptake of inorganic and hydrophobic organic pollutants and their bioaccumulation are influenced in a complex manner by duration of exposure and cell density, by environmental factors such as pH, the concentration of cations and of dissolved and colloidal organic matter, as well as by phytoplankton physiological condition. High concentrations of H⁺, Ca²⁺, and Mg²⁺ ions will reduce trace metal sorption by directly competing for uptake sites on the cell's surface, whereas the presence of dissolved organic carbon such as natural and synthetic chelators and phytoplankton exudates will reduce the bioavailability of both trace metals and hydrophobic organic contaminants. Thus, the impact of toxic contaminants on phytoplankton may be determined as much by the factors influencing uptake and partitioning as by the potency of the toxicants and interspecies differences in sensitivity. Recommendations for improving toxicity assessments are presented.