Stereospecificity and requirements for activity of the respiratory NADH dehydrogenase of Escherichia coli

The respiratory NADH dehydrogenase of Escherichia coli has been further amplified in vivo by genetic methods. The enzyme, a single polypeptide of Mr 47 200 of known amino acid sequence [Young, I. G., Rogers, B. L., Campbell, H. D., Jaworowski, A., & Shaw, D. C. (1981) Eur. J. Biochem. 116, 165-170], constitutes 10-15% of the total protein in the amplified membranes. In situ in the membrane, the enzyme contains 1 mol of FAD/mol of subunit and has a specific NADH:ubiquinone-1 oxidoreductase activity of approximately 1100-1200 units mg-1 at 30 degrees C, pH 7.5. The purified enzyme contains phospholipid, which remains closely associated with it during gel filtration on Sephacryl S-300 in the presence of 0.1% (w/v) cholate at low ionic strength. Under these conditions the enzyme is extensively aggregated (apparent Mr greater than 10(6]. This procedure yielded enzyme with a specific activity of 980 units mg-1, similar to the value observed in the membrane. This preparation contained less than 0.1 mol of Fe/mol of enzyme, confirming that Fe is not involved in reduction of ubiquinone 1 catalyzed by the enzyme. Neutron activation analysis of purified enzyme has demonstrated the absence of 35 trace elements including Se, Zn, Mn, Co, W, Cu, and Fe. The enzyme polypeptide, prepared completely free of phospholipid, FAD, and ubiquinone by gel filtration in the presence of sodium dodecyl sulfate, has been reactivated. The results show that the only components necessary for catalysis of ubiquinone-1 reduction by NADH in this system are the enzyme polypeptide, FAD, and phospholipid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular and Extracellular Uptake of Cadmium by the Moss Rhytidiadelphus squarrosus

Intra- and extracellular Cd uptake were investigated in themoss Rhytidiadelphus squarrosus. Intracellular Cd uptake displayedMichaelis–Menten kinetics and showed that the moss hada high uptake rate and high affinity for Cd. Extracellular Cdbinding capacity was also high. The anion used had little effecton Cd uptake to either location but both intra- and extracellularCd uptake were reduced by equimolar concentrations of Ca, Mgor Zn, although inhibition was not wholly competitive. IntracellularCd uptake was not significantly affected by the supply of energygenerating reserves but, like extracellular Cd uptake, showedaltered characteristics following growth under controlled laboratoryconditions. The results are compared with data on Cd uptakeby the lichen genus Peltigera. Rhytidiadelphus squarrosus, moss, cadmium, intra- and extracellular uptake characteristics, kinetics     

Characterization of the specific binding of rat apolipoprotein E-deficient HDL to rat hepatic plasma membranes

We have used a preparation of rat liver plasma membranes to study the binding of rat apolipoprotein E-deficient HDL to rat liver. The membranes were found to bind HDL by a saturable process that was competed for by excess unlabeled HDL. The binding was temperature-dependent and was 85% receptor-mediated when incubated at 4, 22 and 37 degrees C. The affinity of the binding site for the HDL was consistent at all temperatures, while the maximum binding capacity increased at higher temperatures. The specific binding of HDL to the membranes did not require calcium and was independent of the concentration of NaCl in the media. The effect of varying the pH of the media on HDL binding was small, being 30% higher at pH 6.5 than at pH 9.0. Both rat HDL and human HDL3 were found to compete for the binding of rat HDL to the membranes, whereas rat VLDL remnants and human LDL did not compete. At 4 degrees C, complexes of dimyristoylphosphatidylcholine (DMPC) and apolipoproteins A-I, A-IV and the C apolipoproteins, but not apolipoprotein E, competed for HDL binding to the membranes. At 22 and 37 degrees C, all DMPC-apolipoprotein complexes competed to a similar extent, DMPC vesicles that contained no protein did not compete for the binding of HDL. These results suggest that the rat liver possesses a specific receptor for apolipoprotein E-deficient HDL that recognizes apolipoproteins A-I, A-IV and the C apolipoproteins as ligands.     

Human sperm surface mapping with lectins.

Ten fluorescein isothiocyanate (FITC)-linked lectins [Bauhimia purpurea, Concanavalin A, Dolichos biflorus (DBA), Griffonia simplicifolia I, Griffonia simplicifolia II, Maclura pomifera, Arachis hypogea (PNA), Glycine max, Ulex europaeus (UEA) and Triticum vulgaris agglutinin] have been used to study their binding features on the human ejaculate spermatozoa. Qualitative changes in the labeling pattern have been observed in unfixed and acetone-treated spermatozoa. Furthermore, ultrastructural localization of some of the colloidal gold-linked lectins, namely PNA, UEA and DBA, has been attempted to delineate the binding domains of the specific sugars on the sperm surface. It needs to be emphasized that flow-cytometric methods employed in our study, which provide quantitative slant to qualitative data, should be utilized to evaluate the functional status of the spermatozoa.     

Membrane-binding sites for acyl carrier protein in Escherichia coli

We report that membrane vesicles of Escherichia coli contain protein-binding sites for acyl carrier protein. Scatchard analysis of the binding indicates a dissociation constant around 0.35 micrometers and a maximum number of protein-binding sites around 50 pmol per mg of membrane protein. Binding is on the inner membrane while the outer membrane is devoid of binding sites. These results are consistent with the fact that some acyl carrier protein-dependent enzymes implicated in phospholipid- and membrane-derived oligosaccharide biosynthesis are localized in the cytoplasmic membrane.     

The nitrogen cycle in shallow water sediment systems of rice fields Part III: The influence of N-application on the yield of rice

Fertilizer application to rice-fields in the river-deltas in the Mediterranean area is a potential menace for wildlife protection, through eutrophication.Fertilizer use shows a trend of increasing rates of N application. A rate for N of 200 kg ha^–1 has become normal and a rate of 400 kg ha^–1 has already been recorded.Denitrification causes large losses of N with the result that more fertilizer is applied. This is especially true for the Camargue (S-France), where N is applied long before the rice (Aryza sativa) can take it up.Therefore we have tried to develop techniques which need the application of smaller amounts of N which are used more efficiently. In order to do this we tried to establish a N budget for rice-fields.Experiments were therefore set up in the field (plots of 550 m²) and in pots (2–3 l). Our results suggest that a late application of N (e.g. when the rice shows signs of N-deficiency by becoming yellowish), but at lower concentrations (70 kg ha^–1) can produce the same ultimate yield. The introduction of carp without any further input of N produced the same final yield.The N budget shows that 15±1.5 g m^–2 of N is needed for a normal crop. N losses due to denitrification may be as high as 12.2–13.6 g m^–2 of N. The input by irrigation water may provide up to about 20% of the input; N fixation is negligible. We estimate that 25–50% of the N missing in the budget comes from minderalization of the organic N pool in the soil. Denitrification may render part of this pool bio-available by oxidation. In sum, this work has revealed some surprising effects with potentially important consequences for farming practice and, in consequence, for conservation.