Further characterization of type 2 and type 3 chain blood group A glycosphingolipids from human erythrocyte membranes

Blood group A glycosphingolipids with slow chromatographic mobilities have been separated systematically with an improved chromatographic procedure, and their structures have been analyzed by application of a panel of monoclonal antibodies defining A determinants carried by type 1, type 2, type 3, and type 4 carbohydrate chains as well as by 1H NMR spectroscopy and methylation analysis. Of several A-active fractions, previously termed Aa, Ab, Ac, and Ad, in decreasing order of thin-layer chromatographic mobility, the third fraction (Ac) was characterized as containing one type 3 chain A component and one type 2 chain A component without branching, which have been termed type 3 chain Ab and nor-Ac, respectively. (Formula: see text). The major component present in the fourth A-active fraction (Ad) was isolated and characterized as a branched type 2 chain glycolipid formerly termed Ac. The major component in the fifth A-active fraction (Ae) was identified as a branched type 2 chain A previously termed Ad. The structures of Ac (n = 1) and Ad (n = 2) are (Formula: see text).     

Membrane electrical parameters in turtle bladder measured using impedance-analysis techniques

Summary Equivalent-circuit impedance analysis experiments were performed on the urinary bladders of freshwater turtles in order to quantify membrane ionic conductances and areas, and to investigate how changes in these parameters are associated with changes in the rate of proton secretion in this tissue. In all experiments, sodium reabsorption was inhibited thereby unmasking the electrogenic proton secretion process. We report the following: (1) transepithelial impedance is represented exceptionally well by a simple equivalent-circuit model, which results in estimates of the apical and basolateral membrane ionic conductances and capacitances; (2) when sodium transport is inhibited with mucosal amiloride and serosal ouabain, the apical and basolateral membrane conductances and capacitances exhibit a continual decline with time; (3) this decline in the membrane parameters is most likely caused by subtle time-dependent changes in cell volume, resulting in changes in the areas of the apical and basolateral membranes; (4) stable membrane parameters are obtained if the tissue is not treated with ouabain, and if the oncotic pressure of the serosal solution is increased by the addition of 2% albumin; (5) inhibition of proton secretion using acetazolamide in CO₂ and HCO ₃ ^– -free bathing solutions results in a decrease in the area of the apical membrane, with no significant change in its specific conductance; (6) stimulation of proton transport with CO₂ and HCO ₃ ^– -containing serosal solution results in an increase in the apical membrane area and specific conductance. These results show that our methods can be used to measure changes in the membrane electrophysiological parameters that are related to changes in the rate of proton transport. Notably, they can be used to quantify in the live tissue, changes in membrane area resulting from changes in the net rates of endocytosis and exocytosis which are postulated to be intimately involved in the regulation of proton transport.     

Response of winter chemical defense in Alaska paper birch and green alder to manipulation of plant carbon/nutrient balance

Summary Plant carbon/nutrient balance has been implicated as an important factor in plant defensive chemistry and palatability to herbivores. We tested this hypothesis by fertilizing juvenile growth form Alaska paper birch and green alder with N, P and N-plus-P in a balanced 2x2 factorial experiment. Additionally, we shaded unfertilized plants of both species. Fertilization with N and N-plus-P increased growth of Alaska paper birch, reduced the concentration of papyriferic acid in internodes and increased the palatability of birch twigs to snowshoe hares. Shading decreased birch growth, decreased the concentration of papyriferic acid in internodes and increased twig palatability. These results indicate that the defensive chemistry and palatability of winter-dormant juvenile Alaska paper birch are sensitive to soil fertility and shade. Conversely the defensive chemistry and palatability of green alder twigs to snowshoe hares were not significantly affected by soil fertility or shade. The greater sensitivity of Alaska paper birch defensive chemistry and palatability to snowshoe hares in comparison to green alder is in agreement with the hypothesis that early successional woody plants that are adapted to high resource availability are more plastic in their chemical responses to the physical environment than are species from less favorable environments.     

The crystal structure of cystathionine gamma-synthase from Nicotiana tabacum reveals its substrate and reaction specificity.

Cystathionine gamma-synthase catalyses the committed step of de novo methionine biosynthesis in micro-organisms and plants, making the enzyme an attractive target for the design of new antibiotics and herbicides. The crystal structure of cystathionine gamma-synthase from Nicotiana tabacum has been solved by Patterson search techniques using the structure of Escherichia coli cystathionine gamma-synthase. The model was refined at 2.9 A resolution to a crystallographic R -factor of 20.1 % (Rfree25.0 %). The physiological substrates of the enzyme, L-homoserine phosphate and L-cysteine, were modelled into the unliganded structure. These complexes support the proposed ping-pong mechanism for catalysis and illustrate the dissimilar substrate specificities of bacterial and plant cystathionine gamma-synthases on a molecular level. The main difference arises from the binding modes of the distal substrate groups (O -acetyl/succinyl versusO -phosphate). Central in fixing the distal phosphate of the plant CGS substrate is an exposed lysine residue that is strictly conserved in plant cystathionine gamma-synthases whereas bacterial enzymes carry a glycine residue at this position. General insight regarding the reaction specificity of transsulphuration enzymes is gained by the comparison to cystathionine beta-lyase from E. coli, indicating the mechanistic importance of a second substrate binding site for L-cysteine which leads to different chemical reaction types.