Mechanism and dynamics of conformational ordering in xanthan polysaccharide

The thermally induced order-disorder transition of xanthan (extracellular bacterial polysaccharide from Xanthomonas campestris) has been investigated by optical rotation, differential scanning calorimetry, stopped-flow reaction kinetics and low-angle laser light scattering, and the results have been analysed in terms of Zimm -Bragg helix-coil transition theory. The reciprocal of the transition midpoint temperature (Tm) varies linearly with the logarithm of cation (K+) the salt dependence of Tm, is in agreement with Manning polyelectrolyte theory the ordered structure. The associated increase in cation binding, calculated from the salt dependence of tm, is in agreement with the Manning polyelectrolyte theory for one of the candidate structures from X-ray diffraction, a 5(1) single helix stabilized by packing of side-chains along the polymer backbone, but not for the alternative double-helix structure that has also been proposed. At each salt concentration, the two fundamental parameters of the Zimm -Bragg theory, s and sigma, were calculated. The equilibrium constant for growth of the ordered structure (s) is derived directly from calorimetric measurement of transition enthalpy (delta Hcal ), and sigma, which quantifies the relative instability of the helix nucleus, is derived from the ratio of delta Hcal to the apparent transition enthalpy (delta Happ ) obtained by van't Hoff analysis of the optical rotation data. The temperature course of conformational ordering calculated theoretically is in good quantitative agreement with experimental results from both optical rotation and scanning calorimetry. The calculated average length of stable, ordered chain-sequences increases with decreasing temperature, but equals or exceeds the total chain length from light scattering only at temperatures more than approximately equal to 70 K below Tm, suggesting that ordered and disordered regions may co-exist within the same xanthan molecule. Consistent with this interpretation, the observed rate of conformational ordering increases sharply under conditions where the starting solution for dynamic measurements is partially ordered, suggesting that ordered sequences within each chain may act as helix nuclei for adjacent disordered regions, so that helix growth, rather than the slower nucleation process, becomes rate limiting.     

Nutritional interaction in an alga-barnacle association

Nitrogen is the major growth-limiting nutrient for marine algae. One potential source of nitrogen for marine algae is ammonium released by invertebrates. Many mid-intertidal reefs in northeastern New Zealand are dominated by a close association between the honeycomb barnacle Chamaesipho columna and an encusting brown alga Pseudolithoderma sp. Growth of Pseudolithoderma was enhanced in the presence of live C. columna, which released ammonium at a greater rate than the maximum rate of ammonium uptake by Pseudolithoderma. Algal tissue on barnacle tests had a lower C:N ratio than tissue located more than 2 cm from the nearest barnacle, suggesting the barnacle is an important source of nitrogen for the alga. The role of nutrient exchange in determining ecological patterns of species in marine communities is discussed.     

Photolabelling of the hexose transporter at external and internal sites: fragmentation patterns and evidence for a conformational change

The human erythrocyte sugar transporter has been labelled at its internal site with cytochalasin B and at its outside site by the azidosalicoyl derivative of bis(D-mannose) (ASA-BMPA). The cleavage of the transporter by various proteinases has been studied. Chymotrypsin, subtilisin and V8 proteinase give parallel fragmentation patterns for the two labels down to fragments as small as 7 kDa. Thus the binding sites for the two labels can only be separated by a small span of protein. 2-Nitro-5-thiocyanobenzoic acid (NTCB) cleaves at cysteines to give a 15 kDa fragment from the two labels. N-Bromosuccinimide (a reagent which preferentially cleaves at tryptophan residues) has revealed differences in fragmentation of the transporter labelled with either cytochalasin B or with ASA-BMPA. A major cleavage site is proposed to occur at tryptophan 186 which leaves a C-terminal fragment containing both labels. A tryptophan cleavage at residue 388 divides the cytochalasin B site and the ASA-BMPA site. A further tryptophan cleavage gives a cytochalasin B labelled 3 kDa fragment probably from residues 388-412. This gives an assignment of the cytochalasin B site as the inside of the hydrophobic span H 10. Since the ASA-BMPA site is probably only 7 kDa from residue 388 and is on the same 15 kDa NTCB fragment as cytochalasin B we assign this to the outside of hydrophobic span H 9. Thermolysin only cleaves the transporter labelled with cytochalasin B and not with ASA-BMPA. A 18 kDa cytochalasin B labelled fragment is formed. This is indicative of a change in conformation of the transporter when an outside ligand is bound such that the inside of the hydrogen bonding transmembrane segments H 7 and H 8 (and containing the proposed thermolysin cleavage site) are withdrawn from the cytosolic surface. Thus it appears that the core of the transporter (including the external and internal sites plus the transmembrane channel) is located between segments H 7 and H 10.     

Apnoeic episodes induced by smothering: two cases identified by covert video surveillance.

Recurrent cyanotic episodes associated on some occasions with loss of consciousness due to cerebral hypoxia were investigated by long term tape recordings of breathing activity, oxygen saturation, air flow, electrocardiographic activity, and in some cases electroencephalographic activity. In 51 infants and children the mechanisms for the cyanotic episodes were identified (prolonged expiratory apnoea in 45, sleep related airway obstruction in three, seizure induced apnoea in one, behaviour induced apnoea in one). In one child apnoea was suspected as being caused by suffocation (smothering) by the mother. This was confirmed after enlisting the help of the police, who undertook covert video surveillance during cyanotic episodes. Each cyanotic episode was associated with a pattern of disturbance on the multichannel tape recordings which may be pathognomonic of this type of apnoea. A second infant with cyanotic episodes in whom smothering was suspected was referred for similar investigation after the availability of video recordings became established. Maternal smothering was again supported by specific patterns on multichannel tape recordings and confirmed by video surveillance. Diagnosis by video surveillance produces unequivocal evidence in these cases and avoids the need for medical and nursing staff to confront the mother with a possibly incorrect suspicion or in a court of law.     

Phosphoribosyl pyrophosphate and the measurement on inorganic pyrophosphate in plant tissues

This work provides further evidence that plants contain appreciable amounts of inorganic pyrophosphate (PPi), and that breakdown of phosphoribosyl pyrophosphate (PPRibP) does not contribute significantly to the PPi detected in plant extracts. Inorganic pyrophosphate in extracts of the roots of Pisum sativum L., clubs of the spadices of Arum maculatum L., and the developing endosperm of Zea mays L. was assayed with pyrophosphate fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90), and with sulphate adenyltransferase (EC 2.7.7.4). The two different assays gave the same value for PPi content, and for recovery of added PPi. It was shown that PPRibP is converted to PPi during the extraction of PPi. However, the amounts of PPRibP in clubs of A. maculatum and the developing endosperm of Z. mays were negligible in comparison with the contents of PPi.Abbreviations EDTA ethylenediaminetetraacetic acid - PFK(PPi) pyrophosphate fructose 6-phosphate 1-phosphotransferase - PPi inorganic pyrophosphate - PPRibP phosphoribosyl pyrophosphate     

The role of beta-hydroxyaspartate and adjacent carboxylate residues in the first EGF domain of human factor IX.

beta-Hydroxyaspartic acid is a post-translationally modified amino acid found in a number of plasma proteins in a domain homologous to epidermal growth factor. Its presence can be correlated with a high affinity Ca2+ binding site, with a dissociation constant of 10-100 microM. We describe a system for the expression of human coagulation factor IX in dog kidney cells in tissue culture, in which the post-translational modifications and the biochemical activity are indistinguishable from factor IX synthesized in vivo. This system has been used to express eight different point mutations of human factor IX in the first epidermal growth factor domain in order to study the role of beta-hydroxyaspartate at residue 64, and the adjacent carboxylate residues at positions 47, 49 and 78. We conclude that this domain is essential for factor IX function and suggest that Ca2+ binds to carboxylate ions in this domain and stabilizes a conformation necessary for the interaction of factor IXa with factor X, factor VIII and phospholipid in the next step of the clotting cascade.