Modulation of functionally significant conformational equilibria in adenylate kinase by high concentrations of trimethylamine oxide attributed to volume exclusion

The effect of an inert small molecule osmolyte, trimethyl amine N-oxide (TMAO), upon the conformational equilibria of Escherichia coli adenylate kinase was studied using time-resolved FRET. The relative populations of open and closed clefts between the LID and the CORE domains were measured as functions of the concentrations of the substrate ATP over the concentration range 0–18 mM and TMAO over the concentration range 0–4 M. A model was constructed according to which the enzyme exists in equilibrium among four conformational states, corresponding to combinations of open and closed conformations of the LID-CORE and AMP-CORE clefts. ATP is assumed to bind only to those conformations with the closed LID-CORE cleft, and TMAO is assumed to be differentially excluded as a hard spherical particle from each of the four conformations in accordance with calculations based upon x-ray crystallographic structures. This model was found to describe quantitatively the dependence of the fraction of the closed LID-CORE cleft upon the concentrations of both ATP and TMAO over the entire range of concentrations with just five undetermined parameters.     

Essential role of the plasmid hik31 operon in regulating central metabolism in the dark in Synechocystis sp. PCC 6803

The plasmid hik31 operon (P3, slr6039‐slr6041) is located on the pSYSX plasmid in Synechocystis sp. PCC 6803. A P3 mutant (ΔP3) had a growth defect in the dark and a pigment defect that was worsened by the addition of glucose. The glucose defect was from incomplete metabolism of the substrate, was pH dependent, and completely overcome by the addition of bicarbonate. Addition of organic carbon and nitrogen sources partly alleviated the defects of the mutant in the dark. Electron micrographs of the mutant revealed larger cells with division defects, glycogen limitation, lack of carboxysomes, deteriorated thylakoids and accumulation of polyhydroxybutyrate and cyanophycin. A microarray experiment over two days of growth in light‐dark plus glucose revealed downregulation of several photosynthesis, amino acid biosynthesis, energy metabolism genes; and an upregulation of cell envelope and transport and binding genes in the mutant. ΔP3 had an imbalance in carbon and nitrogen levels and many sugar catabolic and cell division genes were negatively affected after the first dark period. The mutant suffered from oxidative and osmotic stress, macronutrient limitation, and an energy deficit. Therefore, the P3 operon is an important regulator of central metabolism and cell division in the dark.     

Growth optimization of an ectomycorrhizal fungus with respect to pH and temperature in vitro, using Design of Experiments

The effect of external factors like pH of the medium, temperature and incubation period on the growth of the ectomycorrhizal fungus, Pisolithus tinctorius was studied as a function of the total fungal biomass produced, extracellular protein content and residual pH of the medium. The effect of the three parameters on the three responses was found to be significant and interdependent. Therefore, an attempt was made to optimize the growth of the fungus under varying conditions of pH, temperature and incubation period using the Box-Behnken Design of Experiments which is a second order model involving Response Surface Methodology and a second order quadratic equation. With this design expert, the optimum conditions of the three parameters that favoured the maximum growth of the fungus in vitro were found. The experimental values corroborated with the predicted values got from the model with the correlation coefficients 0.9349 for biomass, 0.9913 for protein and 0.9959 for final pH.     

Conformational studies of proteoglycans: theoretical studies on the conformation of heparin.

Various models proposed for heparin have been examined by a stereochemical approach involving contact distance criteria and potential energy calculations. The present study suggests that the model favored by Atkins and coworkers [Biochem. J. (1973) 135 , 729–733 and (1974) 143 , 251–252] is not stereochemically satisfactory. An alternative model has been proposed with N-acetyl-D -glucosamine and one of the uronides in the ⁴C₁ conformation and the other uronide (probably sulfated) in the ¹C₄ conformation. The observed variations in the tetrasaccharide periodicities (16.5–17.3 Å) in different crystalline modifications of heparin have been attributed to possible changes in the rotational angles about the interunit glycosidic bonds rather than a change in the pyranose ring conformation. The proposed model is also independent of the observed variation in the relative composition of uronic acid residues in heparin. These conclusions are in disagreement with those of earlier workers.     

Expression and characterization of glycolipid-anchored B7-1 (CD80) from baculovirus-infected insect cells: protein transfer onto tumor cells.

Tumor cells can be modified to express immunostimulatory molecules such as B7-1 by protein transfer using purified glycosylphosphatidylinositol-anchored B7-1 (GPI-B7-1). In this study recombinant baculovirus encoding GPI-B7-1 (vBacB7-1(GPI)) was established to obtain large quantities of purified GPI-B7-1 to modify tumor cells by protein transfer. vBacB7-1(GPI)-infected insect cells showed high-level cell surface expression of GPI-B7-1 that was susceptible to PIPLC treatment. GPI-B7-1 expressed in insect cells (Bac-GPI-B7-1) mediated T cell proliferation, indicating that the GPI-B7-1 retains costimulatory activity. Moreover, Bac-GPI-B7-1 was completely solubilized in Triton X-100 at 4 degrees C compared to 22% solubilization of GPI-B7-1 expressed in CHOK1 cells, suggesting that GPI-anchored proteins expressed in insect cells may not be clustered into the detergent-insoluble fraction. SDS-PAGE analysis of Bac-GPI-B7-1 showed faster mobility (45 kDa) compared to GPI-B7-1 from CHOK1 (68 kDa) and this difference may be due to a difference in glycosylation. Cell binding assays showed that immunoaffinity-purified Bac-GPI-B7-1 retained its functional ability to bind CD28(+) cells. Moreover, when human tumor cells were incubated with this functionally active purified GPI-B7-1, an efficient transfer of B7-1 onto tumor cells was observed. These results demonstrate that GPI-B7-1 can be expressed in insect cells in a functionally active form and can be used to modify tumor cells for immunotherapeutic applications.     

Release and recovery of porcine pepsin and bovine chymosin from reverse micelles: a new technique based on isopropyl alcohol addition.

After complete solubilization by the direct method, porcine pepsin was not released from AOT in isooctane reverse micelles even under aqueous-phase conditions which would not ordinarily allow uptake. Similarly, bovine chymosin, once forward-transferred at a pH below its isoelectric point, was not back-transferred into an aqueous contact phase buffered at a pH value above its isoelectric point. These results show that there is significant hysteresis in the forward- and backward-transfer processes and further imply that kinetics, and not equilibrium, control uptake or release processes for these enzymes. The addition of 10-15% isopropyl alcohol to the aqueous phase increases the rate of protein release dramatically and allows for nearly complete back-transfer of porcine pepsin and 70% back-transfer of bovine chymosin. IPA addition does not destroy the functional integrity of the system since forward transfer of bovine chymosin still occurs at pH values below (but not above) the pI of the protein.