Directed mutagenesis of the redox-active disulphide bridge in glutathione reductase from Escherichia coli

Directed mutagenesis of the gor gene from Escherichia coli encoding the flavoprotein glutathione reductase was used to convert the two cysteine residues that comprise its redox-active disulphide bridge to alanine (C42A) and serine (C47S) residues. A double mutant (C42AH439A) was also created in which His-439, the proton donor/acceptor in the glutathione-binding site, was additionally converted into an alanine residue. The C42A and C47S mutants were both unable to catalyse the reduction of glutathione by NADPH. The C42A mutant retained the transhydrogenase activity of the wild-type enzyme, whereas the C47S mutant was also inhibited in this reaction. These results support the view that in the catalytic mechanism of E. coli glutathione reductase, the thiolate form of Cys-42 acts as a nucleophile to initiate disulphide exchange with enzyme-bound glutathione and that the thiolate form of Cys-47 generates an essential charge-transfer complex with enzyme-bound FAD. Titration of the C42A and C42AH439A mutants indicated that the imidazole side-chain of His-439 lowered the pKa of the charge-transfer thiol (Cys-47) from 7.7 to 5.7, enhancing its ability to act as an anion at neutral pH. Several important differences between these mutants of E. coli glutathione reductase and similar mutants (or chemically modified forms) of other members of the flavoprotein disulphide oxidoreductase family were noted, but these could be explained in terms of the different redox chemistries of the enzymes concerned.     

Brain lesions in a Pacific white-sided dolphin (Lagenorhynchus obliquidens)

A young, male, free-ranging Pacific white-sided dolphin (Lagenorhynchus obliquidens) was found disoriented and died after being held in captivity for several months. Malacic lesions in several areas of the brain were associated with helminth eggs. The appearance and location of these eggs suggested they were of the genus Nasitrema.     

Effect of growing conditions of recombinantE.coli in carrageenan gel beads upon biomasse production and plasmid stability

Summary The biomass production and the plasmid stability of immobilizedE. coli cells in K-carrageenan gel beads were investigated in continuous cultures. Several factors, such as inoculum size, gel bead volume and gel concentration were examined in order to increase the cell concentration inside the immobilized cell reactor, and therefore to increase the overall productivity.     

Cytokinin Secretion by Frankia sp. HFP ArI3 in Defined Medium

Frankia sp. HFP ArI3 (host plant Alnus rubra Bong.) was grown in defined medium and the culture solution was analyzed for the presence of various cytokinins and related compounds. N⁶- (Δ²-isopentenyl) adenosine was the only cytokinin detected by both high performance liquid chromatography and gas chromatography-mass spectrometry, at levels of approximately 1 ng/ml culture medium.     

Stability fluctuations of plasmid-bearing cells: immobilization effects

The maintenance of the plasmid vectors pTG201 and pTG206 (which both carry the Pseudomonas putida xylE gene) and pB lambda H3 in Escherichia coli hosts was studied in free and immobilized continuous cultures. pTG201, containing the strong lambda PR promoter, was more quickly lost than plasmid pTG206, containing the tetracycline resistance gene promoter. The instability of pTG201 seems to be related to high expression of the cloned xylE genet. Fluctuations in the proportion of pTG201-containing cells were observed in the free system, suggesting the appearance of adaptive descendants (with and without plasmid) from the initial strains. The loss of plasmid vectors from E. coli cells and the fluctuations in the proportion of plasmid-containing cells could be prevented by immobilizing plasmid-containing bacteria in carrageenan gel beads.     

Interaction of peptide boronic acids with elastase: circular dichroism studies

Boronic acid derivatives of good peptide substrates of the serine proteases cause slow-binding inhibition, manifested as biphasic binding (Kettner and Shenvi: J. Biol Chem. 259:15106-15114, 1984). These inhibitors are thought to act as reaction-intermediate analogs. Three peptide boronic acids--Ac-Pro-boro-Val-OH, DNS-Ala-Pro-boro-Val-OH, and Ac-Ala-Ala-Pro-boro-Val-OH--were chosen for far-ultraviolet circular dichroism (CD) studies in order to determine whether the second phase involves a conformational change of pancreatic elastase. The dipeptide is a simple competitive inhibitor (Ki = 0.27 microM) and the latter are slow-binding inhibitors (Ki = 16.4 and 0.25 nM, respectively). Spectral deconvolution and correction for the formation of antiparallel beta-sheet by the peptide inhibitor itself indicate that there is no significant change in the secondary structure of the enzyme in either the initial or final inhibitor complex. A kinetic experiment confirmed that the slow-binding step was not associated with a CD spectral change, and that therefore a protein conformational change was not responsible for the slow binding.     

Differential down-regulation of protein kinase C subspecies in KM3 cells

The down-regulation of protein kinase C (PKC) subspecies in KM3 cells (a pre-B, pre-T cell line) has been examined. The PKC from KM3 cells was resolved into two subspecies, type II (mainly beta II) and type III (alpha), upon hydroxyapatite column chromatography. Biochemical and immunocytochemical analysis revealed that, when these cells were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA), the time course of down-regulation of the PKC subspecies was different; type II PKC was translocated and depleted from the cell more quickly than type III enzyme. The results suggest that each PKC subspecies plays a different role in the cellular response to TPA and probably to other external stimuli.     

Preparation of cytoplasmic bodies (cytospheres) from isolated hepatocytes and their biochemical properties

The controlled centrifugation of isolated rat hepatocytes at 260 000 g results in the formation of membrane-bounded cell fragments that we have termed 'cytospheres'. A method is described for the isolation of these cytospheres. Cytospheres are spherical, have a mean diameter of 9.2 +/- 3.2 microns (SD) and a protein content of 225 +/- 12 mg/g wet wt. About 3% of the protein from the original isolated hepatocyte suspension is recoverable. Transmission electron microscopy (TEM) shows cytospheres to possess a trilaminar membrane, and a finely granular hyaloplasm generally devoid of organelles, filaments and microtubules. Freeze-fracture studies reveal a membrane structure typical of a plasma membrane. Ouabain and wheat germ agglutinin (WGA)-binding studies indicate that the original orientation of the plasma membrane is maintained throughout the formation of the cytospheres. The cytospheres have also been characterized biochemically. Cytospheres are enriched in the enzymes normally associated with the hyaloplasm, whereas the activities of enzymes localized in organelles are greatly diminished. Lipid analysis of the cytosphere membrane indicates that it is derived from the plasma membrane of the hepatocyte. Cytospheres are sensitive to changes in the osmolarity and ionic composition of their environment. Cytospheres should therefore prove a useful preparation for the study of hyaloplasm metabolism and of plasma membrane receptor and permeability properties.     

Effects of pH on Activity and Activation of Ribulose 1,5-Bisphosphate Carboxylase at Air Level CO(2)

The effects of pH on catalysis and activation characteristics of spinach ribulose 1,5-bisphosphate (RuBP) carboxylase were examined at air level of CO₂. Catalysis at limiting CO₂ was independent of pH over the range of pH 8.2 to 8.8 However, the kinetics of activation and the apparent equilibrium between the activated and inactivated forms of the enzyme were strongly dependent upon the pH and the presence or absence of the substrate RuBP. When incubated at air level of CO₂ at pH 8.2 in the absence of RuBP, the enzyme activation state was approximately 75% of that achieved with saturating CO₂ at that pH. The extent of activation increased with pH reaching 100% at pH values of 8.6 or higher. Adding RuBP to the activation medium after equilibrium activation state had been established decreased the apparent equilibrium activation level at pH values below 8.6. This effect was reversed at pH values above 8.6. Activation of inactive enzyme by CO₂ and Mg²⁺ was inhibited dramatically at pH values below 8.6 and less so at pH values above 8.6. Studies showed that binding of RuBP to the inactive form of the enzyme was pH dependent with tighter binding occurring at lower pH values. It is suggested that the tight binding of RuBP to the inactive enzyme tends to decrease the equilibrium concentration of the activated form at pH values less than 8.6. These studies indicate that stromal pH could have a strong effect on the activation state of this enzyme in vivo, and possible feedback interactions which might adjust the apparent V_max to match the rate of RuBP regeneration are discussed.