Effects of dibromothymoquinone on mung bean mitochondrial electron transfer and membrane fluidity

The effects of the quinone analog dibromothymoquinone on electron transfer in isolated mung bean mitochondria are described. Both the main, cyanidesensitive and the alternate, cyanide-insensitive pathways are inhibited by dibromothymoquinone but in markedly different fashions. Half-maximal inhibition appeared at 40 μM and 20 μM dibromothymoquinone for the cyanide-sensitive and alternate pathways, respectively. With succinate as the electron donor, dibromothymoquinone inhibited the alternate pathway at a single site; showing a mixed, non-competitive type inhibition. On the succinate, cyanide-sensitive pathway dibromothymoquinone showed two sites of inhibition and neither coincides with the site of inhibition associated with the alternate pathway. With malate as the electron donor, two sites of inhibition by dibromothymoquinone were observed regardless of the pathway measured.Dibromothymoquinone also inhibited the rate of valinomycin-induced swelling of isolated mung bean mitochondria. Steady-state kinetics showed the inhibition to be non-competitive with respect to valinomycin. Additionally dibromothymoquinone was observed to increase the fluorescence polarization associated with the hydrophobic probe 1,6-diphenylhexatriene. The results indicated that dibromothymoquinone decreased the fluidity of the inner mitochondrial membrane and suggested that the inhibition of mitochondrial electron transfer by dibromothymoquinone may be associated with this decrease in membrane fluidity.The relationship of the multisite nature of the inhibition of electron transfer by dibromothymoquinone and the possible role of mobile electron carriers such as ubiquinone on the main and alternate respiratory pathways of higher plants is discussed.     

Sequential conversion of the glutathionyl side chain of slow reacting substance (SRS) to cysteinyl-glycine and cysteine in rat basophilic leukemia cells stimulated with A-23187

In rat basophilic leukemia (RBL-1) cells stimulated with A-23187, the major slow reacting substance (SRS) species contain glutathione, cysteinyl-glycine, or cysteine in their side chains, corresponding or closely related to leukotrienes LTC4, LTD4, and LTE4, respectively. Evidence is presented that most of the SRS produced during the first few minutes of stimulation by the ionophore has a glutathionyl side chain which is sequentially converted to cysteinyl-glycine and cysteine.     

Aryl sulfatase inactivation of slow reacting substance. Evidence for proteolysis as a major mechanism when ordinary commercial preparations of the enzyme are used

Type II B arylsulfatases are known to inactivate slow reacting substance (SRS), but the mechanism is unclear. In the present study, ordinary commercial preparations of Sigma limpet arylsulfatase largely inactivated the glutathionyl and cysteinyl-glycyl forms of SRS, but the cysteinyl form of SRS was largely resistant to the enzyme. Evidence is presented which established that a major mechanism for the inactivation of the glutathionyl and cysteinyl-glycyl SRS types, at least by the particular enzyme preparations we have studied, involves cleavage of the glycine moiety from the sulfur containing side chain. This was confirmed by digestion studies with glutathione itself. In addition, there is some evidence to indicate that the enzyme may destabilize the double bond structure of the SRS molecule, contributing to the overall inactivation.     

Effect of photosynthetic intermediates on the magnesium inhibition of oxygen evolution by barley chloroplasts

Millimolar concentrations of Mg²⁺ inhibited CO₂-dependent O₂ evolution by barley (Hordeum vulgare L.) chloroplasts and also prevented the activation of NADP-glyceraldehyde-3-phosphate dehydrogenase, ribulose-5-phosphate kinase, and fructose-1,6-diphosphatase by light in intact chloroplasts. When added in the dark, 3-phosphoglycerate prevented the inhibition of O₂ evolution by Mg²⁺ and reduced the Mg²⁺ inhibition of enzyme activation by light. Fructose 1,6-diphosphate and ribulose 5-phosphate also prevented the inhibition of O₂ evolution by Mg²⁺ whereas glucose 1-phosphate, glucose 6-phosphate, ribulose 1,5-diphosphate, and citrate had no effect. Phosphoenolpyruvate gave an intermediate response. Metabolites that prevented the Mg²⁺ inhibition of O₂ evolution shortened the lag phase of CO₂-dependent O₂ evolution in the absence of M²⁺. Loading chloroplasts in the dark with 3-phosphoglycerate reduced both the lag phase of O₂ evolution and the inhibition of O₂ evolution by Mg²⁺. The results suggested that Mg²⁺ inhibition was lessened either by external metabolites that compete with inorganic phosphate for transport into the chloroplast or by a high concentration of internal metabolites.     

Permeability Properties of the Inner Membrane of Mung Bean Mitochondria and Changes during Energization

The permeability properties of the inner membrane of mung bean mitochondria were studied by osmotic swelling techniques. Rapid mitochondrial swelling was observed in isotonic ammonium phosphate, which indicated that an active phosphate/hydroxyl antiporter was present. The phosphate carrier was specifically inhibited by sulfhydryl reagents. Mitochondria did not swell in isotonic ammonium salts of malate, succinate, or fumarate, either in the presence or absence of 10 millimolar phosphate. Additionally, no swelling was observed in ammonium citrate upon addition of malate plus phosphate. Consequently, no evidence was obtained with the osmotic swelling technique for a coupled exchange of phosphate for dicarboxylic acids across the membrane.     

The effect of tobacco smoke on the metabolism and function of rat alveolar macrophages.

Alveolar macrophages harvested by bronchopulmonary lavage from rats exposed to tobacco smoke for 30 days ("smokers") showed alterations in oxidative metabolism, lactate production and phagocytosis of inert starch particles when compared with control macrophages. Phagocytosis of viable Staphylococcus aureus was unaffected by tobacco smoke. Glucose oxidation measured by conversion of glucose-1-14C to 14CO2 moderately affected while oxidation of glucose-6-14C to 14CO2 was not. Smokers routinely yielded fewer cells than controls, though these cells contained approximately 17% more protein than did controls. Opsonization of particles was not necessary for macrophages from either smoker or control animals to manifest a respiratory burst and increased superoxide and hydrogen peroxide release during phagocytosis. The glycolytic inhibitors, sodium fluoride and iodoacetamide, while effectively blocking glycolysis, did not inhibit phagocytosis by macrophages from either group. The results reported clearly distinguish alveolar macrophages from other phagocytic cells (peritoneal macrophages and polymorphonuclear leukocytes) and suggest a state of non-specific activation caused by exposure to tobacco smoke.     

Crystal structure of the human Fab fragment Kol and its comparison with the intact Kol molecule

The crystal structure of the Fab³ fragment of the IgG protein Kol was analysed and an electron density map calculated at 3 Å resolution based on phases obtained from multiple isomorphous replacement. An atomic model was constructed but the lack of amino acid sequence data causes some uncertainty, in particular concerning the amino acid side-chains. Several cycles of constrained crystallographic refinement (Deisenhofer &; Steigemann, 1975) produced an R value of 0.36. The resulting model was compared with the intact Kol IgG crystal structure (Colman et al., 1976), which had also been subjected to constrained crystallographic refinement, and two Fab fragments (Davies et al., 1975). The elbow angle was found open and only 8 ° more bent than in intact Kol, in contrast to the other Fab fragments, which show a strongly bent elbow angle. The intramolecular longitudinal V-C contacts are very similar in Kol Fab and Kol IgG and considerably fewer in number than in the other Fab fragments. The lateral V-V and C-C association is virtually identical in Kol Fab and Kol IgG. Although Kol Fab and Kol IgG crystallize in different lattices, they exhibit a nearly identical head to tail packing of the Fab parts, with the C modules touching the hypervariable segments of the V modules. This strongly conserved particular mode of aggregation might be reflected in the property of cold precipitation of the Kol cryoglobulin.     

Population differentiation within Festuca rubra L. with regard to soil salinity and soil water

Summary Seed and transplanted adult plants from populations of Festuca rubra, collected from inland, salt-marsh and sand-dune sites were grown on culture solution with added sodium chloride. The growth of the populations of the three habitats was reduced differentially by salt. The salt marsh ecotype Festuca rubra ssp. litoralis was only slightly affected and the inland ecotype F. rubra ssp. rubra was severely retarded at 60 mM NaCl. The dune ecotype F. rubra ssp. arenaria had an intermediate tolerance. The tolerant ecotypes accumulated less sodium chloride as compared to the sensitive ecotype, suggesting that salt tolerance is caused in part by salt exclusion.In addition, the dune ecotype F.r. arenaria appeared to be more drought tolerant than the salt marsh ecotype. Abscission of salt-saturated leaves does not function as an adaptation to salinity in Festuca rubra.All three ecotypes accumulated proline with increased salinity. The response was most pronounced in the drought tolerant F.r. arenaria, indicating that proline accumulation is a response to osmotic stress rather than to ion-specific effects of salinity. The observed differences in salt tolerance may be explained by differential sensitivity to toxic effects of sodium chloride.The occurrence on a beach plain of closely adjacent populations of F.r. arenaria and F.r. litoralis, differing markedly in salt tolerance, is briefly discussed.     

Class pi glutathione S-transferase from pig lung. Purification, biochemical characterization, primary structure and crystallization

A cytosolic glutathione S-transferase from pig lung was purified 210-fold to apparent homogeneity. The enzyme was classified as a class pi isoenzyme on the basis of its physical and chemical properties. It is homodimeric with a subunit Mr of 23,500, has a pI of 7.2, and shows a high specific activity towards ethacrynic acid. The glutathione analogues, S-hexylglutathione and glutathione sulfonate, were strong reversible inhibitors. The enzyme's primary structure, established entirely by protein chemical methods, consists of 203 amino acids and is highly similar (82-84% residue identity) to the rat and human class pi isoenzymes. Furthermore, there was no evidence of microheterogeneity or post-translational modifications. Each subunit contains a highly reactive cysteine residue, the modification of which leads to enzyme inactivation. None of the cysteine residues in the pig enzyme appear to form intramolecular disulfide bonds. Singel crystals of the glutathione-S-transferase-glutathione-sulfonate complex were obtained by the hanging-drop method of vapour diffusion from poly(ethylene glycol) 4000 solutions. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions of a = 10.125 nm, b = 8.253 nm and c = 5.428 nm and diffract to better than 0.22 nm.