Probing the Structural Basis for Enzyme-Substrate Recognition In Cu,Zn Superoxide Dismutase

A full understanding of enzyme-substrate interactions requires a detailed knowledge of their structural basis at atomic resolution. Crystallographic and biochemical data have been analyzed with coupled computational and computer graphic approaches to characterize the molecular basis for recognition of the superoxide anion substrate by Cu. Zn superoxide dismutase (SOD). Detailed analysis of the bovine SOD structure aligned with SOD sequences from 15 species provides new results concerning the significance and molecular basis for sequence conservation. Specific roles have been assigned for all 23 invariant residues and additional residues exhibiting functional equivalence. Sequence invariance is dominated by 15 residues that form the active site stcreochemistry. supporting a primary biological function of superoxide dismutation. Using data from crystallographic structures and site-directed mutants, we are testing the role of individual residues in the active site channel, including (in human SOD) Glu132, Glu133, Lys136, Thr137, and Arg 143. Electrostatic calculations incorporating molecular flexibility suggest that the region of positive electrostatic potential in and over the active site channel above the Cu ion sweeps through space during molecular motion to enhance the facilitated diffusion responsible for the enzyme's rapid catalytic rate.     

Effect of algal inoculation on the yield and vitamin C content of two varieties of tomato

Summary Two varieties of tomato (Pusa Rubi and Selection 120) positively responded to algal inoculation in terms of the yield of fruits and shoots, but there was no significant effect on the vitamin C content of the fruits. A combined application of urea and algae was more effective than the application of urea alone.     

Isolation and characterization of Zymomonas mobilis mutants resistant to octadecyltrimethylammonium chloride,a detergent acting on hopanoid-producing bacteria

Growth of the hopanoid-producing bacterium Zymomonas mobilis was inhibited at low concentrations of the cationic detergent octadecyltrimethylammoniumchloride (OTAC). A relationship between sensitivity of Zymomonas mobilis to OTAC, presence of hopanoids and ethanol tolerance was postulated. Mutants resistant to OTAC were isolated from strains ZM1 and ZM4. They did not present any alteration of the hopanoid content and their squalene cyclases showed the same sensitity to OTAC as the parent enzymes. Resistance to OTAC paralleled pleiotropic effects including, enhanced accessibility of the membrane-bound alkaline phosphatase, important release of proteins from cells by Tris/HCl treatment, increased resistance to antibiotics and increased sensitivity to ethanol. In addition, OTAC^R mutants were also characterized by the synthesis or the overproduction of an outer membrane protein (F53) not detected on 2D-PAGE maps of parent strains and by a normal heat shock response. The role of hopanoids, heat shock proteins, protein F53 and membrane organization in ethanol tolerance is discussed.Abbreviations OTAC octadecyltrimethylammoniumchloride - SLS sodium lauryl sarcosinate     

Early stomatal closure in waterlogged pea plants is mediated by abscisic acid in the absence of foliar water deficits

Abstract Soil waterlogging decreased leaf conductance (interpreted as stomatal closure) of vegetative pea plants (Pisuin sativum L. cv. ‘Sprite’) approximately 24 h after the start of flooding, i.e. from the beginning of the second 16 h-long photo-period. Both adaxial and abaxial surfaces of leaves of various ages and the stipules were affected. Stomatal closure was sustained for at least 3 d with no decrease in foliar hydration measured as water content per unit area, leaf water potential or leaf water saturation deficit. Instead, leaves became increasingly hydrated in association with slower transpiration. These changes in the waterlogged plants over 3 d were accompanied by up to 10-fold increases in the concentration of endogenous abscisic acid (ABA). Waterlogging also increased foliar hydration and ABA concentrations in the dark. Leaves detached from non-waterlogged plants and maintained in vials of water for up to 3 d behaved in a similar way to leaves on flooded plants, i.e. stomata closed in the absence of a water deficit but in association with increased ABA content. Applying ABA through the transpiration stream to freshly detached leaflets partially closed stomata within 15 min. The extractable concentrations of ABA associated with this closure were similar to those found in flooded plants. When an ABA-deficient ‘wilty’ mutant of pea was waterlogged, the extent of stomatal closure was less pronounced than that in ordinary non-mutant plants, and the associated increase in foliar ABA was correspondingly smaller. Similarly, waterlogging closed stomata of tomato plants within 24 h, but no such closure was seen in ‘flacca’, a corresponding ABA-deficient mutant. The results provide an example of stomatal closure brought about by stress in the root environment in the absence of water deficiency. The correlative factor operating between the roots and shoots appeared to be an inhibition of ABA transport out of the shoots of flooded plants, causing the hormone to accumulate in the leaves.     

Tn10 mutagenesis in Azotobacter vinelandii

Summary The tetracycline-resistant transposon Tn10 and its high-hopper derivative Tn10HH104 were introduced into the Azotobacter vinelandii genome using suicide conjugative plasmids derived from pRK2013. Several types of mutants induced by either of these elements are described. Nif^- mutants (deficient in nitrogen fixation) were easily isolated, whereas the isolation of other mutant types (auxotrophs, sugar non-users) required special selection conditions. The characterization of the mutations as transposon insertions was often complicated and sometimes required a combination of genetic and physical tests. A common source of complication, the existence of double inserts, was found among the mutants induced by Tn10HH104 but not among those induced by Tn10. Both the high-hopper and the wild-type element proved to undergo secondary transpositions, albeit at different frequencies. Another type of complication, the existence of heterozygotes, occurred because of the high level of redundancy of the A. vinelandii genome.     

Chloroplast photooxidation inhibits the expression of a set of nuclear genes

Summary Mutations or herbicides which inhibit the accumulation of carotenoid pigments in higher plants also result in the arrest of chloroplast development at a very early stage. The cause is extensive photooxidative damage within the chloroplast in the absence of protective carotenoids. Because the extent of photooxidation is dependent upon light intensity, normal chloroplast development can occur when carotenoid-deficient seedlings are grown in very dim light. Normal accumulation of chloroplastic and cytosolic mRNAs encoding chloroplast proteins proceeds only under permissive dim light conditions. Illumination with higher intensity light causes rapid chlorophyll photooxidation and the loss of two cytosolic mRNAs coding for proteins destined for the chloroplast, but does not affect another light-regulated cytosolic mRNA encoding a cytosolic protein. This experimental system may have uncovered a mechanism which coordinates the expression of genes in different cellular compartments.Abbreviations LHCP light-harvesting chlorophyll a/b protein - SSu small subunit - RuBP fibulose 1,5-bisphoshate - PEP phosphoenolpyruvate     

Somaclonal variation versus chemically induced mutagenesis in tomato (Lycopersicon esculentum L.)

Summary A comparison was made of the type and frequency of mutational events found in the progeny of tomato plants regenerated after one passage in vitro with those induced by chemical mutagenesis with ethyl methane sulphonate. Several mutants were recovered in the progeny of regenerated and mutagenized plants of two cultivars of tomato. They can be grouped into the following categories: seedling lethality, male sterility, resistance to Verticillium, short stature, change in number of lateral shoots or in leaf shape. The results indicate that the two sources of variability differ in their effect, changing the spectrum and frequency of the mutants as well as, at least in some cases, their pattern of segregation.     

Sources and inheritance of resistance to leaf curl virus in Lycopersicon

Summary One hundred and twenty-two varieties, lines and wild accessions of Lycopersicon were screened under three different regimes during the autumn/winter season of 1982–83 and 1983–84 for resistance to tomato leaf curl virus (TLCV). L. hirsutum f. glabratum (B6013) and L. hirsutum f. typicum (A1904) proved to be highly resistant to TLCV in all three environments. Various accessions of L. peruvianum were also highly resistant. L. pimpinellifolium (A1921) exhibited no TLCV symptoms within 90 days. Of the cultivated varieties, Acc 99 exhibited the minimim score for susceptibility; AC 142, Collection No. 2, Kalyanpur Angurlata and HS 101 had a low rating for virus incidence. The inheritance of resistance was studied in the interspecific crosses between a TLCV resistant line of L. pimpinellifolium (A1921) and five (HS 101, HS 102, HS 110, Pusa Ruby and Punjab Chhuhara) susceptible cultivars of L. esculentum. Parents, F₁, F₂ and backcross progenies were artificially inoculated with local strains of TLCV using vector the viruliferious whitefly, Bemisia tabaci (Genn.). Data indicated that the resistance of L. pimpinellifolium (A 1921) is monogenic and incompletely dominant over susceptibility.     

Glutathione status and sensitivity to GSH-reacting compounds of Escherichia coli strains deficient in glutathione metabolism and/or catalase activity

The intracellular concentrations of total glutathione, GSSG and protein · S-SG, the total excreted glutathione concentration, and the susceptibility towards GSH-reacting compounds were assayed in strains of Escherichia coli deficient in biosynthesis and/or reduction of glutathione. A deficiency in glutathione reductase displaced the glutathione status towards the oxidized forms. This displacement was more clearly appreciated in strains additionally deficient in glutathione biosynthesis. A deficiency in catalase activity also produced an increase in the oxidation of glutathione. The most severe changes were observed in the concentrations of protein-glutathione mixed disulfides and in the amount of glutathione excreted to the medium. Increased sensitivities towards compounds known to interact with cellular GSH were observed in glutathione reductase deficient strains, although these effects were enhanced in strains additionally deficient in GSH biosynthesis