Inactivation of human arginine-143, lysine-143, and isoleucine-143 Cu,Zn superoxide dismutases by hydrogen peroxide: multiple mechanisms for inactivation

Site-specific mutants of human Cu,Zn superoxide dismutase (Cu,ZnSOD) have been prepared in which the active-site arginine at position 143 (i.e., SODR143) has been replaced by either lysine (SODK143) or isoleucine (SODI143). As reported previously (W.F. Beyer, Jr., et al. (1987) J. Biol. Chem. 262, 11182-11187), SODK143 and SODI143 have 43 and 11%, respectively, of the catalytic activity of SODR143. H2O2, at low concentrations, acts as an affinity reagent for the inactivation of SODR143. At pH 9.0 and 25 degrees C, the process is characterized by a half-saturation constant for H2O2, K50, of 5.1 mM and a maximum pseudo-first-order rate constant for inactivation, Kmax, of 0.53 min-1. At pH 11.5, the corresponding values are 0.63 mM and 1.23 min-1. The active species in the inactivation is likely HO2-, as previously found with yeast and bovine Cu,ZnSODs (see C.L. Borders, Jr., and I. Fridovich (1985) Arch. Biochem. Biophys. 241, 472-476). SODK143 is also inactivated by HO2- by an affinity mechanism, i.e., one where reversible binding of H2O2 (HO2-) is a prerequisite for inactivation. At pH values of 9.0 and 11.5, the kmax values are 0.92 and 1.08 min-1, respectively; however, the corresponding K50 values increase to 42.5 and 15.8 mM, respectively. SODI143 is also inactivated by H2O2, but no evidence for an affinity mechanism was found; instead, a second-order kinetic mechanism was observed. Inactivation of each of the three enzymes is accompanied by the loss of one histidine per subunit. At elevated concentrations of H2O2, a second nonaffinity mechanism of inactivation of both SODR143 and SODK143 was found, in which a second equivalent of H2O2 reacts with the Cu,ZnSOD.HO2- complex to give a competing second-order inactivation. It appears that the positive charge of arginine-143 plays a role in the binding of HO2- at the active site of human Cu,ZnSOD, and that replacement of the arginine by lysine gives an enzyme with a similar affinity mechanism of inactivation, but with a greatly reduced affinity for HO2-. However, replacement with isoleucine causes an entirely different mechanism of inactivation; this raises the possibility that the mechanism of enzyme catalysis of superoxide dismutation by SODI143 is also different.     

Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction

Gene Splicing by Overlap Extension or "gene SOEing" is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro. By modifying the sequences incorporated into the 5'-ends of the primers, any pair of polymerase chain reaction products can be made to share a common sequence at one end. Under polymerase chain reaction conditions, the common sequence allows strands from two different fragments to hybridize to one another, forming an overlap. Extension of this overlap by DNA polymerase yields a recombinant molecule. This powerful and technically simple approach offers many advantages over conventional approaches for manipulating gene sequences.     

Cardenolides from Asclepias asperula subsp. Capricornu and A. Viridis

6′-O-(E-4-hydroxycinnamoyl) Desglucouzarin, the first cardenolide containing a cinnamoyl ester moiety, has been isolated from the ethanolic extract of the milkweed, Asclepias asperula. In addition, five known cardenolides were isolated and identified from A. asperula and A. viridis.     

Differentiated analysis of the secondary structure of hydrophilic and hydrophobic regions in alpha- and beta-subunits of Na+,K+-ATPase by Raman spectroscopy

Raman spectra of active Na+,K+-ATPase from pig kidney and membrane-bound products of its two-stage trypsinolysis, including alpha-subunit hydrophobic regions as well as the intact beta-subunit and hydrophobic regions of alpha- and beta-subunits, were measured to calculate the secondary structure of hydrophilic and hydrophobic regions of the enzyme. Consequent comparison demonstrated unambiguously that (i) membrane-bound hydrophobic parts of polypeptide chains of Na+,K+-ATPase subunits are in the alpha-helical conformation; (ii) essential contents of the alpha-helix as well as beta-sheet are estimated to form the hydrophilic (mainly cytoplasmic) domain of the Na+,K+-ATPase alpha-subunit; (iii) the exoplasmic hydrophilic domain of the beta-subunit is shown to include several antiparallel beta-pleated sheets and a small amount of the alpha-helix and unordered conformations. The model of the secondary structure organization of hydrophilic domains as well as 8 hydrophobic transmembrane segments of the enzyme molecule was proposed on the basis of experimental results and predictional calculations.     

An X-autosome fusion chromosome of Caenorhabditis elegans

Summary The translocation mnT12(IV;X) is a fusion of holocentric chromosomes IV and X, the breakpoints occurring near the left end of IV and the right end of X. Animals homozygous for mnT12 are viable and fertile; they contain five pairs of chromosomes rather than the normal set of six pairs. The mnT12 chromosome is larger than all wild-type chromosomes and thus identifies linkage groups IV and X cytologically. Hermaphrodites heterozygous for mnT12 show high frequency meiotic nondisjunction both between mnT12 and the X chromosome, which results in a high incidence of male self progeny (27% compared to the wild-type incidence of 0.2%), and between mnT12 and chromosome IV, which results in a high incidence of self progeny essentially trisomic for chromosome IV (karyotype IV/mnT12/mnT12). The viability of chromosome IV trisomics has been confirmed by constructing animals trisomic for only normal copies of chromosome IV; these animals are morphologically wild type. Meiotic chromosome disjunction in mnT12 homozygotes appears to be normal, although the frequency of recombination between markers that are normally X-linked is significantly reduced. Males of genotype IV/mnT12/0 are fertile. They can be thought of as having a neo-X(mnT12) neo-Y(normal IV) karyotype since it is possible to maintain a male-hermaphrodite stock of C. elegans consisting of such males and hermaphrodites carrying two neo-X chromosomes and no neo-Y; the organism is thus converted from an XO:XX type of sex determination to an XX:XX system.     

Selective acylation of 6-deoxyglycals

L-Rhamnal was acylated under a variety of conditions with various acylating reagents. Substitution of the hydroxyl group in the allylic position was favored when acetyl chloride, N-acetylimidazole, benzoyl chloride, and N-benzoylimidazole were used (40-60% net yields), whereas the homoallylic group of L-rhamnal was selectively protected when acetic anhydride-pyridine was employed for the acylation. The monoacetates of L-fucal underwent O-3----O-4 migration of the acetyl group, and selective acylation of this glycal could not be achieved.     

Phosphorylation of chloroplast membrane proteins partially protects against photoinhibition

Thylakoids isolated from peas (Pisum sativum cv. Kelvedon Wonder) and phosphorylated by incubation with ATP have been compared with non-phosphorylated thylakoids in their sensitivity to photoinhibition by exposure to illumination in vitro. Assays of the kinetics of fluorescence induction at 20° C and the fluorescence emission spectra at-196° C indicate a proportionally larger decrease in fluorescence as a result of photoinhibitory treatment of non-phosphorylated compared with phosphorylated thylakoids. It is concluded that protein phosphorylation can afford partial protection to thylakoids exposed to photoinhibitory conditions.Abbreviations and symbols DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F ₀ Level of chlorophyll fluorescence when photosystem 2 traps are open - F _m Level of chlorphyll fluorescence when photosystem 2 traps are closed - P Maximum level of fluorescence reached in the absence of DCMU - PSI (II) photosystem I(II)     

Augmentation of wheat common root rot by Fusarium acuminatum

Wheat was inoculated with Bipolaris sorokiniana and Fusarium acuminatum individually and in combination under controlled conditions. Only B. sorokininana produced significant disease when used alone; however, F. acuminarum significantly augmented the effects of B. sorokiniana when plants are inoculated with both fungi.