Effects of Photosystem II Herbicides on the Photosynthetic Membranes of the Cyanobacterium Aphanocapsa 6308

The effects of the photosystem II herbicides diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) on the photosynthetic membranes of a cyanobacterium, Aphanocapsa 6308, were compared to the effects on a higher plant, Spinacia oleracea. The inhibition of photosystem II electron transport by these herbicides was investigated by measuring the photoreduction of the dye 2,6-dichlorophenol-indophenol spectrophotometrically using isolated membranes. The concentration of herbicide that caused 50% inhibition of electron transport (I₅₀ value) in Aphanocapsa membranes for diuron was 6.8 × 10⁻⁹ molar and the I₅₀ value for atrazine was 8.8 × 10⁻⁸ molar. ¹⁴C-labeled diuron and atrazine were used to investigate herbicide binding with calculated binding constants (K) being 8.2 × 10⁻⁸ molar for atrazine and 1.7 × 10⁻⁷ molar for diuron. Competitive binding studies carried out on Aphanocapsa membranes using radiolabeled [¹⁴C]atrazine and unlabeled diuron revealed that diuron competed with atrazine for the herbicide-binding site. Experiments involving the photoaffinity label [¹⁴C]azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-2-triazine) and autoradiography of polyacrylamide gels indicated that the herbicide atrazine binds to a 32-kilodalton protein in Aphanocapsa 6308 cell extracts.     

Comparison of Triazine-Resistant and -Susceptible Biotypes of Senecio vulgaris and Their F(1) Hybrids

The relationship of triazine resistance to decreased plant productivity was investigated in Senecio vulgaris L. F₁ reciprocal hybrids were developed from pure-breeding susceptible (S) and resistant (R) lines. The four biotypes (S, S × R, R, R × S) were compared in terms of atrazine response, electron transport, carbon fixation, and biomass production. Atrazine response, carbon fixation rate, and PSII and whole-chain electron transport rates of hybrids were nearly identical to those of their respective maternal parents. Significant differences occurred between the two susceptible (S, S × R) and two resistant (R, R × S) biotypes in atrazine response (I₅₀), carbon fixation rate, and PSII and whole-chain electron transport rates; PSI rates were identical in all four biotypes. Coupled and uncoupled, whole-chain electron transport rates of thylakoids of the two susceptible biotypes were approximately 50% greater than those of the two resistant biotypes at photon flux densities greater than 215 micromoles per square meter per second. Carbon exchange rates of the two susceptible biotypes were 23% greater than those of the two resistant biotypes. Hybrid biotypes (S × R, R × S) were not identical to their maternal parents in biomass production. The S, S × R, and R × S plants all achieved greater biomass than R plants. These results suggest that while the resistance mutation influences thylakoid performance, reduced productivity of triazine-resistant plants cannot be ascribed solely to decreases in electron transport or carbon assimilation rates brought about by the altered binding protein. Since the F₁ hybrids differed from their maternal parents only in nuclear genes, it appears that the detrimental effects of the triazine resistance mutation on plant growth may be attenuated by interactions of the plastid and nuclear genomes.     

Photosynthesis of Euglena gracilis under Cobalamin-Sufficient and -Limited Growing Conditions

Cobalamin is essential for growth of Euglena gracilis and photosynthesis. Methylcobalamin in Euglena chloroplasts (Y Isegawa, Y Nakano, S Kitaoka, 1984 Plant Physiol 76: 814-818) functions as a coenzyme of methionine synthetase. The requirement of cobalamin for photosynthesis appeared remarkably high in Euglena grown under the dark-precultured condition. The required amount of cobalamin for normal photosynthetic activity was 7.4 × 10⁻¹¹ molar, while 7.4 × 10⁻¹⁰ molar cobalamin was required for normal growth. The lowered photosynthetic activity in cobalamin-limited cells was restored 20 hours after feeding cyanocobalamin or methionine to cobalamin-limited cells. Lowering of photosynthetic activity was due to loss of photosystem I activity. This photosynthetic activity was recovered after supplementation by methionine or cobalamin. The results suggest that methionine serves for the stabilization of photosystem I. This paper is the first report of the physiological function of cobalamin in chloroplasts of photosynthetic eukaryotes.     

5'-Azido-N-1-Napthylphthalamic Acid, a Photolabile Analog of N-1-Naphthylphthalamic Acid : Synthesis and Binding Properties in Curcurbita pepo L

A photolabile analog of N-1-naphthylphthalamic acid (NPA), 5′-azido-N-1-naphthylphthalamic acid (Az-NPA), has been synthesized and characterized. This potential photoaffinity label for the plasma membrane NPA binding protein competes with [³H]NPA for binding sites on Curcurbita pepo L. (zucchini) hypocotyl cell membranes with K_0.5 = 2.8 × 10⁻⁷ molar. The K_0.5 for NPA under these conditions is 2 × 10⁻⁸ molar, indicating that the affinity of Az-NPA for the membranes is only 14-fold lower than NPA. While the binding of Az-NPA to NPA binding sites is reversible in the dark, exposure of the Az-NPA treated membranes to light results in a 30% loss in [³H]NPA binding ability. Pretreatment of the membranes with NPA protects the membranes against photodestruction of [³H]NPA binding sites by Az-NPA supporting the conclusion that Az-NPA destroys these sites by specific covalent attachment.     

Pyrophosphorylases in Solanum tuberosum: II. CATALYTIC PROPERTIES AND REGULATION OF ADP-GLUCOSE AND UDP-GLUCOSE PYROPHOSPHORYLASE ACTIVITIES IN POTATOES

Pyrophosphorylytic kinetic constants (S_0.5, V_max) of partially purified UDP-glucose- and ADP-glucose pyrophosphorylases from potato tubers were determined in the presence of various intermediary metabolites. The S_0.5 of UDP-glucose pyrophosphorylase for UDP-glucose (0.17 millimolar) or pyrophosphate (0.30 millimolar) and the V_max were not influenced by high concentrations (2 millimolar) of these substances. The most efficient activator of ADP-glucose pyrophosphorylase was 3-P-glycerate (A_0.5 = 4.5 × 10⁻⁶ molar). The S_0.5 for ADP-glucose and pyrophosphate was increased 3.5-fold (0.83 to 0.24 millimolar) and 1.8-fold (0.18 to 0.10 millimolar), respectively, with 0.1 millimolar 3-P-glycerate while the V_max was increased nearly 4-fold. The magnitude of 3-P-glycerate stimulation was dependent upon the integrity of key sulfhydryl groups (−SH) and pH. Oxidation or blockage of −SH groups resulted in a marked reduction of enzyme activity. Stimulations of 3.1-, 2.9-, 4.8-, and 9.5-fold were observed at pH 7.5, 8.0, 8.5, and 9.0, respectively, in the presence of 3-P-glycerate (2 millimolar). The most potent inhibitor of ADP-glucose pyrophosphorylase was orthophosphate (I_0.5 = 8.8 × 10⁻⁵. molar). This inhibition was reversed with 3-P-glycerate (1.2 × 10⁻⁴ molar), resulting in an increased I_0.5 value of 1.5 × 10⁻³ molar. Likewise, orthophosphate (7.5 × 10⁻⁴ molar) caused a decrease in the activation efficiency of 3-P-glycerate (A_0.5 from 4.5 × 10⁻⁶ molar to 6.7 × 10⁻⁵ molar). The significance of 3-P-glycerate activation and orthophosphate inhibition in the regulation of α-glucan biosynthesis in Solanum tuberosum is discussed.     

Laser activation of rapid absorption changes in spinach chloroplasts and chlorella

The kinetics of the 520 mμ absorption change in spinach chloroplasts and Chlorella vulgaris following a flash from the ruby laser have been determined as follows: rise halftime ≤ 0.3 × 10⁻⁶ second; rapid recovery halftime = 5 to 6 × 10⁻⁶ second; intermediate recovery halftime = 4 × 10⁻⁴ second (spinach chloroplasts only); slow recovery halftime = 12 to 170 × 10⁻³ second, dependent on the measuring light intensity and aerobicity of the suspension.

The rapid phase of the 520 mμ reaction is approximately independent of temperature, from 295° to 77° Absolute.

With increasing oxygenation of the sample, the extent of the rapid phase decreases, the extent of the slow phase increases, while the extent of the intermediate phase in spinach chloroplasts remains constant.

In spinach chloroplasts, no recovery halftime of the 3 recovery phases for the 520 mμ absorption change was observed to correspond to the halftime for oxidation of cytochrome f (t_½ = 1.3 × 10⁻³ second).

     

Characteristics of the Inhibition of Potato (Solanum tuberosum) Invertase by an Endogenous Proteinaceous Inhibitor in Potatoes

Effect of several parameters on inhibition of potato (Solanum tuberosum) invertase by its endogenous proteinaceous inhibitor was determined using homogeneous preparations of both proteins. The inhibitor and invertase formed an inactive complex with an observed association rate constant at pH 4.70 and 37°C of 8.82 × 10² per molar per second and a dissociation rate constant of 3.3 × 10⁻³ per minute. The inhibitor appeared to bind to invertase in more than one step. Initial interaction (measured by loss of invertase activity) was rapid, relatively weak, readily reversible (K_i of 2 × 10⁻⁶ molar) and noncompetitive with substrate at pH 4.70. Initial interaction was probably followed by isomerization to a tighter (K_i of 6.23 × 10⁻⁸ molar) complex, which dissociated slowly with a half-time of 3.5 hour. Interaction between enzyme and inhibitor appeared to be of ionic character and essentially pH independent between pH 3.5 and 7.4.     

Biodegradation of Atrazine by Agrobacterium radiobacter J14a and Use of This Strain in Bioremediation of Contaminated Soil

We examined the ability of a soil bacterium, Agrobacterium radiobacter J14a, to degrade the herbicide atrazine under a variety of cultural conditions, and we used this bacterium to increase the biodegradation of atrazine in soils from agricultural chemical distribution sites. J14a cells grown in nitrogen-free medium with citrate and sucrose as carbon sources mineralized 94% of 50 μg of [¹⁴C-U-ring]atrazine ml⁻¹ in 72 h with a concurrent increase in the population size from 7.9 × 10⁵ to 5.0 × 10⁷ cells ml⁻¹. Under these conditions cells mineralized the [ethyl-¹⁴C]atrazine and incorporated approximately 30% of the ¹⁴C into the J14a biomass. Cells grown in medium without additional carbon and nitrogen sources degraded atrazine, but the cell numbers did not increase. Metabolites produced by J14a during atrazine degradation include hydroxyatrazine, deethylatrazine, and deethyl-hydroxyatrazine. The addition of 10⁵ J14a cells g⁻¹ into soil with a low indigenous population of atrazine degraders treated with 50 and 200 μg of atrazine g⁻¹ soil resulted in two to five times higher mineralization than in the noninoculated soil. Sucrose addition did not result in significantly faster mineralization rates or shorten degradation lag times. However, J14a introduction (10⁵ cells g⁻¹) into another soil with a larger indigenous atrazine-mineralizing population reduced the atrazine degradation lag times below those in noninoculated treatments but did not generally increase total atrazine mineralization.     

Effects of Salicylic and Acetylsalicylic Acids on the Scotonastic and Photonastic Leaflet Movements of Cassia fasciculata

Salicylic and acetylsalicylic acids applied on excised leaves of Cassia fasciculata modify the dark-induced (scotonastic) and light-induced (photonastic) leaflet movements. They inhibit the scotonastic movements in a dose-dependent manner from 1 × 10⁻⁴ to 1 × 10⁻³ molar and they promote the photonastic movements at an optimum concentration of 5 × 10⁻⁴ molar. These results suggest that these phenolic compounds do not act specifically on the K⁺ uptake, which was shown to be inhibited by their action on other materials.     

Inhibition of coral and algal photosynthesis by ca-antagonist phenothiazine drugs

The effects of various calcium ion antagonists and ion transport inhibitors on photosynthetic O₂ evolution of corals, isolated zooxanthellae, sea anemone tentacles, and Chlorococcum oleofaciens were measured. Only the phenothiazine drugs were effective at inhibiting photosynthesis. Trifluoperazine, a calcium ion antagonist drug, inhibited at low concentrations, with 10⁻⁴ molar and 8 × 10⁻⁶ molar completely abolishing photosynthesis in the intact corals and isolated zooxanthellae, respectively. Net photosynthetic O₂ evolution of C. oleofaciens was eliminated by concentrations of trifluoperazine as low as 2.8 × 10⁻⁵ molar.     

Effects of abscisic Acid treatment on the thermostability of the photosynthetic apparatus in barley chloroplasts

Thermostability of the photosynthetic apparatus of abscisic acid (ABA)-treated seedlings of barley (Hordeum vulgare) was studied by light-scattering and by fluorescence measurements of isolated chloroplasts. ABA treatment markedly decreased heat damage of the chloroplast ultrastructure; an exogenous ABA concentration of 10⁻⁵ molar was most effective. Heat-induced increase of the 77 kilodalton fluorescence ratio F₇₄₀/F₆₈₅ was also smaller at this ABA concentration. The heat-induced increase of the initial chlorophyll fluorescence level (F_o) was virtually eliminated in ABA-treated (10⁻⁵ molar) chloroplasts up to 45°C and slightly increased at 50°C, relative to control chloroplasts where F_o increased even at 35°C and reached its maximal value at 45°C. In control chloroplasts, F_o increased with a 5-minute pretreatment temperature, an effect observed as low as 35°C. F_o was maximal at 45°C. In contrast, chloroplasts treated with 10⁻⁵ molar ABA did not exhibit a heat-induced increase in F_o until 50°C.     

Inhibition of HCO(3) Binding to Photosystem II by Atrazine at a Low-Affinity Herbicide Binding Site

In maize chloroplasts, the ratio of HCO₃⁻ (anion) binding sites to high-affinity atrazine binding sites is unity. In the dark, atrazine noncompetitively inhibits the binding of half of the HCO₃⁻ to the photosystem II (PSII) complexes. The inhibition of binding saturates at 5 micromolar atrazine, little inhibition is seen at 0.5 micromolar atrazine, although the high-affinity herbicide binding sites are nearly filled at this concentration. This means that HCO₃⁻ and atrazine interact noncompetitively at a specific low-affinity herbicide binding site that exists on a portion of the PSII complexes. Light abolishes the inhibitory effects of atrazine on HCO₃⁻ binding. Based on the assumption that there is one high-affinity atrazine binding site per PSII complex, we conclude that there is also only one binding site for HCO₃⁻ with a dissociation constant near 80 micromolar. The location of the HCO₃⁻ binding site, and the low-affinity atrazine binding site, is not known.     

Triazine Resistance in Senecio vulgaris Parental and Nearly Isonuclear Backcrossed Biotypes Is Correlated with Reduced Productivity

Isonuclear triazine-susceptible and triazine-resistant Senecio vulgaris L. biotypes were developed by making reciprocal crosses between susceptible and resistant biotypes to obtain F₁ hybrids and backcrossing the hybrids to the appropriate pollen parent. The electrophoretic isozyme patterns of the enzyme aconitase obtained from leaf extracts of triazine-susceptible parental (S) and backcrossed (S×R_BC6) biotypes, and triazine-resistant parental (R) and backcrossed (R×S_BC6) biotypes verified that the biotypes had the expected nuclear genomes. Atrazine inhibition of chloroplast whole chain electron transport from water to methyl viologen was measured to verify susceptibility or resistance to triazine herbicides. The photosynthetic rate and biomass accumulation of greenhouse grown susceptible and resistant S. vulgaris biotypes were measured 28, 35, 42, 50, 57, and 64 days after planting to determine the effect of altered chloroplast function. S and S×R_BC6 biotypes had CO₂ assimilation rates of 16.2 and 16.6 micromoles CO₂ per square meter per second, respectively, and I₅₀ values (herbicide concentration producing 50% inhibition) of about 0.49 micromolar atrazine. The corresponding values for the R and R×S_BC6 biotypes were 14.7 and 14.6 micromoles CO₂ per square meter per second with I₅₀ values of 65.0 micromolar atrazine. The S biotype was larger and more productive than the R biotype at all harvests. At the harvest 57 days after planting, mean shoot dry weight was 33.2 and 8.7 grams for the S and R biotypes, respectively. The growth effect associated with chloroplast differences was shown in comparisons of the S biotype with the R×S_BC6 biotype and of the S×R_BC6 biotype with the R biotype. The R×S_BC6 biotype had 72% of the shoot dry weight of the S biotype while the R biotype had 55% of the shoot dry weight of the S×R_BC6 biotype. The R×S_BC6 and R biotypes produced about 73 and 62% of the leaf area of the S and S×R_BC6 biotypes, respectively. Relative growth rate was similar in biotypes with the same nuclear genome; however, instantaneous unit leaf rate was higher in the S compared to the R×S_BC6 biotype and in the S×R_BC6 compared to the R biotype. At 57 days after planting, the cumulative leaf area duration (i.e. photosynthetic opportunity) of the R×S_BC6 and R biotypes was 86 and 66% of that of the S and S×R_BC6 biotypes, respectively. Our data indicate that impaired chloroplast function in triazine resistant S. vulgaris biotypes limits growth and productivity at the whole plant level.     

Binding Specificity and Possible Analytical Applications of the Cytokinin-binding Antibody, Anti-N-Benzyladenosine

Antibodies elicited in rabbits by immunization with an N⁶-benzyladenosine-bovine serum albumin conjugate bound N⁶-benzyladenosine specifically. The affinity constants and specific binding site concentrations for a number of cytokinins and related compounds were estimated by nonlinear least squares analysis of direct or competitive ultrafiltration data. The antisera contained 230 to 330 nanomoles of cytokinin binding sites per gram protein. Affinity constants were 8.8 × 10⁸ molar⁻¹ for 6-benzylaminopurine, 8.4 × 10⁷ molar⁻¹ for kinetin, 9.1 × 10⁷ molar⁻¹ for 6-(3-methyl-2-butenylamino)purine, 6.6 × 10⁶ molar⁻¹ for 6-(4-hydroxy-3-methyl-trans-2-butenylamino)purine, and 2.0 × 10⁴ molar⁻¹ for 6-methylaminopurine. Affinity constants were below the limit of detectability (<10⁴ molar⁻¹) for benzylamine, adenine, and other adenine derivatives without an N⁶-side chain. The N⁶-substituent was thus immunodominant, but the purine moiety was also necessary for binding affinity. The antibodies were immobilized on cyanogen bromide-activated Sepharose with 95% retention of binding capacity and without apparent change in affinity constants. Columns of the immobilized antibody retained 64% of the [³H]6-(3-methyl-2-butenylam-ino)purine from 2 nanomolar solutions and readily trapped [¹⁴C]6-benzylaminopurine that had been added to crude extracts of cabbage. Aqueous 10% pyridine adjusted to pH 7.3 with formic acid effectively eluted bound cytokinins from gel columns without loss of binding capacity of the immobilized antibody.     

Immobilised Histidine Tagged β 2-Adrenoceptor Oriented by a Diazonium Salt Reaction and Its Application in Exploring Drug-Protein Interaction Using Ephedrine and Pseudoephedrine as Probes

A new oriented method using a diazonium salt reaction was developed for linking β ₂-adrenoceptor (β ₂-AR) on the surface of macroporous silica gel. Stationary phase containing the immobilised receptor was used to investigate the interaction between β ₂-AR and ephedrine plus pseudoephedrine by zonal elution. The isotherms of the two drugs best fit the Langmuir model. Only one type of binding site was found for ephedrine and pseudoephedrine targeting β ₂-AR. At 37 °C, the association constants during the binding were (5.94±0.05)×10³/M for ephedrine and (3.80±0.02) ×10³/M for pseudoephedrine, with the binding sites of (8.92±0.06) ×10⁻⁴ M. Thermodynamic studies showed that the binding of the two compounds to β ₂-AR was a spontaneous reaction with exothermal processes. The ΔG^θ, ΔH^θ and ΔS^θ for the interaction between ephedrine and β ₂-AR were −(22.33±0.04) kJ/mol, −(6.51±0.69) kJ/mol and 50.94±0.31 J/mol·K, respectively. For the binding of pseudoephedrine to the receptor, these values were −(21.17±0.02) kJ/mol, −(7.48±0.56) kJ/mol and 44.13±0.01 J/mol·K. Electrostatic interaction proved to be the driving force during the binding of the two drugs to β ₂-AR. The proposed immobilised method will have great potential for attaching protein to solid substrates and realizing the interactions between proteins and drugs.     

Binding of β-lactam antibiotics to the exocellular dd-carboxypeptidase–transpeptidase of Streptomyces R39

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their β-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its β-lactam ring. In Tris–NaCl–MgCl₂ buffer at pH7.7 and 37°C, the rate constants for the dissociation of the antibiotic–enzyme complexes were 2.8×10⁻⁶, 1.5×10⁻⁶ and 0.63×10⁻⁶s⁻¹ (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [¹⁴C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a β-lactam-antibiotic-destroying enzyme but did not function as a β-lactamase. Incubation at 37°C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [¹⁴C]benzylpenicillin–enzyme complex. The rate constants were 1.6×10⁻⁵s⁻¹ and 0.8×10⁻⁴s⁻¹ respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site.     

High-Frequency Phage-Mediated Gene Transfer among Escherichia coli Cells, Determined at the Single-Cell Level

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3 × 10⁻⁸ to 2 × 10⁻⁶, 1 × 10⁻⁸ to 4 × 10⁻⁸, and <4 × 10⁻⁹ to 4 × 10⁻⁸ per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7 × 10⁻⁴ to 1 × 10⁻³, 9 × 10⁻⁴ to 3 × 10⁻³, and 5 × 10⁻⁴ to 4 × 10⁻³ for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.     

A th-1 Mutant of Arabidopsis thaliana Is Defective for a Thiamin-Phosphate-Synthesizing Enzyme: Thiamin Phosphate Pyrophosphorylase

We have examined the activity of the thiamin phosphate pyrophosphorylase in Arabidopsis thaliana wild type and in a mutant (th-1) which requires exogenous thiamin for growth. Mutant and wild-type plants grown in 1 × 10⁻⁷ molar thiamin were used for the examination of the production of thiamin and thiamin monophosphate (TMP) using 4-methyl-5-hydroxyethylthiazole phosphate and 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate as substrates. While the wild-type strain formed both thiamin and TMP, the th-1 mutant did not. When TMP was added to the extracts, the th-1 mutant, as well as wild type, produced thiamin. Accordingly, it was concluded that the th-1 mutant was defective in the activity of TMP pyrophosphorylase. Some of the characteristics of the enzyme from the wild-type plant were examined. The optimum temperature for the reaction is 45°C, and the K_m values for the substrates are 2.7 × 10⁻⁶ molar for 4-methyl-5-hydroxyethylthiazole phosphate and 1.8 × 10⁻⁶ molar for 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate.     

The Enzymatic Synthesis of Rubber Polymer in Parthenium argentatum Gray

Washed rubber particles isolated from stem homogenates of Parthenium argentatum Gray by ultracentrifugation and gel filtration on columns of LKB Ultrogel AcA34 contain rubber transferase which catalyzes the polymerization of isopentenyl pyrophosphate into rubber polymer. The polymerization reaction requires Mg²⁺ isopentenyl pyrophosphate, and an allylic pyrophosphate. The K_m values for Mg²⁺, isopentenyl pyrophosphate, and dimethylallyl pyrophosphate were 5.2 × 10⁻⁴ molar, 8.3 × 10⁻⁵ molar, and 9.6 × 10⁻⁵ molar, respectively. The molecular characteristics of the rubber polymer synthesized from [¹⁴C]isopentenyl pyrophosphate were examined by gel permeation chromatography on three linear columns of 1 × 10⁶ to 500 Ångstroms Ultrastyragel in a Waters 150C Gel Permeation Chromatograph. The peak molecular weight of the radioactive polymer increased from 70,000 in 15 minutes to 750,000 in 3 hours. The weight average molecular weight of the polymer synthesized over a 3 hour period was 1.17 × 10⁶ compared to 1.49 × 10⁶ for the natural rubber polymer extracted from the rubber particles. Over 90% of the in vitro formation of the rubber polymer was de novo from dimethylallyl pyrophosphate and isopentenyl pyrophosphate. Treatment of the washed rubber particles with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilized the rubber transferase. The solubilized enzyme(s) catalyzed the polymerization of isopentenyl pyrophosphate into rubber polymer with a peak molecular weight of 1 × 10⁵ after 3 hours of incubation with Mg²⁺ and dimethylallyl pyrophosphate. The data support the conclusion that the soluble preparation of rubber transferase is capable of catalyzing the formation of a high molecular weight rubber polymer from an allylic pyrophosphate initiator and isopentenyl pyrophosphate monomer.     

Genome-wide Association Study Identifies TNFSF15 and POU2AF1 as Susceptibility Loci for Primary Biliary Cirrhosis in the Japanese Population

For the identification of susceptibility loci for primary biliary cirrhosis (PBC), a genome-wide association study (GWAS) was performed in 963 Japanese individuals (487 PBC cases and 476 healthy controls) and in a subsequent replication study that included 1,402 other Japanese individuals (787 cases and 615 controls). In addition to the most significant susceptibility region, human leukocyte antigen (HLA), we identified two significant susceptibility loci, TNFSF15 (rs4979462) and POU2AF1 (rs4938534) (combined odds ratio [OR] = 1.56, p = 2.84 × 10⁻¹⁴ for rs4979462, and combined OR = 1.39, p = 2.38 × 10⁻⁸ for rs4938534). Among 21 non-HLA susceptibility loci for PBC identified in GWASs of individuals of European descent, three loci (IL7R, IKZF3, and CD80) showed significant associations (combined p = 3.66 × 10⁻⁸, 3.66 × 10⁻⁹, and 3.04 × 10⁻⁹, respectively) and STAT4 and NFKB1 loci showed suggestive association with PBC (combined p = 1.11 × 10⁻⁶ and 1.42 × 10⁻⁷, respectively) in the Japanese population. These observations indicated the existence of ethnic differences in genetic susceptibility loci to PBC and the importance of TNF signaling and B cell differentiation for the development of PBC in individuals of European descent and Japanese individuals.