Identification of a 5′-Deoxyadenosine Deaminase in Methanocaldococcus jannaschii and Its Possible Role in Recycling the Radical S-Adenosylmethionine Enzyme Reaction Product 5′-Deoxyadenosine

We characterize here the MJ1541 gene product from Methanocaldococcus jannaschii, an enzyme that was annotated as a 5′-methylthioadenosine/S-adenosylhomocysteine deaminase (EC 3.5.4.31/3.5.4.28). The MJ1541 gene product catalyzes the conversion of 5′-deoxyadenosine to 5′-deoxyinosine as its major product but will also deaminate 5′-methylthioadenosine, S-adenosylhomocysteine, and adenosine to a small extent. On the basis of these findings, we are naming this new enzyme 5′-deoxyadenosine deaminase (DadD). The K_m for 5′-deoxyadenosine was found to be 14.0 ± 1.2 μM with a k_cat/K_m of 9.1 × 10⁹ M⁻¹ s⁻¹. Radical S-adenosylmethionine (SAM) enzymes account for nearly 2% of the M. jannaschii genome, where the major SAM derived products is 5′-deoxyadenosine. Since 5′-dA has been demonstrated to be an inhibitor of radical SAM enzymes; a pathway for removing this product must be present. We propose here that DadD is involved in the recycling of 5′-deoxyadenosine, whereupon the 5′-deoxyribose moiety of 5′-deoxyinosine is further metabolized to deoxyhexoses used for the biosynthesis of aromatic amino acids in methanogens.     

Stimulation of human 8-oxoguanine-DNA glycosylase by AP-endonuclease: potential coordination of the initial steps in base excision repair

8-Oxoguanine-DNA glycosylase 1 (OGG1), with intrinsic AP lyase activity, is the major enzyme for repairing 7,8-dihydro-8-oxoguanine (8-oxoG), a critical mutagenic DNA lesion induced by reactive oxygen species. Human OGG1 excised the damaged base from an 8-oxoG·C-containing duplex oligo with a very low apparent k_cat of 0.1 min^–1 at 37°C and cleaved abasic (AP) sites at half the rate, thus leaving abasic sites as the major product. Excision of 8-oxoG by OGG1 alone did not follow Michaelis–Menten kinetics. However, in the presence of a comparable amount of human AP endonuclease (APE1) the specific activity of OGG1 was increased ~5-fold and Michaelis–Menten kinetics were observed. Inactive APE1, at a higher molar ratio, and a bacterial APE (Nfo) similarly enhanced OGG1 activity. The affinity of OGG1 for its product AP·C pair (K_d ~ 2.8 nM) was substantially higher than for its substrate 8-oxoG·C pair (K_d ~ 23.4 nM) and the affinity for its final β-elimination product was much lower (K_d ~ 233 nM). These data, as well as single burst kinetics studies, indicate that the enzyme remains tightly bound to its AP product following base excision and that APE1 prevents its reassociation with its product, thus enhancing OGG1 turnover. These results suggest coordinated functions of OGG1 and APE1, and possibly other enzymes, in the DNA base excision repair pathway.     

Control of 5′,5′-Dinucleoside Triphosphate Catabolism by APH1, a Saccharomyces cerevisiae Analog of Human FHIT

The putative human tumor suppressor gene FHIT (fragile histidine triad) (M. Ohta et al., Cell 84:587–597, 1996) encodes a protein behaving in vitro as a dinucleoside 5′,5′′′-P¹,P³-triphosphate (Ap₃A) hydrolase. In this report, we show that the Saccharomyces cerevisiae APH1 gene product, which resembles human Fhit protein, also hydrolyzes dinucleoside 5′,5′-polyphosphates, with Ap₃A being the preferred substrate. Accordingly, disruption of the APH1 gene produced viable S. cerevisiae cells containing reduced Ap₃A-hydrolyzing activity and a 30-fold-elevated Ap₃N concentration.     

The Reaction Mechanism of Methyl-Coenzyme M Reductase: HOW AN ENZYME ENFORCES STRICT BINDING ORDER*

Methyl-coenzyme M reductase (MCR) is a nickel tetrahydrocorphinoid (coenzyme F430) containing enzyme involved in the biological synthesis and anaerobic oxidation of methane. MCR catalyzes the conversion of methyl-2-mercaptoethanesulfonate (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoB₇SH) to CH₄ and the mixed disulfide CoBS-SCoM. In this study, the reaction of MCR from Methanothermobacter marburgensis, with its native substrates was investigated using static binding, chemical quench, and stopped-flow techniques. Rate constants were measured for each step in this strictly ordered ternary complex catalytic mechanism. Surprisingly, in the absence of the other substrate, MCR can bind either substrate; however, only one binary complex (MCR·methyl-SCoM) is productive whereas the other (MCR·CoB₇SH) is inhibitory. Moreover, the kinetic data demonstrate that binding of methyl-SCoM to the inhibitory MCR·CoB₇SH complex is highly disfavored (K_d = 56 mm). However, binding of CoB₇SH to the productive MCR·methyl-SCoM complex to form the active ternary complex (CoB₇SH·MCR(Ni^I)·CH₃SCoM) is highly favored (K_d = 79 μm). Only then can the chemical reaction occur (k_obs = 20 s⁻¹ at 25 °C), leading to rapid formation and dissociation of CH₄ leaving the binary product complex (MCR(Ni^II)·CoB₇S⁻·SCoM), which undergoes electron transfer to regenerate Ni(I) and the final product CoBS-SCoM. This first rapid kinetics study of MCR with its natural substrates describes how an enzyme can enforce a strictly ordered ternary complex mechanism and serves as a template for identification of the reaction intermediates.     

Valuable ingredients and feed toxicity evaluation of Microcystis aeruginosa acidolysis product in mice

This research studied the extraction from Microcystis aeruginosa using hydrochloric acid method as a potentially valuable protein resource from eutrophic lakes. Amino acid composition, residual algal toxins, and heavy metals of the acidolysis product were studied. After 18 h of hydrochloric acid treatment, the product of M. aeruginosa contained 17 amino acids, 51.34% of total amino acid requirements, and 30.25% of the livestock and poultry essential amino acid (Eaa). The residual microcystin-LR (MC-LR) was 0.94 µg kg⁻¹, which was less than WHO drinking water limit of microcystins. The removal ratio of microcystins was higher than 99.99% during the process of hydrolysis. The concentration of heavy metals of the product was in compliance with feed standards. Furthermore, using Horn’s method, Mouse Micronucleus Test and Sperm Shape Abnormality Test were conducted to study the forage safety of the product. Half lethal dose (LD₅₀) of acidolysis product in mice was >9.09 g kg⁻¹ body weight, actually belonging to non-toxic grade. Every dose treatment did not significantly increase activities of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and γ-glutamyltransferase (γ-GT). The results of both micronucleus test and sperm shape abnormality test were negative, which suggested the product with no mutagenicity and sperm malformation effects. This study indicated that the acidolysis product of M. aeruginosa was safe to be used as a feed ingredient.     

The quadruplex r(CGG)n destabilizing cationic porphyrin TMPyP4 cooperates with hnRNPs to increase the translation efficiency of fragile X premutation mRNA

The 5′ untranslated region of the FMR1 gene which normally includes 4–55 d(CGG) repeats expands to > 55–200 repeats in carriers of fragile X syndrome premutation. Although the levels of premutation FMR1 mRNA in carrier cells are 5–10-fold higher than normal, the amount of the product FMR protein is unchanged or reduced. We demonstrated previously that premutation r(CGG)_n tracts formed quadruplex structures that impeded translation and lowered the efficiency of protein synthesis. Normal translation could be restored in vivo by the quadruplex r(CGG)_n destabilizing action of CBF-A and hnRNP A2 proteins. Here we report that the quadruplex-interacting cationic porphyrin TMPyP4 by itself and in cooperation with CBF-A or hnRNP A2 also unfolded quadruplex r(CGG)_n and increased the efficiency of translation of 5′-(CGG)₉₉ containing reporter firefly (FL) mRNA. TMPyP4 destabilized in vitro a (CGG)₃₃ intramolecular quadruplex structure and enhanced the translation of 5′-(CGG)₉₉-FL mRNA in a rabbit reticulocyte lysate and in HEK293 cells. The efficiency of translation of (CGG)₉₉-FL mRNA was additively increased in cells exposed to TMPyP4 together with CBF-A. Whereas low doses of TMPyP4, CBF-A or hnRNP A2 by themselves did not affect the in vivo utilization of (CGG)₉₉-FL mRNA, introduction of TMPyP4 together with either protein synergistically augmented its translation efficiency.     

Common Single Nucleotide Polymorphisms in Genes Related to Immune Function and Risk of Papillary Thyroid Cancer

Accumulating evidence suggests that alterations in immune function may be important in the etiology of papillary thyroid cancer (PTC). To identify genetic markers in immune-related pathways, we evaluated 3,985 tag single nucleotide polymorphisms (SNPs) in 230 candidate gene regions (adhesion-extravasation-migration, arachidonic acid metabolism/eicosanoid signaling, complement and coagulation cascade, cytokine signaling, innate pathogen detection and antimicrobials, leukocyte signaling, TNF/NF-kB pathway or other) in a case-control study of 344 PTC cases and 452 controls. We used logistic regression models to estimate odds ratios (OR) and calculate one degree of freedom P values of linear trend (P_SNP-trend) for the association between genotype (common homozygous, heterozygous, variant homozygous) and risk of PTC. To correct for multiple comparisons, we applied the false discovery rate method (FDR). Gene region- and pathway-level associations (P_Region and P_Pathway) were assessed by combining individual P_SNP-trend values using the adaptive rank truncated product method. Two SNPs (rs6115, rs6112) in the SERPINA5 gene were significantly associated with risk of PTC (P_SNP-FDR/P_SNP-trend = 0.02/6×10⁻⁶ and P_SNP-FDR/P_SNP-trend = 0.04/2×10⁻⁵, respectively). These associations were independent of a history of autoimmune thyroiditis (OR = 6.4; 95% confidence interval: 3.0–13.4). At the gene region level, SERPINA5 was suggestively associated with risk of PTC (P_Region-FDR/P_Region =0.07/0.0003). Overall, the complement and coagulation cascade pathway was the most significant pathway (P_Pathway = 0.02) associated with PTC risk largely due to the strong effect of SERPINA5. Our results require replication but suggest that the SERPINA5 gene, which codes for the protein C inhibitor involved in many biological processes including inflammation, may be a new susceptibility locus for PTC.     

Suicide Inactivation of Catechol 2,3-Dioxygenase from Pseudomonas putida mt-2 by 3-Halocatechols

The inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-chloro- and 3-fluorocatechol and the iron-chelating agent Tiron (catechol-3,5-disulfonate) was studied. Whereas inactivation by Tiron is an oxygen-independent and mostly reversible process, inactivation by the 3-halocatechols was only observed in the presence of oxygen and was largely irreversible. The rate constants for inactivation (K₂) were 1.62 × 10⁻³ sec⁻¹ for 3-chlorocatechol and 2.38 × 10⁻³ sec⁻¹ for 3-fluorocatechol. The inhibitor constants (K_i) were 23 μM for 3-chlorocatechol and 17 μM for 3-fluorocatechol. The kinetic data for 3-fluorocatechol could only be obtained in the presence of 2-mercaptoethanol. Besides inactivated enzyme, some 2-hydroxyhexa-2,4-diendioic acid was formed from 3-chlorocatechol, suggesting 5-chloroformyl-2-hydroxypenta-2,4-dienoic acid as the actual suicide product of meta-cleavage. A side product of 3-fluorocatechol cleavage is a yellow compound with the spectral characteristics of a 2-hydroxy-6-oxohexa-2,4-dienoic acid indicating 1,6-cleavage. Rates of inactivation by 3-fluorocatechol were reduced in the presence of superoxide dismutase, catalase, formate, and mannitol, which implies that superoxide anion, hydrogen peroxide, and hydroxyl radical exhibit additional inactivation.     

The Effects of Exercise Intensity vs. Metabolic State on the Variability and Magnitude of Left Ventricular Twist Mechanics during Exercise

Increased left ventricular (LV) twist and untwisting rate (LV twist mechanics) are essential responses of the heart to exercise. However, previously a large variability in LV twist mechanics during exercise has been observed, which complicates the interpretation of results. This study aimed to determine some of the physiological sources of variability in LV twist mechanics during exercise. Sixteen healthy males (age: 22 ± 4 years, V˙O_2peak: 45.5 ± 6.9 ml∙kg^-1∙min^-1, range of individual anaerobic threshold (IAT): 32–69% of V˙O_2peak) were assessed at rest and during exercise at: i) the same relative exercise intensity, 40%_peak, ii) at 2% above IAT, and, iii) at 40%_peak with hypoxia (40%_peak+HYP). LV volumes were not significantly different between exercise conditions (P > 0.05). However, the mean margin of error of LV twist was significantly lower (F_2,47 = 2.08, P < 0.05) during 40%peak compared with IAT (3.0 vs. 4.1 degrees). Despite the same workload and similar LV volumes, hypoxia increased LV twist and untwisting rate (P < 0.05), but the mean margin of error remained similar to that during 40%peak (3.2 degrees, P > 0.05). Overall, LV twist mechanics were linearly related to rate pressure product. During exercise, the intra-individual variability of LV twist mechanics is smaller at the same relative exercise intensity compared with IAT. However, the absolute magnitude (degrees) of LV twist mechanics appears to be associated with the prevailing rate pressure product. Exercise tests that evaluate LV twist mechanics should be standardised by relative exercise intensity and rate pressure product be taken into account when interpreting results.     

The NADP-Dependent Methylene Tetrahydromethanopterin Dehydrogenase in Methylobacterium extorquens AM1

An NADP-dependent methylene tetrahydromethanopterin (H₄MPT) dehydrogenase has recently been proposed to be involved in formaldehyde oxidation to CO₂ in Methylobacterium extorquens AM1. We report here on the purification of this novel enzyme to apparent homogeneity. Via the N-terminal amino acid sequence, it was identified to be the mtdA gene product. The purified enzyme catalyzed the dehydrogenation of methylene H₄MPT with NADP⁺ rather than with NAD⁺, with a specific activity of approximately 400 U/mg of protein. It also catalyzed the dehydrogenation of methylene tetrahydrofolate (methylene H₄F) with NADP⁺. With methylene H₄F as the substrate, however, the specific activity (26 U/mg) and the catalytic efficiency (V_max/K_m) were approximately 20-fold lower than with methylene H₄MPT. Whereas the dehydrogenation of methylene H₄MPT (E₀ = −390 mV) with NADP⁺ (E₀ = −320 mV) proceeded essentially irreversibly, the dehydrogenation of methylene H₄F (E₀ = −300 mV) was fully reversible. Comparison of the primary structure of the NADP-dependent dehydrogenase from M. extorquens AM1 with those of methylene H₄F dehydrogenases from other bacteria and eucarya and with those of methylene H₄MPT dehydrogenases from methanogenic archaea revealed only marginally significant similarity (<15%).     

Components of Sodium and Chloride Flux Across Toad Bladder

The effect of transepithelial potential difference (ψ) on Na and Cl flux across toad bladder was assessed by measuring isotopic flux between identical media at various values of ψ. The contribution of edge damage to ionic permeability was eliminated, resulting in relatively high spontaneous ψ (-97 ±4 mv) and low electrical conductance g. Bidirectional Na fluxes were measured simultaneously. Unidirectional Cl fluxes were measured in paired hemibladders at ψ = 0 mv or -97 mv. Net Na flux J_Na, at ψ = 0 mv, was slightly less than short-circuit current (SCC). At ψ = -97 mv, J_Na averaged 17% of SCC, and was sometimes zero. ΔJ_Na/Δψ (= g₊) averaged 60% of g between -97 mv and +75 mv; at -150 mv, g₊ fell, indicating rectification. Analysis of unidirectional Na fluxes indicates low passive conductance (1.5 μmho/mg wet weight), a bidirectional, electrically neutral flux of approximately 0.13 μa/mg, and relatively large conductance of the active transport path at ψ ≥ -97 mv. The absence of appreciable transstimulation of serosal (S)-to-mucosal (M) Na flux (in response to increasing mucosal Na concentration) indicates that the electrically neutral flux is not exchange diffusion in the usual sense. Analysis of Cl fluxes indicates similar values for passive conductance and neutral flux, suggesting linked neutral flux of Na and Cl. Either the electromotive force of the Na pump E, its conductance g_a, or both are strong functions of ψ. The product of these two quantities, Eg_a, is a measure of the “transport capacity” at any given value of ψ, independent of the direct effect of ψ on J_Na through the pump path. Eg_a varies with ψ. Hence estimation of the net Na flux or current at any one value of ψ, including ψ = 0, fails to reveal the maximal transport capacity of the pump, its resting electromotive force (when J_Na = 0 through the pump), or the dependence of transport capacity on potential.     

Formation of 2'-deoxyoxanosine from 2'-deoxyguanosine and nitrous acid: mechanism and intermediates

The reaction mechanism for the formation of 2′-deoxyoxanosine from 2′-deoxyguanosine by nitrous acid was explored using methyl derivatives of guanosine and an isolated intermediate of the reaction. When 1-methylguanosine was incubated with NaNO₂ under acidic conditions, N⁵-methyloxanosine and 1-methylxanthosine were generated, whereas the same treatment of N²,N²-dimethylguanosine generated no product. In a similar experiment without NO₂^–, participation of a Dimroth rearrangement was ruled out. In the guanosine–HNO₂ reaction system, an intermediate with a half-life of 5.6 min (pH 7.0, 20°C) was isolated and tentatively identified as a diazoate derivative of guanosine. The diazoate intermediate was converted into oxanosine and xanthosine at a molar ratio (oxanosine:xanthosine) of 0.26 at pH 7.0 and 20°C. The ratio was not affected by the incubation pH between 2 and 10, but increased linearly with temperature from 0.22 (0°C) to 0.32 (50°C). The addition of acetone also increased the ratio up to 0.85 (98% acetone). Based on these results, a con-ceivable pathway for the formation of 2′-deoxyoxanosine from 2′-deoxyguanosine by HNO₂ is proposed.     

atp Mutants of Escherichia coli Fail To Grow on Succinate Due to a Transport Deficiency

Escherichia coli atp mutants, which lack a functional H⁺-ATPase complex, are capable of growth on glucose but not on succinate or other C₄-dicarboxylates (Suc⁻ phenotype). Suc⁺ revertants of an atp deletion strain were isolated which were capable of growth on succinate even though they lack the entire H⁺-ATPase complex. Complementation in trans with the yhiF gene suppressed the growth of the Suc⁺ mutants on succinate, which implicates the yhiF gene product in the regulation of C₄-dicarboxylate metabolism. Indeed, when the E. coli C₄-dicarboxylate transporter (encoded by the dctA gene) was expressed in trans, the Suc⁻ phenotype of the atp deletion strain reverted to Suc⁺, which shows that the reason why the E. coli atp mutant is unable to grow aerobically on C₄-dicarboxylates is insufficient transport capacity for these substrates.     

NO(3) Enhances the Kinase Activity for Phosphorylation of Phosphoenolpyruvate Carboxylase and Sucrose Phosphate Synthase Proteins in Wheat Leaves: Evidence from the Effects of Mannose and Okadaic Acid

The aim of this work was to determine which of the two reactions (i.e. phosphorylation or dephosphorylation) involved in the establishment of the phosphorylated status of the wheat leaf phosphoenolpyruvate carboxylase and sucrose phosphate synthase protein responds in vivo to NO₃⁻ uptake and assimilation. Detached mature leaves of wheat (Triticum aestivum L. cv Fidel) were fed with N-free (low-NO₃⁻ leaves) or 40 mm NO₃⁻ solution (high-NO₃⁻ leaves). The specific inhibition of the enzyme-protein kinase or phosphatase activities was obtained in vivo by addition of mannose or okadaic acid, respectively, in the uptake solution. Mannose at 50 mm, by blocking the kinase reaction, inhibited the processes of NO₃⁻-dependent phosphoenolpyruvate carboxylase activation and sucrose phosphate synthase deactivation. Following the addition of mannose, the deactivation of phosphoenolpyruvate carboxylase and the activation of sucrose phosphate synthase, both due to the enzyme-protein dephosphorylation, were at the same rate in low-NO₃⁻ and high-NO₃⁻ leaves, indicating that NO₃⁻ had no effect per se on the enzyme-protein phosphatase activity. Upon treatment with okadaic acid, the higher increase of phosphoenolpyruvate carboxylase and decrease of sucrose phosphate synthase activities observed in high NO₃⁻ compared with low NO₃⁻ leaves showed evidence that NO₃⁻ enhanced the protein kinase activity. These results support the concept that NO₃⁻, or a product of its metabolism, favors the activation of phosphoenolpyruvate carboxylase and deactivation of sucrose phosphate synthase in wheat leaves by promoting the light activation of the enzyme-protein kinase(s) without affecting the phosphatase(s).     

Novel β-1,4-Mannanase Belonging to a New Glycoside Hydrolase Family in Aspergillus nidulans

Many filamentous fungi produce β-mannan-degrading β-1,4-mannanases that belong to the glycoside hydrolase 5 (GH5) and GH26 families. Here we identified a novel β-1,4-mannanase (Man134A) that belongs to a new glycoside hydrolase (GH) family (GH134) in Aspergillus nidulans. Blast analysis of the amino acid sequence using the NCBI protein database revealed that this enzyme had no similarity to any sequences and no putative conserved domains. Protein homologs of the enzyme were distributed to limited fungal and bacterial species. Man134A released mannobiose (M₂), mannotriose (M₃), and mannotetraose (M₄) but not mannopentaose (M₅) or higher manno-oligosaccharides when galactose-free β-mannan was the substrate from the initial stage of the reaction, suggesting that Man134A preferentially reacts with β-mannan via a unique catalytic mode. Man134A had high catalytic efficiency (k_cat/K_m) toward mannohexaose (M₆) compared with the endo-β-1,4-mannanase Man5C and notably converted M₆ to M₂, M₃, and M₄, with M₃ being the predominant reaction product. The action of Man5C toward β-mannans was synergistic. The growth phenotype of a Man134A disruptant was poor when β-mannans were the sole carbon source, indicating that Man134A is involved in β-mannan degradation in vivo. These findings indicate a hitherto undiscovered mechanism of β-mannan degradation that is enhanced by the novel β-1,4-mannanase, Man134A, when combined with other mannanolytic enzymes including various endo-β-1,4-mannanases.     

Photosynthetic Electron Transport Involved in PxcA-Dependent Proton Extrusion in Synechocystis sp. Strain PCC6803: Effect of pxcA Inactivation on CO2, HCO3−, and NO3− Uptake

The product of pxcA (formerly known as cotA) is involved in light-induced Na⁺-dependent proton extrusion. In the presence of 2,5-dimethyl-p-benzoquinone, net proton extrusion by Synechocystis sp. strain PCC6803 ceased after 1 min of illumination and a postillumination influx of protons was observed, suggesting that the PxcA-dependent, light-dependent proton extrusion equilibrates with a light-independent influx of protons. A photosystem I (PS I) deletion mutant extruded a large number of protons in the light. Thus, PS II-dependent electron transfer and proton translocation are major factors in light-driven proton extrusion, presumably mediated by ATP synthesis. Inhibition of CO₂ fixation by glyceraldehyde in a cytochrome c oxidase (COX) deletion mutant strongly inhibited the proton extrusion. Leakage of PS II-generated electrons to oxygen via COX appears to be required for proton extrusion when CO₂ fixation is inhibited. At pH 8.0, NO₃⁻ uptake activity was very low in the pxcA mutant at low [Na⁺] (~100 μM). At pH 6.5, the pxcA strain did not take up CO₂ or NO₃⁻ at low [Na⁺] and showed very low CO₂ uptake activity even at 15 mM Na⁺. A possible role of PxcA-dependent proton exchange in charge and pH homeostasis during uptake of CO₂, HCO₃⁻, and NO₃⁻ is discussed.     

Reprogramming the tRNA-splicing activity of a bacterial RNA repair enzyme

Programmed RNA breakage is an emerging theme underlying cellular responses to stress, virus infection and defense against foreign species. In many cases, site-specific cleavage of the target RNA generates 2′,3′ cyclic phosphate and 5′-OH ends. For the damage to be repaired, both broken ends must be healed before they can be sealed by a ligase. Healing entails hydrolysis of the 2′,3′ cyclic phosphate to form a 3′-OH and phosphorylation of the 5′-OH to form a 5′-PO₄. Here, we demonstrate that a polynucleotide kinase-phosphatase enzyme from Clostridium thermocellum (CthPnkp) can catalyze both of the end-healing steps of tRNA splicing in vitro. The route of tRNA repair by CthPnkp can be reprogrammed by a mutation in the 3′ end-healing domain (H189D) that yields a 2′-PO₄ product instead of a 2′-OH. Whereas tRNA ends healed by wild-type CthPnkp are readily sealed by T4 RNA ligase 1, the H189D enzyme generates ends that are spliced by yeast tRNA ligase. Our findings suggest that RNA repair enzymes can evolve their specificities to suit a particular pathway.     

Control of Seed Germination by Abscisic Acid : III. Effect on Embryo Growth Potential (Minimum Turgor Pressure) and Growth Coefficient (Cell Wall Extensibility) in Brassica napus L

The physical mechanism of seed germination and its inhibition by abscisic acid (ABA) in Brassica napus L. was investigated, using volumetric growth (= water uptake) rate (dV/dt), water conductance (L), cell wall extensibility coefficient (m), osmotic pressure (_i), water potential (Ψ_i), turgor pressure (P), and minimum turgor for cell expansion (Y) of the intact embryo as experimental parameters. dV/dt, _i, and Ψ_i were measured directly, while m, P, and Y were derived by calculation. Based on the general equation of hydraulic cell growth [dV/dt = Lm/(L + m) (Δ - Y), where Δ = _i - of the external medium], the terms (Lm/(L + m) and _i - Y were defined as growth coefficient (k_G) and growth potential (GP), respectively. Both k_G and GP were estimated from curves relating dV/dt (steady state) to of osmotic test solutions (polyethylene glycol 6000).

During the imbibition phase (0-12 hours after sowing), k_G remains very small while GP approaches a stable level of about 10 bar. During the subsequent growth phase of the embryo, k_G increases about 10-fold. ABA, added before the onset of the growth phase, prevents the rise of k_G and lowers GP. These effects are rapidly abolished when germination is induced by removal of ABA. Neither L (as judged from the kinetics of osmotic water efflux) nor the amount of extractable solutes are affected by these changes. _i and Ψ_i remain at a high level in the ABA-treated seed but drop upon induction of germination, and this adds up to a large decrease of P, indicating that water uptake of the germinating embryo is controlled by cell wall loosening rather than by changes of _i or L. ABA inhibits water uptake by preventing cell wall loosening. By calculating Y and m from the growth equation, it is further shown that cell wall loosening during germination comprises both a decrease of Y from about 10 to 0 bar and an at least 10-fold increase of m. ABA-mediated embryo dormancy is caused by a reversible inhibition of both of these changes in cell wall stability.

     

Purification and Characterization of Pea Epicotyl beta-Amylase

The most abundant β-amylase (EC 3.2.1.2) in pea (Pisum sativum L.) was purified greater than 880-fold from epicotyls of etiolated germinating seedlings by anion exchange and gel filtration chromatography, glycogen precipitation, and preparative electrophoresis. The electrophoretic mobility and relative abundance of this β-amylase are the same as that of an exoamylase previously reported to be primarily vacuolar. The enzyme was determined to be a β-amylase by end product analysis and by its inability to hydrolyze β-limit dextrin and to release dye from starch azure. Pea β-amylase is an approximate 55 to 57 kilodalton monomer with a pl of 4.35, a pH optimum of 6.0 (soluble starch substrate), an Arrhenius energy of activation of 6.28 kilocalories per mole, and a K_m of 1.67 milligrams per milliliter (soluble starch). The enzyme is strongly inhibited by heavy metals, p-chloromer-curiphenylsulfonic acid and N-ethylmaleimide, but much less strongly by iodoacetamide and iodoacetic acid, indicating cysteinyl sulfhydryls are not directly involved in catalysis. Pea β-amylase is competitively inhibited by its end product, maltose, with a K_i of 11.5 millimolar. The enzyme is partially inhibited by Schardinger maltodextrins, with α-cyclohexaamylose being a stronger inhibitor than β-cycloheptaamylose. Moderately branched glucans (e.g. amylopectin) were better substrates for pea β-amylase than less branched or non-branched (amyloses) or highly branched (glycogens) glucans. The enzyme failed to hydrolyze native starch grains from pea and glucans smaller than maltotetraose. The mechanism of pea β-amylase is the multichain type. Possible roles of pea β-amylase in cellular glucan metabolism are discussed.