Purification and Characterization of the Soluble F(1)-ATPase of Oat Root Mitochondria

The properties of the soluble moiety (F₁) of the mitochondrial H⁺-ATPase from oat roots were examined and compared to those of the native mitochondrial membrane-bound enzyme. The chloroform soluble preparation was purified by Sephadex G-200 and DEAE-cellulose chromatography. The purified F₁ preparation contained major polypeptides corresponding to α, β, γ, δ, and ε of apparent molecular mass 58, 55, 35, 22, and 14 kilodaltons, respectively. The purified F₁-ATPase, like the native enzyme, was inhibited by azide (I₅₀ = 10 micromolar), nitrate (I₅₀ = 7-10 millimolar), 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (I₅₀ = 1-3 micromolar), and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (I₅₀ = 3 micromolar). F₁-ATPase activity was stimulated by bicarbonate but not by chloride. In both the native and the F₁-form of the ATPase, ATP was hydrolyzed in preference to GTP. The results indicate that these properties of the native membrane-bound mitochondrial ATPase have been conserved in the purified F₁. In contrast to the membrane-bound enzyme, the F₁-ATPase was not inhibited by oligomycin or by N,N′-dicyclohexylcarbodiimide. The mitochondrial F₁-ATPase from oat roots is analogous to other known F₁F₀-ATPases.     

Mechanism of 2-acetyl-1-pyrroline biosynthesis in Bassia latifolia Roxb. flowers

The flowers of Bassia latifolia are known to contain 2-acetyl-1-pyrroline (2AP), the compound responsible for pleasant aroma in basmati and other scented rice. Four growth stages of Bassia flowers were identified and 2AP contents were analysed in each stage. It was found that 2AP (3.30 ppm) gets synthesized only in fleshy corolla of mature flowers (fourth stage). The activity of γ-aminobutyraldehyde dehydrogenase (AADH); an enzyme responsible for synthesis of γ-aminobutyricacid (GABA) from γ-aminobutyraldehyde (GABald) was assessed in these four stages. The AADH activity was absent in the fourth stage. It was concluded that ceased activity of AADH in fourth stage flowers leads to the accumulation of γ-aminobutyraldehyde which is cyclised spontaneously to Δ¹-pyrroline, the key precursor of 2AP. Δ¹-pyrroline further reacts unenzymatically with methylglyoxal to form 2AP.     

Isolation of Amatoxin-Resistant Lines of Chlamydomonas reinhardtii: Evidence for RNA Polymerase Mutants

The inhibitory activities of amatoxins on the growth of Chlamydomonas reinhardtii have been determined using a convenient assay based upon incubation in multiwell tissue culture plates followed by turbidimetric estimates of growth on a multiwell plate reader. Values for the inhibitory dosage at which growth is 50% of untreated culture (ID₅₀) of 5.4, 6.6, and 5.6 micromolar were obtained for α-amanitin, O-methyl-α-amanitin, and amaninamide, respectively. Treatment of liquid cultures with 1 microgram per milliliter N-methyl-N′ -nitro-N-nitrosoguanidine followed by growth in agar pour tubes containing 25 micromolar α-amanitin led to the selection of several lines demonstrating varying resistance to amanitin inhibition, with ID₅₀ values from 36 micromolar to greater than 200 micromolar. Two lines completely resistant to inhibition by 200 micromolar α-amanitin provided partially purified RNA polymerase activities that were 160-fold and 5600-fold more resistant to inhibition than the analogous enzyme activity from the wild-type strain. These studies provide evidence that Chlamydomonas reinhardtii does not contain significant activity capable of inactivating α-amanitin and that this amatoxin may be used to select for RNA polymerase mutants.     

Involvement of the Terminal Oxygenase β Subunit in the Biphenyl Dioxygenase Reactivity Pattern toward Chlorobiphenyls

Biphenyl dioxygenase (BPH dox) oxidizes biphenyl on adjacent carbons to generate 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356 and in Pseudomonas sp. strain LB400. The enzyme comprises a two-subunit (α and β) iron sulfur protein (ISP_BPH), a ferredoxin (FER_BPH), and a ferredoxin reductase (RED_BPH). B-356 BPH dox preferentially catalyzes the oxidation of the double-meta-substituted congener 3,3′-dichlorobiphenyl over the double-para-substituted congener 4,4′-dichlorobiphenyl or the double-ortho-substituted congener 2,2′-dichlorobiphenyl. LB400 BPH dox shows a preference for 2,2′-dichlorobiphenyl, and in addition, unlike B-356 BPH dox, it can catalyze the oxidation of selected chlorobiphenyls such as 2,2′,5,5′-tetrachlorobiphenyl on adjacent meta-para carbons. In this work, we examine the reactivity pattern of BPH dox toward various chlorobiphenyls and its capacity to catalyze the meta-para dioxygenation of chimeric enzymes obtained by exchanging the ISP_BPH α or β subunit of strain B-356 for the corresponding subunit of strain LB400. These hybrid enzymes were purified by an affinity chromatography system as His-tagged proteins. Both types, the chimera with the α subunit of ISP_BPH of strain LB400 and the β subunit of ISP_BPH of strain B-356 (the α_LB400β_B-356 chimera) and the α_B-356β_LB400 chimera, were functional. Results with purified enzyme preparations showed for the first time that the ISP_BPH β subunit influences BPH dox’s reactivity pattern toward chlorobiphenyls. Thus, if the α subunit were the sole determinant of the enzyme reactivity pattern, the α_B-356β_LB400 chimera should have behaved like B-356 ISP_BPH; instead, its reactivity pattern toward the substrates tested was similar to that of LB400 ISP_BPH. On the other hand, the α_LB400β_B-356 chimera showed features of both B-356 and LB400 ISP_BPH where the enzyme was able to metabolize 2,2′- and 3,3′-dichlorobiphenyl and where it was able to catalyze the meta-para oxygenation of 2,2′,5,5′-tetrachlorobiphenyl.     

Partial Purification and Characterization of a 3'- Phosphoadenosine 5' -Phosphosulfate: Desulfoglucosinolate Sulfotransferase from Cress (Lepidium sativum)

A 3′ -phosphoadenosine 5′ -phosphosulfate (PAPS):desulfoglucosinolate sulfotransferase (EC 2.8.2-) was extensively purified from light-grown cress (Lepidium sativum L.) seedlings by gel filtration and concanavalin A-Sepharose 4B, Matrex Gel Green A, and Mono Q fast protein liquid chromatography. The purified enzyme, which required bovine serum albumin for stabilization, had a native molecular weight of 31,000 ± 5,000 and an apparent isoelectric point of 5.2. Using PAPS (K_m 60 micromolar) as sulfur donor, it catalyzed the sulfation of desulfobenzylglucosinolate (K_m 82 micromolar), desulfo-p-hydroxybenzylglucosinolate (K_m 670 micromolar), and desulfoallylglucosinolate (K_m 6.5 millimolar) at an optimal pH of 9.0. All other potential substrates tested, including flavonoids, flavonoid glycosides, cinnamic acids, and phenylacetaldoxime, were not sulfated. Sulfotransferase activity was stimulated by MgCl₂, MnCl₂ and reducing agents and inhibited by ZnCl₂, PbNO₃ NiCl₂ and the reaction product PAP. The thiol reagents N-ethylmaleimide, p-chloromercuriphenylsulfonic acid, and 5,5′ -dithio-bis-(2-nitrobenzoic acid) were also potent inhibitors, but the enzyme was protected from covalent modification by β-mercaptoethanol. The kinetics of desulfobenzylglucosinolate sulfation were consistent with a rapid equilibrium ordered mechanism with desulfobenzylglucosinolate binding first and PAPS second.     

Substrate Specificity of Purified Recombinant Human β-Carotene 15,15′-Oxygenase (BCO1)

Humans cannot synthesize vitamin A and thus must obtain it from their diet. β-Carotene 15,15′-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15–15′ double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (k_cat/K_m). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of β-carotene with a V_max = 197.2 nmol retinal/mg BCO1 × h, K_m = 17.2 μm and catalytic efficiency k_cat/K_m = 6098 m⁻¹ min⁻¹. The enzyme also catalyzed the oxidative cleavage of α-carotene, β-cryptoxanthin, and β-apo-8′-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of β-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of β-carotene. The shorter β-apocarotenals (β-apo-10′-carotenal, β-apo-12′-carotenal, β-apo-14′-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-β-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.     

Galactosyl-Lactose Sialylation Using Trypanosoma cruzi trans-Sialidase as the Biocatalyst and Bovine κ-Casein-Derived Glycomacropeptide as the Donor Substrate

trans-Sialidase (TS) enzymes catalyze the transfer of sialyl (Sia) residues from Sia(α2-3)Gal(β1-x)-glycans (sialo-glycans) to Gal(β1-x)-glycans (asialo-glycans). Aiming to apply this concept for the sialylation of linear and branched (Gal)_nGlc oligosaccharide mixtures (GOS) using bovine κ-casein-derived glycomacropeptide (GMP) as the sialic acid donor, a kinetic study has been carried out with three components of GOS, i.e., 3′-galactosyl-lactose (β3′-GL), 4′-galactosyl-lactose (β4′-GL), and 6′-galactosyl-lactose (β6′-GL). This prebiotic GOS is prepared from lactose by incubation with suitable β-galactosidases, whereas GMP is a side-stream product of the dairy industry. The trans-sialidase from Trypanosoma cruzi (TcTS) was expressed in Escherichia coli and purified. Its temperature and pH optima were determined to be 25°C and pH 5.0, respectively. GMP [sialic acid content, 3.6% (wt/wt); N-acetylneuraminic acid (Neu5Ac), >99%; (α2-3)-linked Neu5Ac, 59%] was found to be an efficient sialyl donor, and up to 95% of the (α2-3)-linked Neu5Ac could be transferred to lactose when a 10-fold excess of this acceptor substrate was used. The products of the TcTS-catalyzed sialylation of β3′-GL, β4′-GL, and β6′-GL, using GMP as the sialic acid donor, were purified, and their structures were elucidated by nuclear magnetic resonance spectroscopy. Monosialylated β3′-GL and β4′-GL contained Neu5Ac connected to the terminal Gal residue; however, in the case of β6′-GL, TcTS was shown to sialylate the 3 position of both the internal and terminal Gal moieties, yielding two different monosialylated products and a disialylated structure. Kinetic analyses showed that TcTS had higher affinity for the GL substrates than lactose, while the V_max and k_cat values were higher in the case of lactose.     

Effect of Cadmium on gamma-Glutamylcysteine Synthesis in Maize Seedlings

Cysteine, γ-glutamylcysteine, and glutathione and the extractable activity of the enzymes of glutathione biosynthesis, γ-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3), were measured in roots and leaves of maize seedlings (Zea mays L. cv LG 9) exposed to CdCl₂ concentrations up to 200 micromolar. At 50 micromolar Cd²⁺, γ-glutamylcysteine contents increased continuously during 4 days up to 21-fold and eightfold of the control in roots and leaves, respectively. Even at 0.5 micromolar Cd²⁺, the concentration of γ-glutamylcysteine in the roots was significantly higher than in the control. At 5 micromolar and higher Cd²⁺ concentrations, a significant increase in γ-glutamylcysteine synthetase activity was measured in the roots, whereas in the leaves this enzyme activity was enhanced only at 200 micromolar Cd²⁺. Labeling of isolated roots with [³⁵S]sulfate showed that both sulfate assimilation and glutathione synthesis were increased by Cd. The accumulation of γ-glutamylcysteine in the roots did not affect the root exudation rate of this compound. Our results indicate that maize roots are at least in part autonomous in providing the additional thiols required for phytochelatin synthesis induced by Cd.     

1H n.m.r. of the DNA-dependent RNA polymerase from Escherichia coli

The ¹H n.m.r. study of the DNA-dependent RNA polymerase from Escherichia coli has revealed that the holoenzyme (ββ′α₂σ) displays two mobile regions: one, observable also in the core enzyme (ββ′α₂), is characterized by basic amino acids and its appearance and form depend on ionic strength; the other, specific to the holoenzyme, is characterized by threonine residues and its appearance does not depend on ionic strength.     

Batroxobin Binds Fibrin with Higher Affinity and Promotes Clot Expansion to a Greater Extent than Thrombin

Batroxobin is a thrombin-like serine protease from the venom of Bothrops atrox moojeni that clots fibrinogen. In contrast to thrombin, which releases fibrinopeptide A and B from the NH₂-terminal domains of the Aα- and Bβ-chains of fibrinogen, respectively, batroxobin only releases fibrinopeptide A. Because the mechanism responsible for these differences is unknown, we compared the interactions of batroxobin and thrombin with the predominant γ_A/γ_A isoform of fibrin(ogen) and the γ_A/γ′ variant with an extended γ-chain. Thrombin binds to the γ′-chain and forms a higher affinity interaction with γ_A/γ′-fibrin(ogen) than γ_A/γ_A-fibrin(ogen). In contrast, batroxobin binds both fibrin(ogen) isoforms with similar high affinity (K_d values of about 0.5 μm) even though it does not interact with the γ′-chain. The batroxobin-binding sites on fibrin(ogen) only partially overlap with those of thrombin because thrombin attenuates, but does not abrogate, the interaction of γ_A/γ_A-fibrinogen with batroxobin. Furthermore, although both thrombin and batroxobin bind to the central E-region of fibrinogen with a K_d value of 2–5 μm, the α(17–51) and Bβ(1–42) regions bind thrombin but not batroxobin. Once bound to fibrin, the capacity of batroxobin to promote fibrin accretion is 18-fold greater than that of thrombin, a finding that may explain the microvascular thrombosis that complicates envenomation by B. atrox moojeni. Therefore, batroxobin binds fibrin(ogen) in a manner distinct from thrombin, which may contribute to its higher affinity interaction, selective fibrinopeptide A release, and prothrombotic properties.     

Pyrrole-imidazole hairpin polyamides with high affinity at 5'-CGCG-3' DNA sequence; influence of cytosine methylation on binding

To investigate the binding of 5′–CpG–3′ sequences by small molecules, two pyrrole (Py)–imidazole (Im) hairpin polyamides, PyImPyIm–γ–PyImPyIm–β–Dp (1) and PyIm–β–Im–γ–PyIm–β–Im–β–Dp (2), which recognize the sequence 5′–CGCG–3′, were synthesized. The binding affinities of the 5′–CGCG–3′ sequence to the Py–Im hairpin polyamides were measured by surface plasmon resonance (SPR) analysis. SPR data revealed that dissociation equilibrium constants (K_d) of polyamides 1 and 2 were 1.1 (± 0.3) × 10^–6 M and 1.7 (± 0.4) × 10^–8 M, respectively. Polyamide 2 possesses great binding affinity for this sequence, 65-fold higher than polyamide 1. Moreover, when all cytosines in 5′–CpGpCpG–3′ were replaced with 5-methylcytosines (^mCs), the K_d value of polyamide 2 increased to 5.8 (± 0.7) × 10^–9 (M), which indicated about 3-fold higher binding than the unmethylated 5′–CGCG–3′ sequence. These results suggest that polyamide 2 would be suitable to target CpG-rich sequences in the genome.     

Additive Expression of Consolidated Memory through Drosophila Mushroom Body Subsets

Associative olfactory memory in Drosophila has two components called labile anesthesia-sensitive memory and consolidated anesthesia-resistant memory (ARM). Mushroom body (MB) is a brain region critical for the olfactory memory and comprised of 2000 neurons that can be classified into αβ, α′β′, and γ neurons. Previously we demonstrated that two parallel pathways mediated ARM consolidation: the serotonergic dorsal paired medial (DPM)–αβ neurons and the octopaminergic anterior paired lateral (APL)–α′β′ neurons. This finding prompted us to ask how this composite ARM is retrieved. Here, we showed that blocking the output of αβ neurons and that of α′β′ neurons each impaired ARM retrieval, and blocking both simultaneously had an additive effect. Knockdown of radish and octβ2R in αβ and α′β′ neurons, respectively, impaired ARM. A combinatorial assay of radish mutant background rsh¹ and neurotransmission blockade confirmed that ARM retrieved from α′β′ neuron output is independent of radish. We identified MBON-β2β′2a and MBON-β′2mp as the MB output neurons downstream of αβ and α′β′ neurons, respectively, whose glutamatergic transmissions also additively contribute to ARM retrieval. Finally, we showed that α′β′ neurons could be functionally subdivided into α′β′m neurons required for ARM retrieval, and α′β′ap neurons required for ARM consolidation. Our work demonstrated that two parallel neural pathways mediating ARM consolidation in Drosophila MB additively contribute to ARM expression during retrieval.     

Further characterization of the magnesium chelatase in isolated developing cucumber chloroplasts : substrate specificity, regulation, intactness, and ATP requirements

Mg-chelatase catalyzes the first step unique to the chlorophyll branch of tetrapyrrole biosynthesis, namely the insertion of Mg into protoporphyrin IX (Proto). Mg-chelatase was assayed in intact chloroplasts from semi-green cucumber (Cucumis sativus, cv Sumter) cotyledons. In the presence of Proto and MgATP, enzyme activity was linear for 50 minutes. Plastid intactness was directly related to (and necessary for) Mg-chelatase activity. Uncouplers and ionophores did not inhibit Mg-Chelatase in the presence of ATP. The nonhydrolyzable ATP analogs, β,γ-methylene ATP and adenylylimidodiphosphate, could not sustain Mg-chelatase activity alone and were inhibitory in the presence of ATP (I₅₀ 10 and 3 millimolar, respectively). Mg-chelatase was also inhibited by N-ethylmaleimide (I₅₀, 50 micromolar) and the metal ion chelators 2,2′-dipyridyl and 1, 10 phenanthroline (but not to the same degree by their nonchelating analogs). In addition to Proto, the following porphyrins acted as Mg-chelatase substrates, giving comparable specific activities: deuteroporphyrin, mesoporphyrin, 2-ethyl, 4-vinyl Proto and 2-vinyl, 4-ethyl Proto. Mg-chelatase activity and freely exchangeable heme levels increased steadily with greening, reaching a maximum and leveling off after 15 hours in the light. Exogenous protochlorophyllide, chlorophyllide, heme, and Mg-Proto had no measurable effect on Mg-chelatase activity. The potent ferrochelatase inhibitors, N-methylmesoporphyrin and N-methylprotoporphyrin, inhibited Mg-chelatase at micromolar concentrations.     

Warm growth temperatures decrease soybean cholinephosphotransferase activity

The activity of cytidine 5′-diphosphate (CDP) choline: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) in developing soybean (Glycine max L. var Williams 82) seeds was 3 to 5 times higher in cotyledons grown at 20°C than in those grown at 35°C. Some characteristics of the enzyme from cotyledons cultured at 20 and 35°C were compared. In preparations from both growth temperatures, the enzyme showed a pH optimum of 7, K_m of 7.0 micromolar for CDP-choline, and an optimum assay temperature of 45°C. Both enzyme preparations were stimulated by increasing concentrations of Mg²⁺ or Mn²⁺, up to 10 millimolar and 50 micromolar, respectively, though Mn²⁺ produced lower activities than Mg²⁺. Enzymes from both 20 and 35°C show the same specificity for exogenous diacylglycerol. No metabolic effectors were detected by addition of heat treated extracts to the assay mixture. The above findings suggest that the higher enzyme activity at 20°C can be attributed to a higher level of the enzyme rather than to the involvement of isozymes or metabolic effectors. Enzyme activity decreased rapidly during culture at 35°C, indicating a rapid turnover of the enzyme. The level of temperature modulation was found to be a function of seed developmental stage.     

Identification,Purification, and Characterization of a Novel Amino Acid Racemase,Isoleucine 2-Epimerase,from Lactobacillus Species

Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of l-isoleucine to d-allo-isoleucine and d-allo-isoleucine to l-isoleucine. In contrast, the enzyme showed no γ-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The K_m and V_max values of the enzyme for l-isoleucine were 5.00 mM and 153 μmol·min⁻¹·mg⁻¹, respectively, and those for d-allo-isoleucine were 13.2 mM and 286 μmol·min⁻¹·mg⁻¹, respectively. Hydroxylamine and other inhibitors of pyridoxal 5′-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5′-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.     

Purification and characterization of active-site components of the putative p-cresol methylhydroxylase membrane complex from Geobacter metallireducens

p-Cresol methylhydroxylases (PCMH) from aerobic and facultatively anaerobic bacteria are soluble, periplasmic flavocytochromes that catalyze the first step in biological p-cresol degradation, the hydroxylation of the substrate with water. Recent results suggested that p-cresol degradation in the strictly anaerobic Geobacter metallireducens involves a tightly membrane-bound PCMH complex. In this work, the soluble components of this complex were purified and characterized. The data obtained suggest a molecular mass of 124 ± 15 kDa and a unique αα′β₂ subunit composition, with α and α′ representing isoforms of the flavin adenine dinucleotide (FAD)-containing subunit and β representing a c-type cytochrome. Fluorescence and mass spectrometric analysis suggested that one FAD was covalently linked to Tyr³⁹⁴ of the α subunit. In contrast, the α′ subunit did not contain any FAD cofactor and is therefore considered to be catalytically inactive. The UV/visible spectrum was typical for a flavocytochrome with two heme c cofactors and one FAD cofactor. p-Cresol reduced the FAD but only one of the two heme cofactors. PCMH catalyzed both the hydroxylation of p-cresol to p-hydroxybenzyl alcohol and the subsequent oxidation of the latter to p-hydroxybenzaldehyde in the presence of artificial electron acceptors. The very low K_m values (1.7 and 2.7 μM, respectively) suggest that the in vivo function of PCMH is to oxidize both p-cresol and p-hydroxybenzyl alcohol. The latter was a mixed inhibitor of p-cresol oxidation, with inhibition constants of a K_ic (competitive inhibition) value of 18 ± 9 μM and a K_iu (uncompetitive inhibition) value of 235 ± 20 μM. A putative functional model for an unusual PCMH enzyme is presented.     

Metabolism of Cytokinin : DEPHOSPHORYLATION OF CYTOKININ RIBONUCLEOTIDE BY 5'-NUCLEOTIDASES FROM WHEAT GERM CYTOSOL

Two forms (F-I and F-II) of 5′-nucleotidases (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) which catalyze the dephosphorylation of N⁶-(Δ²-isopentenyl)adenosine 5′-monophosphate and AMP to form the corresponding nucleosides were partially purified from the cytosol of wheat (Triticum aestivum) germ. Both the F-I (molecular weight, 57,000) and F-II (molecular weight, 110,000) 5′-nucleotidases dephosphorylate the ribonucleotides at an optimum pH of 7. The K_m values for the cytokinin nucleotide are 3.5 micromolar (F-I enzyme) and 12.8 micromolar (F-II enzyme) in 100 millimolar Tris-maleate buffer (pH 7) at 37 C. The F-I enzyme is less rapidly inactivated by heating than is the F-II enzyme. Both nucleotidases hydrolyze purine ribonucleoside 5′-phosphates, AMP being the preferred substrate. N⁶-(Δ²-isopentenyl)Adenosine 5′-monophosphate is hydrolyzed at a rate 72 and 86% that of AMP by the F-I and F-II nucleotides, respectively. Phenylphosphate and 3′-AMP are not substrates for the enzymes. It is proposed that dephosphorylation of cytokinin nucleotide by cytosol 5′-nucleotidases may play an important role in regulating levels of “active cytokinin” in plant cells.     

DNA polymerases κ and ζ cooperatively perform mutagenic translesion synthesis of the C8–2′-deoxyguanosine adduct of the dietary mutagen IQ in human cells

The roles of translesion synthesis (TLS) DNA polymerases in bypassing the C8–2′-deoxyguanosine adduct (dG-C8-IQ) formed by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a highly mutagenic and carcinogenic heterocyclic amine found in cooked meats, were investigated. Three plasmid vectors containing the dG-C8-IQ adduct at the G₁-, G₂- or G₃-positions of the NarI site (5′-G₁G₂CG₃CC-3′) were replicated in HEK293T cells. Fifty percent of the progeny from the G₃ construct were mutants, largely G→T, compared to 18% and 24% from the G₁ and G₂ constructs, respectively. Mutation frequency (MF) of dG-C8-IQ was reduced by 38–67% upon siRNA knockdown of pol κ, whereas it was increased by 10–24% in pol η knockdown cells. When pol κ and pol ζ were simultaneously knocked down, MF of the G₁ and G₃ constructs was reduced from 18% and 50%, respectively, to <3%, whereas it was reduced from 24% to <1% in the G₂ construct. In vitro TLS using yeast pol ζ showed that it can extend G₃*:A pair more efficiently than G₃*:C pair, but it is inefficient at nucleotide incorporation opposite dG-C8-IQ. We conclude that pol κ and pol ζ cooperatively carry out the majority of the error-prone TLS of dG-C8-IQ, whereas pol η is involved primarily in its error-free bypass.     

Identification of Propofol Binding Sites in a Nicotinic Acetylcholine Receptor with a Photoreactive Propofol Analog

Propofol, a widely used intravenous general anesthetic, acts at anesthetic concentrations as a positive allosteric modulator of γ-aminobutyric acid type A receptors and at higher concentration as an inhibitor of nicotinic acetylcholine receptors (nAChRs). Here, we characterize propofol binding sites in a muscle-type nAChR by use of a photoreactive analog of propofol, 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol (AziPm). Based upon radioligand binding assays, AziPm stabilized the Torpedo nAChR in the resting state, whereas propofol stabilized the desensitized state. nAChR-rich membranes were photolabeled with [³H]AziPm, and labeled amino acids were identified by Edman degradation. [³H]AziPm binds at three sites within the nAChR transmembrane domain: (i) an intrasubunit site in the δ subunit helix bundle, photolabeling in the nAChR desensitized state (+agonist) δM2-18′ and two residues in δM1 (δPhe-232 and δCys-236); (ii) in the ion channel, photolabeling in the nAChR resting, closed channel state (−agonist) amino acids in the M2 helices (αM2-6′, βM2-6′ and -13′, and δM2-13′) that line the channel lumen (with photolabeling reduced by >90% in the desensitized state); and (iii) at the γ-α interface, photolabeling αM2-10′. Propofol enhanced [³H]AziPm photolabeling at αM2-10′. Propofol inhibited [³H]AziPm photolabeling within the δ subunit helix bundle at lower concentrations (IC₅₀ = 40 μm) than it inhibited ion channel photolabeling (IC₅₀ = 125 μm). These results identify for the first time a single intrasubunit propofol binding site in the nAChR transmembrane domain and suggest that this is the functionally relevant inhibitory binding site.     

Occurrence of a putative ancient-like isomerase involved in histidine and tryptophan biosynthesis

We report the occurrence of an isomerase with a putative (βα)₈-barrel structure involved in both histidine and trypto-phan biosynthesis in Streptomyces coelicolor A3(2) and Mycobacterium tuberculosis HR37Rv. Deletion of a hisA homologue (SCO2050) putatively encoding N′-[(5′-phosphoribosyl)-formimino]-5 amino-imidazole-4-carboxamide ribonucleotide isomerase from the chromosome of S. coelicolor A3(2) generated a double auxotrophic mutant for histidine and tryptophan. The bifunctional gene SCO2050 and its orthologue Rv1603 from M. tuberculosis complemented both hisA and trpF mutants of Escherichia coli. Expression of the E. coli trpF gene in the S. coelicolor mutant only complemented the tryptophan auxo-trophy, and the hisA gene only complemented the histidine auxotrophy. The discovery of this enzyme, which has a broad-substrate specificity, has implications for the evolution of metabolic pathways and may prove to be important for understanding the evolution of the (βα)₈-barrels.