Carbohydrate binding studies on the lectin from Datura stramonium seeds

The carbohydrate-binding properties of the Datura stramonium seed lectin were studied by equilibrium dialysis, quantitative precipitation of natural and synthetic glycoproteins, and hapten inhibition of precipitation. The dimeric lectin (M_r = 86,000) possesses two carbohydrate-binding sites for N,N′,N′',N?-tetraacetylchitotetritol/mol protein, with an apparent K_a = 8.7 × 10³M^?1 at 4 °C. Whereas fetuin and orosomucoid reacted poorly with the Datura lectin, the asialo derivatives of these glycoproteins gave strong precipitation with the lectin. Carcinoembryonic antigen, type 14 pneumococcal capsular polysaccharide, and bovine serum albumin, highly substituted with N,N′- diacetylchitobiose units, also precipitated the lectin. Of the homologous series of chitin oligosaccharides tested, N,N′,N?-triacetylchitotriose was over 6-fold more potent than the disaccharide (N′,N′-diacetylchitobiose) which, in turn, was 90 times more reactive than N-acetyl-d-glucosamine.N-Acetyllactosamine [β-d-Gal-(1 → 4)-d-GlcNAc] was also a potent inhibitor of Datura lectin being equivalent to N,N′-diacetylchitobiose. The requirement for an N-acetyl-d-glucosaminyl unit linked at the C-4 position was established. The biantennary pentasaccharide (penta-2,6) was a 500-fold more potent inhibitor than N-acetyllactosamine, suggesting that it might interact with both saccharide-binding sites of the Datura lectin simultaneously.     

Further chemical characterization and biological activities of fluorescent I,N6-ethenoadenosine derivatives

The fluorescent 1,N⁶-ethenoadenosine derivatives of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, 3′:5′-cyclic adenosine monophosphate, adenosine and nicotinamide adenine dinucleotide have been prepared. Paper and thin layer chromatographic purification methods have been developed. Nuclear magnetic resonance and mass spectrum data indicate that only the purine ring has been modified.The 1,N⁶-ethenoadenosine triphosphate had about 70% of the activity of adenosine triphosphate as a substrate for total adenosine triphosphatase activity of hypophysectomized rat liver membranes. The 1,N⁶-ethenoadenosine diphosphate had about 86% of the activity of adenosine diphosphate as a substrate for adenosine diphosphatase of hypophysectomized rat liver membranes. The 1,N⁶-etheno derivative of nicotinamide adenine dinucleotide had about 8% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide glycohydrolase and about 54% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide pyrophosphatase of hypophysectomized rat liver membranes.K_m's for the ATPase, ADPase and yeast alcohol dehydrogenase using ε-ATP and ε-ADP and ε-NAD as substrates are presented.     

Protective effect of nicotinamide against poly(ADP-ribose) polymerase-1-mediated astrocyte death depends on its transporter-mediated uptake

AimPoly(ADP-ribose) polymerase-1 (PARP-1) is a DNA repair enzyme, and its excessive activation, following ischemia, trauma, etc., depletes cellular nicotinamide adenine dinucleotide (NAD⁺) as a substrate and eventually leads to brain cell death. Nicotinamide, an NAD⁺ precursor and a PARP-1 inhibitor, is known to prevent PARP-1-triggered cell death, but there is no available information on the mechanisms involved in its transport. Here we clarified the transport characteristics of nicotinamide in primary cultured mouse astrocytes.Main methodsUptake characteristics of [¹⁴C]nicotinamide were assessed by a conventional method with primary cultured mouse astrocytes. Cell viability and PARP-1 activity were determined with intracellular LDH activity and immunocytochemical detection of PAR accumulation, respectively.Key findingsPARP-1 activation was induced by treatment of astrocytes with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), an alkylating agent. MNNG-triggered astrocyte death and PAR accumulation were completely inhibited by treatment with nicotinamide as with DPQ (3,4-dihydro-5-(4-(1-piperidinyl)butoxy)-1(2H)-isoquinolinone), a second generation PARP inhibitor. The uptake of [¹⁴C]nicotinamide was time-, temperature-, concentration- and pH-dependent, and was inhibited and stimulated by co- and pre-treatment with N-methylnicotinamide, a representative substrate of an organic cation transport system, respectively. Co-treatment of astrocytes with nicotinamide and N-methylnicotinamide resulted in a decrease in PAR accumulation and absolute prevention of cell death.SignificanceThese findings suggest that nicotinamide has a protective effect against PARP-1-induced astrocyte death and that its transporter-mediated uptake, which is extracellular pH-sensitive and common to N-methylnicotinamide, is critical for prevention of PARP-1-triggered cell death.     

Replacement of light by dibutyryl-CAMP and CAMP in betacyanin synthesis

The effect of adenosine 3′,5′-cyclic monophosphate and its N⁶,O^2′- dibutyryl derivative (Bu₂-CAMP) on betacyanin formation in etiolated Amaranthus paniculatus seedlings was investigated. Both substances can replace the action of light in the synthesis of these pigments, the formation of which is controlled by phytochrome. The specificity of this mimicry is underlined by the observations that sodium butyrate does not promote any betacyanin formation and that theophylline enhances the effect of Bu₂-AMP. Puromycin inhibits the induction of betacyanin synthesis by Bu₂-CAMP just as it does the light-induced pigment formation. These findings suggest that phytochrome exerts its controlling role in the synthesis of betacyanins through the agency of CAMP.     

Targeting radiopharmaceuticals: Comparative biodistribution studies of gallium and indium complexes of multidentate ligands

New multidentate ligands with structures similar to N,N′-bis[2-hydroxybenzyl]ethylenediamme-N,N′-diacetic acid (HBED) and N,N′-bis(pyridoxyl)ethylenediamine-N,N′-diacetic acid (PLED) were synthesized. The in vitro lipophilicity, electrophoretic behavior, and the in vivo biodistribution were studied for the ¹¹¹In- and ^67,68Ga-complexes of N,N′-bis(2-hydroxy-3,5-dimethylbenzyl)ethylenediamine-N,N′-diacetic acid (Me₄HBED); N,N′-bis(5-t-butyl-2-hydroxy-3-methylbenzyl)ethylenediamine-N,N′-diacetic acid (t-butyl HBED); N,N′-bis[2-hydroxy-5-sulfobenzyl)ethylenediamine-N,N′-diacetic acid (SHBED); N,N′-bis(2-hydroxy-3,5-dimethylbenzyl)ethylenediamine-N-(2-hydroxyethyl)-N′-acetic acid (HBMA); and N,N′-bis(5-deoxypyridoxyl)ethylenediamine-N,N′-diacetic acid (DPLED). The biodistribution of the radiometal complexes were carried out in rats and an imaging study was performed in a non-human primate. The rapid clearance of the lipophilic complexes from blood and through the hepatobiliary system was easily demonstrated; as well, the hydrophilic complexes were cleared rapidly through the urinary tract. Positron emission tomographic images were generated from a study in a primate after administration of ⁶⁸Ga-t-butyl HBED. These images well demonstrate the efficient liver accumulation and rapid hepatobiliary clearance (< 1 h) and well differentiate images of the liver and gall bladder.     

Isolation of transfer RNA isoacceptors by chromatography on dihydroxyboryl-substituted cellulose,polyacrylamide, and glass

Polyacrylamide and porous-glass supports containing the dihydroxyborylphenyl group can be prepared by a method similar to that used in the synthesis of N-[N′-(m-dihydroxyborylphenyl)succinamyl]aminoethylcellulose. The reaction of aminoethylpolyacrylamide or amino-substituted glass with N-(m-dihydroxyborylphenyl)succinamic acid in the presence of N-cyclohexyl-N′-β-(4-methyl-morpholinium) ethylcarbodiimide yields products which, together with the cellulose derivative, are all capable of binding tRNA dissolved in buffers at pH 8.7. The demonstration that bound tRNA can be released with sorbitol solutions or with low pH buffers, together with studies on the binding of tRNA species that contain chemically modified 3′-terminals, indicate that the predominant binding mechanism consists of cyclic complex formation between the immobilized dihydroxyboryl groups and the 3′-terminal cis-diol groups of the tRNA molecules. Aminoacylated tRNA does not bind under the conditions necessary to bind tRNA and this permits the isolation of specific tRNA isoacceptors. The purification of tRNA^Phe and the partial purification of tRNA^Glu and tRNA^Trp are described.     

Fast cleavage of a diselenide induced by a platinum(II)-methionine complex and its biological implications

Platinum-based anticancer drugs such as cisplatin induce increased oxidative stress and oxidative damage of DNA and other cellular components, while selenium plays an important role in the antioxidant defense system. In this study, the interaction between a platinum(II) methionine (Met) complex [Pt(Met)Cl₂] and a diselenide compound selenocystine [(Sec)₂] was studied by electrospray ionization mass spectrometry, high performance liquid chromatography mass spectrometry, and ¹H NMR spectroscopy. The results demonstrate that the diselenide bond in (Sec)₂ can readily and quickly be cleaved by the platinum complex. Formation of the selenocysteine (Sec) bridged dinuclear complex [Pt₂(Met-S,N)₂(μ-Sec-Se,Cl)]³⁺ and Sec chelated species [Pt(Met-S,N)(Sec-Se,N)]²⁺ was identified at neutral and acidic media, which seems to result from the intermediate [Pt(Met-S,N)(Sec-Se)Cl]⁺. An accelerated formation of S-Se and S-S bonds was also observed when (Sec)₂ reacted with excessive glutathione in the presence of [Pt(Met)Cl₂]. These results imply that the mechanism of activity and toxicity of platinum drugs may be related to their fast reaction with seleno-containing biomolecules, and the chemoprotective property of selenium agents against cisplatin-induced toxicity could also be connected with such reactions.     

Targeting radiopharmaceuticals—II. Evaluation of new trivalent metal complexes with different overall charges

Three new multidentate ligands designed to have high affinity for Ga³⁺, In³⁺ and Fe³⁺ have been synthesized, N-(2-hydroxybenzyl)-N′-pyridoxylethylenediamine-N,N′-diacetic acid (HBPLED), N-(2-hydroxy-3,5-dimethylbenzyl)-N′-(3-hydroxy-1,2,5-trimethyl-4-pyridylmethyl)ethylenediamine-N,N′-diacetic acid (Me₄HBPLED) and N,N′-bis(3-hydroxy-1,2,5-trimethyl-4-pyridylmethyl) ethylenediamine-N,N′-diacetic acid (DMPLED). These ligands give metal-ligand (M–L) complexes with (M = Ga³⁺, In³⁺, Fe³⁺) overall charges of −1, 0 and + 1, respectively. The ⁶⁷Ga, ⁶⁸Ga and ¹¹¹In complexes of each of the three ligands and the ⁵⁹Fe complex of Me₄HBPLED were prepared, characterized and their biodistributions determined in rats after intravenous injection. Despite the differences in overall charge, the biological behavior of all three ¹¹¹In complexes were similar, in that the radioactivity cleared rapidly via the kidney. The biodistributions of the ⁶⁸Ga and ⁶⁷Ga complexes were comparable to that of the ¹¹¹In complex counterpart. Also, the ⁵⁹Fe-Me₄HBPLED biodistribution was not significantly different from those of the ⁶⁸Gaand ¹¹¹In-Me₄HBPLED. The renal clearance rate seems insensitive to the overall M-L charge; suggesting that the hydrophilic periphery of the ligand rather than the overall molecular charge determines the biological fate (with respect to renal clearance).     

Gas chromatography-mass spectrometry determination of [15N]ammonia enrichment in blood and urine

A rapid gas chromatography-mass spectrometry method for [¹⁵N]ammonia analysis is deseribed which is based on the formation of [¹⁵N]glutamic acid from ammonia and analysis of isotopic abundance in the N-trifluoroacetyl-n-butylester glutamate derivative. Mean recovery of [¹⁵N]ammonia added to either plasma or urine was greater than 99% with a relative standard deviation of less than 10%. The method can be applied to the determination of extremely low levels of ammonia through an isotope dilution technique. The [¹⁵N]ammonia abundance of blood and urine was determined in an adult following on oral dose (500 mg) of ¹⁵NH₄Cl. A peak isotopic abundance of 13 atoms% excess was reached by 30 min. Urinary excretion of [¹⁵N]ammonia during the first 4 h after administration of the isotope amounted to 4.1% of the isotope administered.     

Synthesis,evaluation of anti-HIV-1 and anti-HCV activity of novel 2′,3′-dideoxy-2′,2′-difluoro-4′-azanucleosides

A series of 2′,3′-dideoxy-2′,2′-difluoro-4′-azanucleosides of both pyrimidine and purine nucleobases were synthesized in an efficient manner starting from commercially available L-pyroglutamic acid via glycosylation of difluorinated pyrrolidine derivative 15. Several 4′-azanucleosides were prepared as a separable mixture of α- and β-anomers. The 6-chloropurine analogue was obtained as a mixture of N⁷ and N⁹ regioisomers and their structures were identified based on NOESY and HMBC spectral data. Among the 4′-azanucleosides tested as HIV-1 inhibitors in primary human lymphocytes, four compounds showed modest activity and the 5-fluorouracil analogue (18d) was found to be the most active compound (EC₅₀ = 36.9 μM) in this series. None of the compounds synthesized in this study demonstrated anti-HCV activity.     

Extended N6 substitution of rigid C2-arylethynyl nucleosides for exploring the role of extracellular loops in ligand recognition at the A3 adenosine receptor

2-Arylethynyl-(N)-methanocarba adenosine 5′-methyluronamides containing rigid N⁶-(trans-2-phenylcyclopropyl) and 2-phenylethynyl groups were synthesized as agonists for probing structural features of the A₃ adenosine receptor (AR). Radioligand binding confirmed A₃AR selectivity and N⁶-1S,2R stereoselectivity for one diastereomeric pair. The environment of receptor-bound, conformationally constrained N⁶ groups was explored by docking to an A₃AR homology model, indicating specific hydrophobic interactions with the second extracellular loop able to modulate the affinity profile. 2-Pyridylethynyl derivative 18 was administered orally in mice to reduce chronic neuropathic pain in the chronic constriction injury model.     

A dolichol-linked trisaccharide from central nervous tissue: structure and biosynthesis.

Calf brain membranes have previously been shown to enzymatically transfer N-acetyl[¹⁴C]glucosamine from UDP-N-acetyl[¹⁴C]glucosamine into N-acetyl[¹⁴C]glucosami-nylpyrophosphoryldolichol, N,N′-diacetyl[¹⁴C]chitobiosylpyrophosphoryldolichol and a minor labeled product with the chemical and chromatographic properties of a [¹⁴C]trisaccharide lipid (Waechter, C. J., and Harford, J. B. (1977) Arch. Biochem. Biophys.181, 185–198). This paper demonstrates that incubating calf brain membranes containing endogenous, prelabeled N-acetyl[¹⁴C]glucosaminyl lipids with unlabeled GDP-mannose enhances the formation of the [¹⁴C]trisaccharide lipid. The intact [¹⁴C]trisaccharide lipid behaves like a dolichol-bound trisaccharide, in which the glycosyl group is linked via a pyrophosphate bridge, when chromatographed on SG-81 paper or DEAE-cellulose. Mild acid treatment releases a water-soluble product that comigrates with authentic β-Man-(1→4)-β-GlcNAc(1→4)-GlcNAc. The free [¹⁴C]trisaccharide is converted to N,N′-diacetyl[¹⁴C]chitobiose by incubation with a highly purified β-mannosidase. These findings indicate that the trisaccharide lipid formed by calf brain membranes is β-mannosyl-N,N′-diacetylchito-biosylpyrophosphoryldolichol. The two glycosyltransferases responsible for the enzymatic conversion of the N-acetylglucosaminyl lipid to the trisaccharide lipid have been studied using exogenous, purified [¹⁴C]glycolipid substrates. Calf brain membranes enzymatically transfer N-acetylglucosamine from UDP-N-acetylglucosamine to exogenous N-acetyl[¹⁴C] glucosaminylpyrophosphoryldolichol to form [¹⁴C]disaccharide lipid. The biosynthesis of [¹⁴C]disaccharide lipid is stimulated by unlabeled UDP-N-acetylglucosamine under conditions that inhibit N-acetylglucosaminylpyrophosphoryldolichol synthesis. Unlike the formation of N-acetylglucosaminylpyrophosphoryldolichol the enzymatic addition of the second N-acetylglucosamine residue is not inhibited by tunicamycin. Exogenous purified [¹⁴C] disaccharide lipid is enzymatically mannosylated by calf brain membranes to form the [¹⁴C] trisaccharide lipid. The formation of the [¹⁴C]trisaccharide lipid from exogenous [¹⁴C] disaccharide lipid is stimulated by unlabeled GDP-mannose and Mg²⁺, and inhibited by EDTA. Exogenous dolichyl monophosphate is also inhibitory. These results strongly suggest that the calf brain mannosyltransferase involved in the synthesis of the trisaccharide lipid requires a divalent cation and utilizes GDP-mannose, not mannosylphosphoryldolichol, as the direct mannosyl donor.     

4-N-Alkanoyl and 4-N-alkyl gemcitabine analogues with NOTA chelators for 68-gallium labelling

The conjugation of 4-N-(3-aminopropanyl)-2′-deoxy-2′,2′-difluorocytidine with 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bn-NOTA) ligand in 0.1?M Na₂CO₃ buffer (pH 11) at ambient temperature provided 4-N-alkylgemcitabine-NOTA chelator. Incubation of latter with excess of gallium(III) chloride (GaCl₃) (0.6?N AcONa/H₂O, pH?=?9.3) over 15?min gave gallium 4-N-alkylgemcitabine-NOTA complex which was characterized by HRMS. Analogous [⁶⁸Ga]-complexation of 4-N-alkylgemcitabine-NOTA conjugate proceeded with high labeling efficiency (94%–96%) with the radioligand almost exclusively found in the aqueous layer (～95%). The high polarity of the gallium 4-N-alkylgemctiabine-NOTA complex resulted in rapid renal clearance of the ⁶⁸Ga-labelled radioligand in BALB/c mice.     

Evidence for the enzymatic transfer of N-acetylglucosamine from UDP-N-acetylglucosamine into dolichol derivatives and glycoproteins by calf brain membranes

Incubating white matter membranes with UDP-N-acetyl-[¹⁴C]glucosamine in the presence of Mg²⁺ and AMP resulted in the labeling of two major glycolipids, a minor glycolipid and several membrane-associated glycoproteins. The addition of AMP protected the labeled sugar nucleotide from degradation by a membrane-bound sugar nucleotide pyrophosphatase activity. While no labeled oligosaccharide lipid was recovered in a CHCl₃CH₃OHH₂O (10:10:3) extract after incubating with only UDP-N-acetyl-[¹⁴C] glucosamine, Mg²⁺, and AMP, the inclusion of unlabeled GDP-mannose led to the formation of an N-acetyl-[¹⁴C]glucosamine-labeled oligosaccharide lipid that was soluble in CHCl₃CH₃OHH₂O (10:10:3). The [GlcNAc-¹⁴C]oligosaccharide unit was released by treatment with 0.1 N HCl in 80% tetrahydrofuran at 50 °C for 30 min and appears to have the same molecular size as the lipid-linked [mannose-¹⁴C] oligosaccharide, formed enzymatically by white matter membranes as judged by their elution behavior on Bio-Gel P-6. The incorporation of N-acetyl-[¹⁴C]glucosamine into glycolipid was stimulated by exogenous dolichol monophosphate, but inhibited by UMP or tunicamycin, a glucosamine-containing antibiotic. Although UMP and tunicamycin drastically inhibited the labeling of glycolipid, these compounds had very little effect on the labeling of glycoproteins. The major glycolipids have the chemical and Chromatographic characteristics of N-acetylglucosaminylpyrophosphoryldolichol and N,N′-diacetylchitobiosylpyrophosphoryldolichol. When the labeled glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, four labeled polypeptides were observed, having apparent molecular weights of 145,000, 105,000, 54,000, and 35,000. Virtually all of the N-acetyl-[¹⁴C]glucosamine was released when the labeled glycopeptides, produced by pronase digestion, were incubated with an exo-β-N-acetylglucosaminidase, indicating that all of the N-acetyl-[¹⁴C]glucosamine incorporated under these conditions is attached to white matter membrane glycoproteins at nonreducing termini.     

Synthesis and evaluation of two series of 4′-aza-carbocyclic nucleosides as adenosine A2A receptor agonists

The synthesis of two series of 4′-aza-carbocyclic nucleosides are described in which the 4′-substituent is either a reversed amide, relative to the carboxamide of NECA, or an N-bonded heterocycle. Using established purine substitution patterns, potent and selective examples of agonists of the human adenosine A_2A receptor have been identified from both series. The propionamides 14–18 and the 4-hydroxymethylpyrazole 32 were determined to be the most potent and selective examples from the 4′-reversed amide and 4′-N-bonded heterocyclic series, respectively.     

Active transport of chloride by Anacystis nidulans

Chloride uptake by the cyanobacterium Anacystis nidulans at 38°C is energy dependent showing maximum rate (around 5.10^-7 mol Cl^-xml cell water^-1xmin^-1) and accumulation (up to 160 fold) in light and air. The respective values in air and darkness were 40–70% lower. In the dark under N₂ no uptake was found. Chloride transport had an optimum at pH 6.7 and a K _M of 2.10^-5 M which was pH-independent. It was inhibited by carbonyl cyanide m-chlorophenylhydrazone and N,N′-dicyclohexylcarbodiimide in the light and in the dark, and also to a lesser extent by valinomycin. 3-(3,4-dichlorophenyl)-1,1-dimethylurea in the light caused a moderate stimulation. To obtain information about the energy source of active chloride transport the action of the four inhibitors on membrane potential (determined through the distribution of triphenylmethylphosphonium) and ATP level (determined by the firefly method) was examined. It was found that a high negative membrane potential was unfavorable for chloride accumulation probably by stimulating passive efflux. On the other hand a good correlation between ATP level and chloride transport activity was obtained. Attempts to induce chloride uptake by sudden acidification of the external medium in presence of N,N′-dicyclohexyl-carbodiimide or during anaerobiosis were not successful. Two mechanisms of chloride uptake are discussed:

1. primary active transport by an ATP-dependent pump, and
2. “chemiosmotic” secondary active transport linked to a proton gradient, the present data favoring mechanism a.

     

Nuclear magnetic resonance studies on pyridine dinucleotides. The pH dependence of the carbon-13 nuclear magnetic resonance of NAD+ analogs.

The pH dependence of the ¹³C chemical shifts for nicotinamide adenine dinucleotide (NAD⁺), thionicotinamide adenine dinucleotide (TNAD⁺), pyridine adenine dinucleotide (PyrAD⁺), N-methyl-nicotinamide adenine dinucleotide (N-Me-NAD⁺), acetylpyridine adenine dinucleotide (AcPyAD⁺), nicotinamide hypoxanthine dinucleotide (NHD⁺), and nicotinamide adenine dinucleotide phosphate (NADP⁺) are reported. In these analogs the ¹³C chemical shifts of the pyridinium moiety reflect the pK_a of the opposing purine base, while the ¹³C chemical shift dependence on pD for the pyridinium carbons of nicotinamide mononucleotide (NMN⁺) and adenosine monophosphate (AMP), 1,4-dihydronicotinamide adenine dinucleotide (NADH), 1,4-dihydronicotinamide adenine dinucleotide phosphate (NADPH), and nicotinic acid adenine dinucleotide (N(a)AD⁺) are not influenced by the adenine ring in the pD range tested. Through the use of ¹³C-labeled NAD⁺, the source of the pH dependence of the ¹³C chemical shifts was shown to be intramolecular in origin. However, serious doubt is cast on the utility of employing the pD dependence of chemical shift data to determine the nature of solution conformers or their relative populations.     

New Method of Denitrification Analysis of Bradyrhizobium Field Isolates by Gas Chromatographic Determination of 15N-Labeled N2

To evaluate the denitrification abilities of many Bradyrhizobium field isolates, we developed a new ¹⁵N-labeled N₂ detection methodology, which is free from interference from atmospheric N₂ contamination. ³⁰N₂ (¹⁵N¹⁵N) and ²⁹N₂ (¹⁵N¹⁴N) were detected as an apparent peak by a gas chromatograph equipped with a thermal conductivity detector with N₂ gas having natural abundance of ¹⁵N (0.366 atom%) as a carrier gas. The detection limit was 0.04% ³⁰N₂, and the linearity extended at least to 40% ³⁰N₂. When Bradyrhizobium japonicum USDA110 was grown in cultures anaerobically with ¹⁵NO₃⁻, denitrification product (³⁰N₂) was detected stoichiometrically. A total of 65 isolates of soybean bradyrhizobia from two field sites in Japan were assayed by this method. The denitrification abilities were partly correlated with filed sites, Bradyrhizobium species, and the hup genotype.