Specificity towards oligomannoside and hybrid type glycans of the endo-β-N-acetylglucosaminidase B from the basidiomy ceteSporotrichum dimorphosporum

We have previously shown that an endo--N-acetylglucosaminidase (EC 3.2.1.96) named Endo B, isolated from culture filtrates of the basidiomyceteSporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of theN-acetyllactosamine type linked to the asparagine residue of peptide or protein moieties [Bouquelet S, Strecker G, Montreuil J, Spik G (1980) Biochimie 62:43–49]. In the present paper, the substrate specificity of the enzyme towards oligomannoside and hybrid type glycans has been analyzed. The results obtained indicate that ovalbumin glycopeptides containing four to seven mannose residues and bovine lactotransferrin glycopeptides containing four to nine mannose residues were completely hydrolyzed by the enzyme. The degree of cleavage was variable among hybrid type structures, since glycopeptides containing the following glycans: (Gal)₁(GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₄(GlcNAc)₂ were not hydrolyzed by the enzyme while the percentage of hydrolysis of a glycopeptide containing (GlcNAc)₂(Man)₅(GlcNAc)₂ glycan reached 90%. The bovine lactotransferrin was partially deglycosylated (40%) in the absence of non-ionic detergent while native ovalbumin glycoprotein was not hydrolyzed by the enzyme.The oligomannoside-and theN-acetyllactosamine-type degrading activities present in the culture filtrates were not separated at any step of the purification procedure. Both activities were eluted as a single component with an apparent molecular mass of 89 kDa suggesting that they are located on the same enzyme molecule.Endo B represents a powerful tool for removing oligomannoside-andN-acetyllactosamine-type glycans fromN-glycopeptides andN-glycoproteins. Moreover, advantages in the use of Endo B in a soluble form as well as in an immobilized form result in its high activity and in its stability to heat denaturation and storage.Abbreviations Gal d-galactose - Man d-mannose - GlcNAc N-acetyl-d-glucosamine - Con A concanavalin A - Asn asparagine - GLC gas liquid chromatography - TLC thin layer chromatography - Endo endo--N-acetylglucosaminidase - Endo B endo--N-acetylglucosaminidase isolated fromSporotrichum dimorphosporum - PBE polybuffer exchanger - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Selective preparation of N-acetyl-D-glucosamine and N,N'-diacetylchitobiose from chitin using a crude enzyme preparation from Aeromonas sp

A bacterium, Aeromonas sp. GJ-18, having strong chitinolytic activity was isolated from coastal soil and used for crude enzyme preparations. This enzyme preparation contained N-acetyl-D-glucosaminidase and N,N-diacetylchitobiohydrolase. N-Acetyl-D-glucosaminidase was inactive above 50 °C, but N,N-diacetylchitobiohydrolase was stable at this temperature. Utilizing the temperature sensitivities of the chitin degradation enzymes in crude enzyme preparation, N-acetyl-D-glucosamine (GlcNAc) and N,N-diacetylchitobiose [(GlcNAc)₂] were selectively produced from chitin. At 45 °C, GlcNAc was produced as a major hydrolytic product (94% composition) with a yield of 74% in 5 d, meanwhile at 55 °C (GlcNAc)₂ was the major product (86%) with a yield of 35% within 5 d.Revisions requested 29 September 2004; Revisions received 1 November 2004     

Interaction of α-Chymotrypsin with Dimethyl Sulfoxide: A Change of Substrate Could “Change” the Interaction Mechanism

The kinetic behavior of -chymotrypsin was studied in water–DMSO mixtures at concentrations of the organic solvent that do not cause irreversible denaturation of the enzyme. Various substrates (N-substituted derivatives of L-tyrosine) were found to display substantially different kinetic patterns of interaction with -chymotrypsin, which can be described by totally different kinetic schemes. The differences were ascribed to competition between the N-acyl group of the substrate and the DMSO molecule at the S ₂ site of substrate binding to the active site of the enzyme.     

Properties of the deglycosylatedβ-fructofuranosidaseP-2 fromAureobasidium sp. ATCC 20524

Summary The carbohydrate moiety of -fructofuranosidaseP-2 fromAureobasidium sp. ATCC 20524 was largely removed by exposure of the enzyme to endo--N-acetylglucosaminidase F; the total carbohydrate content of the enzyme was decreased from 53% (w/w) to 15% (w/w). The stability of the deglycosylated enzyme at pH 4 to 7 and 40 to 50°C was decreased and theK _m value for sucrose was increased from 0.65 to 1.43 M. The deglycosylated enzyme was more sensitive to proteases such as pronase E and subtilisin than the native enzyme. It is concluded that the carbohydrate moiety of -fructofuranosidaseP-2 contributes to the stability of the enzyme as well as its affinity for sucrose.     

The carbohydrate structures ofβ-galactosidase from human liver

Acid -galactosidase (EC3.2.1.23) was obtained from human liver in a pure monomeric state (M_r63 000). The carbohydrate content of the enzyme was established to be, 9% by weight; mannose,N-acetylglucosamine, galactose andN-acetylneuraminic acid were found to be the constituent monosaccharides. The carbohydrate structures of the enzyme were studied at the glycopeptide level by employing 500 MHz¹H-NMR spectroscopy, carbohydrate composition analysis and methylation analysis involving GLCMS. Based upon the intensities of relevant signals in the¹H-NMR spectrum, approximately 60% of the chains were found to be of theN-acetyllactosamine type, having the structure The rest appeared to be of the oligomannoside type (Man₅-₆GlcNAc₂Asn). The carbohydrate composition and methylation analysis results sustained these findings, although the calculation of the distribution based upon these techniques indicated a somewhat lower percentage ofN-acetyllactosamine type chains. There are approximately three oligosaccharide chains per molecule. These findings offer an explanation for the abnormal distribution of -galactosidase in tissues and cultured fibroblasts of patients with I-cell disease.     

Evidence from lectin-binding studies for abnormal glycosylation ofβ-hexosaminidase in the leukaemic cell-line CCRF/CEM

The lectin affinities of -N-acetyl-d-hexosaminidase (E.C.3.2.1.52) from an acute lymphoblastic leukaemic cell-line (CCRF/CEM), a non-malignant lymphoblastic cell-line (SM1) and normal human fibroblasts were studied for both mature and precursor forms of the enzyme. Four immobilised lectins concanavalin A-Sepharose wheat germ agglutinin-Agarose,Ricinus communis agglutinin I-Agarose,Phaseolus vulgaris erythroagglutinin-Agarose and a column of serotonin-Sepharose were used. The activities of -hexosaminidase from fibroblasts and SM1 cells generally behaved similarly while the CCRF/CEM enzyme exhibited different binding patterns. Differences were also noted between precursor and mature enzyme from each cell type consistent with changes in glycosylation between the precursor form and the mature form appearing in the lysosome. These results suggest that changes in the glycosylation of -hexosaminidase, and possibly other lysosomal enzymes, may be associated with malignancy.Abbreviations Con A concanavalin A-Sepharose - RCA-I Ricinus communis agglutinin I-Agarose - WGA wheat germ agglutinin-Agarose - PHA-E Phaseolus vulgaris erythroagglutinin-Agarose - SER serotonin-Sepharose: non-T - non-B ALL non-T, non-B cell acute lyphoblastic leukaemia - 4-MU-GLcNAc 4-methylumbelliferyl 2-acetamido-2-deoxy--D-glucopyranoside     

Strategy and Design of Novel Reagents for the Fluorometric Analysis of Biomolecules

This article covers molecular designs to develop several new fluorometric reagents and their applications to increase the sensitivities up to the picomole level using HPLC for the measurement of biomolecules. The methods were designed to demonstrate the physiological activities, for example (1) N-(9-acridinyl)maleimide (NAM) for the measurement of SH, –S–S–, and sulfite such as cysteine, (2) diphenyl-1-pyrenylphosphine (DPPP) for the hydroperoxides in lipids, serum, tissues, and foodstuffs, (3) 9-bromomethylacridine (9-BrMA), (4) 2-(anthracene-2,3-dicarboxylimide)ethyltrifluoromethane sulfonate (AE-OTf) for carboxylic acids, and (5) The chiral fluorometric labelling reagent (S)-( + )-2-tert-butyl-2-methyl-1,3-benzodioxole-4-carboxylic acid (TBMB) to identify the chiralities of amino acids, sugars, and mono- and diacylglycerols.     

β-Oxidation of fatty acids in algae: Localization of thiolase and acyl-CoA oxidizing enzymes in three different organisms

In the algae Mougeotia, Bumilleriopsis and Eremosphaera, recently shown to possess the enzymes hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and enoyl-CoA hydratase (EC 4.2.1.17), the presence of thiolase (EC 2.3.1.9) and acyl-CoA-oxidizing enzymes can also be demonstrated, indicating that -oxidation of fatty acids is possible in these organisms. The compartmentation of enzymes is different in the various algae. In Mougeotia, both thiolase and the acyl-CoA-oxidizing enzyme are located exclusively in the peroxisomes. The latter enzyme was found to be an oxidase using molecular oxygen as an electron acceptor. On the other hand, in Bumilleriopsis all enzymes of the fatty-acid -oxidation pathway tested are constituents only of the mitochondria, and acyl-CoA is oxidized by a dehydrogenase incapable of reducing oxygen. Finally, in Eremosphaera thiolase and acyl-CoA-oxidizing enzymes were found in the peroxisomes as well as in the mitochondria. In the peroxisomes, oxidation of acyl-CoA is catalyzed by an oxidase, whereas the corresponding enzyme in the mitochondria is a dehydrogenase. The acyl-CoA oxidases/dehydrogenases of the three algae differ not only by their capability for oxidation of acyl-CoA of different chain lengths but also with regard to their K_m values and substrate specificities. Indications were obtained that the oxygen is reduced to water rather than to H₂O₂ by the algal acyl-CoA oxidases. When cells of Eremosphaera were cultured with hypolipodemic substances in the growth medium the activities of the peroxisomal enzymes, but not those of the mitochondrial enzymes of the fatty-acid -oxidation pathway, were increased by a factor of two to three.Abbreviations DPIP 2,6-dichlorophenol indophenol - INT p-iodonitrotetrazolium violet - MEHP monoethylhexylphthalate     

High-performance liquid chromatographic resolution of (R,S)-α-alkyl-α-amino acids as diastereomeric derivatives

Summary A number (27) of racemic-alkyl--amino acids (AAA) were derivatized either witho-phthaldialdehyde (OPA) in combination withN-t-butoxycarbonyl-L-cysteine (Boc-Cys) orN-acetyl-L-cysteine (Ac-Cys), or withN ²-(5-fluoro-2,4-dinitrophenyl)-L-alanine amide (Marfey's reagent). The resolution of the diastereoisomers formed was investigated by reversed-phase (C₁₈) high-performance liquid chromatography (HPLC) using gradient elution conditions employing sodium phosphate buffers of pH 7.2 together with acetonitrile, and fluorescence detection at 344 nm (excitation) and 443 nm (emission) for the OPA/Boc-Cys or OPA/Ac-Cys derivatives. For the diastereomers formed by derivatization with Marfey's reagent triethylammonium phosphate buffers of pH 3.0 (pH 7.2 for acidic AAA) together with acetonitrile, and u.v. detection at 340 nm were used. Whereas with Marfey's reagent all diastereomers of AAA showed complete, or almost complete, resolution, only 8, or 11, respectively of the diastereomers formed by derivatization with OPA/Boc-Cys or OPA/Ac-Cys were resolved under the chromatographic conditions used.     

Penicillinase (β-lactamase) formation by blue-green algae

-Lactamase (penicillinase) activity was found in a number of strains of blue-green algae. In some cases, this enzyme permitted algae to overcome the inhibitory effects of penicillin. Production and localization of -lactamase were studied in a unicellular species, Coccochloris elabens (strain 7003), and in a filamentous, nitrogen-fixing Anabaena species (strain 7120). When cells were grown in a neutral medium with NaNO₃ as N source, the pH rose during growth; at a pH of about 10, most of the enzyme was extracellular and all the cell-bound enzyme was expressed equally well in intact or disrupted cells. If the pH was kept near neutrality during growth by gassing with CO₂ in N₂ or by growth under conditions of N₂ fixation, the enzyme remained cell-bound and cryptic for most of the growth phase, being measurable only after cells were disrupted. The enzymes from strains 7003 and 7120 had greater activity on benzyl penicillin and other penicillins than on cephalo-sporins. Some differences were observed in the substrate profiles of penicillinases from the two strains against different penicillins.A preliminary account of this work was presented at the 1974 meetings of the American Society for Microbiology in Chicago (Abstracts of Meetings, M37)     

Partial purification and characterization of alpha-glucosidase from Pseudomonas fluorescens W

The -glucosidase (-d-glucoside glucohydrolase, EC 3.2.1.20) of Pseudomonas fluorescens W was partially purified by (NH₄)₂SO₄ fractionation, Sephadex G-200 and DEAE-cellulose column chromatography. The enzyme showed great specificity for maltose hydrolysis, with very little action against polymeric forms. Sucrose, isomaltose, -methylglucoside, and maltobionic acid were not hydrolyzed. Turanose was a strong competitive inhibitor, and glucose a weaker one. Tris (2-amino-2-hydroxymethylpropan-1:3-diol) inhibited enzyme activity significantly only at alkaline pH. Mercuric, cupric, and silver cations strongly inhibited, and EDTA (ethylenediaminetetraacetate) weakly inhibited the enzyme. The isolated enzyme was rather unstable even at 4° C, and was destroyed by freezing and lyophilization. Inositol and albumin had a slightly protective effect. Sulfhydryl-binding reagents strongly inhibited the enzyme.Abbreviations PNPG paranitrophenyl--d-glucoside - PCMB parachloromercuribenzoate - DEAE diethylaminoethyl cellulose - NEM N-ethylmaleimide - EDTA ethylenediaminetetraacetate     

Genetics of insect hemolymph β-N-acetylglucosaminidase in the silkworm,Bombyx mori

The distribution of insect hemolymph -N-acetylglucosaminidase was investigated in the silkworm, Bombyx mori. Activity in 115 varieties was 6.92±3.22 units/ml, ranging from 1.4 to 17.0 units/ml. No enzyme-deficient individuals were observed. By selecting individuals showing either high or low enzyme activities, homozygotes were separated with activities varying 10-fold between isolates. No differences in activity of -mannosidase and -galactosidase were observed. Thus, it appears that high- or low-enzyme silkworms (High or Low lines) shared the same genetic background except for the gene concerning the activity of -N-acetylglucosaminidase. Studies on the heredity of the enzyme indicated that the synthesis of the enzyme protein was controlled by an autosomal allele. Examination by immunotitration and CM₅₂-cellulose column chromatography revealed that the difference in activity between High and Low lines was due to the amount of the active enzyme, but not to an endogeneous activator or inhibitor. Furthermore, there was no isozymic difference in -N-acetylglucosaminidase. Slab gel electrophoresis on polyacrylamide showed a species of enzyme (A) that stained more intensely in the High line. For the second species of enzyme (B), the converse was true. This evidence suggests that enzyme levels in hemolymph are under the control of a gene affording association of enzyme subunits to the active enzyme molecule.     

Quantitative and qualitative changes in the endoplasmic reticulum of barley aleurone layers

Changes in the level of the endoplasmicreticulum (ER) marker enzyme cytochrome-c reductase (EC 1.6.2.1) were followed with time of imbibition of de-embryonated half-seeds of barley (Hordeum vulgare L.) and the subsequent incubation of their aleurone layers in gibberellic acid (GA₃) and H₂O. During imbibition there is an increase in the level of cytochrome-c-reductase activity and in the amount of 280-nm absorbance associated with this enzyme. When aleurone layers are incubated for a further 42 h in water, there is a doubling of the cytochrome-c-reductase activity. In GA₃, the activity of cytochrome-c reductase reaches a maximum at 24 h of incubation and thereafter falls to below 70% of its level at the beginning of the incubation period. Changes in the cytochrome-c-reductase activity correlate with changes in the fine structure of the aleurone cell. The ER isolated in low Mg²⁺ from aleurone layers incubated in buffer for up to 18 h has buoyant density of 1.13–1.14 g cc^-1 while that from layers incubated in GA₃ for 7.5–18 h has a density of 1.11–1.12 g cc^-1. The -amylase (EC3.2.1.1) isolated with the organelle fraction by Sepharose gel filtration is associated with the ER on isopycnic and rate-zonal density gradients, and its activity can be enhanced by Triton X-100. The soluble -amylase fraction from Separose-4B columns, on the other hand, is not Triton-activated but is acid-labile. Acid phosphatase (EC3.1.3.2) is distributed in at least three peaks on isopycnic gradients. In low Mg²⁺ the second peak of activity has a density of 1.12 g cc^-1 in GA₃-treated tissue and 1.13–1.14 g cc^-1 in H₂O-treated tissue. With high-Mg²⁺ buffers, this peak of phosphatase activity disappears. Acid-phosphatase activity is not enhanced by Triton X-100 nor is it acid-labile.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid     

Discrimination Against 13CO2 in Leaves,Pod Walls,and Seeds of Water-stressed Chickpea

The rate of photosynthesis (P _N) in leaves and pods as well as carbon isotope content in leaves, pod walls, and seeds was measured in well-watered (WW) and water-stressed (WS) chickpea plants. The P _N, on an area basis, was negligible in pods compared to leaves and was reduced by water stress (by 26%) only in leaves. WS pod walls and seeds discriminated less against ¹³CO₂ than did the controls. This response was not observed for leaves as is usually the case. Pod walls and seeds discriminated less against ¹³CO₂ than did leaves in both WW and WS plants. Measurement of carbon isotope composition in pods may be a more sensitive tool for assessing the impact of water stress on long-term assimilation than is the instantaneous measurement of gas exchange rates.     

Synthesis of optically active lipidicα-amino acids and lipidic 2-amino alcohols

Summary Lipidic-amino acids (LAAs) are a class of compounds combining structural features of amino acids with those of fatty acids. They are non-natural-amino acids with saturated or unsaturated long aliphatic side chains. Synthetic approaches to optically active LAAs and lipidic 2-amino alcohols (LAALs) are summarized in this review. A general approach to enantioselective synthesis of saturated LAAs is based on the oxidative cleavage of 3-amino -1,2-diols obtained by the regioselective opening of enantiomerically enriched long chain 2,3-epoxy alcohols. Unsaturated LAAs are prepared in their enantiomeric forms by Wittig reactionvia methyl (S)-2di-tert-butoxycarbonylamino-5-oxo-pentanoate. This key intermediate aldehyde is obtained by selective reduction of dimethyl N,N-di-Boc glutamate with DIBAL. (R) or (S) LAALs may be prepared starting from D-mannitol or L-serine. LAAs are converted into LAALs by chemoselective reduction of their fluorides using sodium borohydride with retention of optical purity. Replacement of the hydroxyl group of LAALs by the azido group, followed by selective reduction leads to unsaturated optically active lipidic 1,2-diamines.Abbreviations Bn benzyl - Boc tert-butoxycarbonyl - DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone - DET diethyl tartrate - DIBAL diisobutyl aluminum hydride - DMAP 4-dimethylaminopyridine - DMF N,N-dimethylformamide - DMSO dimethyl sulfoxide - EDC N-ethyl-N-(3-dimethylaminopropyl)carbodiimide - Et₃N triethylamine - HMPA hexamethylphosphoramide - HOBt 1-hydroxybenzotriazole - KN(TMS)₂ potassium bis(trimethylsilyl)-amide - LAA lipidic-amino acid - LAAAl lipidic 2-amino alcohol - LDA lipidic 1,2-diamine - LP lipidic peptide - MPM-Cl p-methoxybenzyl chloride - MsCl methanesulphonyl chloride - MTPA -methoxy--(trifluoromethyl)phenylaccitc - PLA₂ phospholipase A₂ - TBIIP tert-butyl hydroperoxide - THF tetrahydrofuran - TMSCl trimethylsilyl chloride - Tr trityl - Z benzyloxycarbonyl     

Synthesis and characterization of branched phosphopeptides: Prototype consolidated ligands for SH(32) domains

Summary This paper details the solid-phase synthesis by N -9-fluorenylmethyloxycarbonyl (Fmoc) chemistry of a series of bivalent consolidated ligands, branched peptides with lengths of 22 to 25 residues. The target peptides were designed to, and in fact do, interact with greater specificity and higher affinity with the SH2 and SH3 domains of Abelson kinase in an SH(32) dual domain construct. Fmoc-O-phospho-l-tyrosine[Fmoc-Tyr(PO₃H₂)-OH] was used to introduce the required phosphotyrosine residues, and Fmoc-N -1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl-l-lysine [Fmoc-Lys(Dde)-OH] was used to introduce a branch point that allowed proper orientation of individual ligands. The resultant product peptides were characterized by amino acid analyses and electrospray mass spectra.This paper is based on a presentation given at the Symposium on Peptide Structure and Design as part of the 31st Annual ACS Western Regional Meeting held in San Diego, CA, USA, October 18–21, 1995.     

Purification and characterization of extracellular β-glucosidase from Myceliophthora thermophila

The extracellular -glucosidase has been purified from culture broth of Myceliophthora thermophila ATCC 48104 grown on crystalline cellulose. The enzyme was purified approximately 30-fold by (NH₄)₂SO₄ precipitation and column chromatography on DEAE-Sephadex A-50, Sephadex G-200 and DEAE-Sephadex A-50. The molecular mass of the enzyme was estimated to be about 120 kD by both sodium dodecyl sulphate gel electrophoresis and gel filtration chromatography. It displayed optimal activity at pH 4.8 and 60°C. The purified enzyme in the absence of substrate was stable up to 60°C and pH between 4.5 and 5.5. The enzyme hydrolysed p-nitrophenyl--d-glucoside, cellobiose and salicin but not carboxymethyl cellulose or crystalline cellulose. The K _m of the enzyme was 1.6mm for p-nitrophenyl--d-glucoside and 8.0mm for cellobiose. d-Glucose was a competitive inhibitor of the enzyme with a K of 22.5mm. Enzyme K activity was inhibited by HgCl₂, FeSO₄, CuSO₄, EDTA, sodium dodecyl sulphate, p-chloromercurobenzoate and iodoacetamide and was stimulated by 2-mercaptoethanol, dithiothreitol and glutathione. Ethanol up to 1.7 m had no effect on the enzyme activity.The authors are with the Department of Microbiology, Bose Institute, 93/1, A.P.C. Road, Calcutta 700 009, India. S.K. Raha is presently with the Department of Medicine, University of Saskatchewan, Saskatoon, Canada S7N OXO.     

Succinate-ethanol fermentation in Clostridium kluyveri: purification and characterisation of 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA Δ3-Δ2-isomerase

Anaerobically prepared cell extracts of Clostridium kluyveri grown on succinate plus ethanol contained high amounts of 4-hydroxybutyryl-CoA dehydratase, which catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under strictly anaerobic conditions to over 95% homogeneity and had a specific activity of 123 nkat mg^-1. The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethanol by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate via 4-hydroxybutyrate to butyrate. Interestingly, the enzyme is almost identical to the previously isolated 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The dehydratase was revealed as being a homotetramer (m=59 kDa/subunit), containing 2±0.2 mol FAD, 13.6±0.8 mol Fe and 10.8±1.2 mol inorganic sulfur. The enzyme was irreversibly inactivated after exposure to air. Reduction by sodium dithionite also yielded an inactive enzyme which could be reactivated, however, up to 84% by oxidation with potassium hexacyanoferrate(III). The enzyme possesses an intrinsic vinylacetyl-CoA isomerase activity which was also found in 4-hydroxybutyryl-CoA dehydratase from C. aminobutyricum. Moreover, the N-terminal sequences of the dehydratases from both organisms were found to be 63% identical.     

Control of nitrogenase inAzospirillum sp.

Many N₂-fixing organisms can turn off nitrogenase activity in the presence of NH₄ ⁺ and turn it on again when the NH₄ ⁺ is exhausted. One of the most interesting systems for accomplishing this is by covalent modification of one subunit of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). The system can be reactivated when NH₄ ⁺ is exhausted, by dinitrogenase reductase activating glycohydrolase (DRAG) which removes the inactivating group. It is fascinating that some species of the genusAzospirillum possess the DRAT and DRAG systems (A. lipoferum andA. brasilense), whereasA. amazonense in the same genus lacks DRAT and DRAG.A. amazonense responds to NH₄ ⁺ but does not exhibit modification of dinitrogenase reductase characteristic of the action of DRAT. However, it has been possible to clone DRAT and DRAG and to introduce them intoA. amazonense, whereupon they become functional in this organism. The DRAT and DRAG system does not appear to function inAcetobacter diazotrophicus, an organism isolated from sugar cane, that fixes N₂ at a pH as low as 3.0.A. diazotrophicus does show a rather sluggish response to NH₄ ⁺. A level of about 10 M NH₄ ⁺ is required to switch off the system. The response to NH₄ ⁺ is influenced by the dissolved oxygen concentration (DOC) as has been reported forAzospirillum sp. A DOC in equilibrium with 0.1 to 0.2 kPa O₂ seems optimal for the response inA. diazotrophicus.