Validation of a prototype DiodeAir for small field dosimetry.

Standard commercial diode detectors over-respond within small radiation fields, an effect largely attributable to the relatively high mass-density of silicon. However, Monte Carlo studies can be used to optimise dosimeter designs and have demonstrated that 'mass-density compensation'-for example, introducing a low-density air-gap upstream of a diode's high-density silicon volume-can substantially improve instrument response. In this work we used egs_chamber Monte Carlo simulations to predict the ideal air-gap thickness for a PTW 60017 unshielded diode detector. We then developed a prototype instrument incorporating that air-gap and, for a 6 MV linac, tested it experimentally against EBT3 film. We also tested a further three prototypes with different air-gap thicknesses. Our results demonstrate that for a 10 × 10 cm(2) reference field the DiodeAir, a PTW 60017 diode with a built-in air-gap of 1 mm, has on-axis correction factors near unity. Laterally the DiodeAir performs very well off-axis and reports FWHM and penumbra values consistent with those measured using EBT3. For PDD measurement, the performance of the DiodeAir matches that of the original PTW 60017. The experimental focus of this work was 6 MV but we also simulated the on-axis response of the DiodeAir within 15 MV beams and found that our modification proved robust to this substantial increase in beam energy. However, the original diode 60017 does exhibit low energy scatter dependencies and may over-respond to high linac dose-rates such that applying the mass-density compensation method to an alternative instrument (particularly a diamond detector) could ultimately take us even closer to the small-field ideal.

Mass-density compensation can improve the performance of a range of different detectors under non-equilibrium conditions.

Dosimeters often consist of several components whose mass densities differ substantially from water. These components cause small-field correction factors to vary significantly as lateral electronic equilibrium breaks down. Even amongst instruments designed for small-field dosimetry, inter-detector variation in the correction factors associated with very small (∼0.5 cm) fields can amount to tens of per cent. For a given dosimeter, small-field correction factors vary not only with field size but also with detector azimuthal angle and position within the field. Furthermore the accurate determination of these factors typically requires time-intensive Monte Carlo simulations. Thus, if achievable, 'correction factor free' small-field dosimetry would be highly desirable. This study demonstrates that a new generation of mass-density compensated detectors could take us towards this goal. Using a 6 MV beam model, it shows that 'mass-density compensation' can be utilized to improve the performance of a range of different detectors under small-field conditions. Non-sensitive material of appropriate mass-density is incorporated into detector designs in order to make the instruments behave as if consisting only of water. The dosimeter perturbative effects are then reduced to those associated with volume averaging. An even better solution-which modifies detectors to obtain profiles that look like those measured by a point-like water structure-is also considered. Provided that adequate sensitivity can be achieved for a small measurement volume, this study shows that it may be possible to use mass-density compensation (and Monte Carlo-driven design) to produce a solid-state dosimeter/ionization chamber with a near-perfect non-equilibrium response.

Detector density and small field dosimetry: integral versus point dose measurement schemes.

PURPOSE: The Alfonso et al. [Med. Phys. 35, 5179-5186 (2008)] formalism for small field dosimetry proposes a set of correction factors (kQclin,Qmsrfclin,fmsr) which account for differences between the detector response in nonstandard (clinical) and machine-specific-reference fields. In this study, the Monte Carlo method was used to investigate the viability of such small field correction factors for four different detectors irradiated under a variety of conditions. Because kQclin,Qmsrfclin,fmsr values for single detector position measurements are influenced by several factors, a new theoretical formalism for integrated-detector-position [dose area product (DAP)] measurements is also presented and was tested using Monte Carlo simulations. METHODS: A BEAMnrc linac model was built and validated for a Varian Clinac iX accelerator. Using the egs++ geometry package, detailed virtual models were built for four different detectors: a PTW 60012 unshielded diode, a PTW 60003 Diamond detector, a PTW 31006 PinPoint (ionization chamber), and a PTW 31018 MicroLion (liquid-filled ionization chamber). The egs_chamber code was used to investigate the variation of kQclin,Qmsrfclin,fmsr with detector type, detector construction, field size, off-axis position, and the azimuthal angle between the detector and beam axis. Simulations were also used to consider the DAP obtained by each detector: virtual detectors and water voxels were scanned through high resolution grids of positions extending far beyond the boundaries of the fields under consideration. RESULTS: For each detector, the correction factor (kQclin,Qmsrfclin,fmsr) was shown to depend strongly on detector off-axis position and detector azimuthal angle in addition to field size. In line with previous studies, substantial interdetector variation was also observed. However, it was demonstrated that by considering DAPs rather than single-detector-position dose measurements the high level of interdetector variation could be eliminated. Under small field conditions, mass density was found to be the principal determinant of water equivalence. Additionally, the mass densities of components outside the sensitive volumes were found to influence the detector response. CONCLUSIONS: kQclin,Qmsrfclin,fmsr values for existing detector designs depend on a host of variables and their calculation typically relies on the use of time-intensive Monte Carlo methods. Future moves toward density-compensated detector designs or DAP based protocols may simplify the methodology of small field dosimetry.

Application of a lectin from the mushroom Polysporus squamosus for the histochemical detection of the NeuAcalpha2,6Galbeta1,4Glc/GlcNAc sequence of N-linked oligosaccharides: a comparison with the Sambucus nigra lectin.

The lectin from the mushroom Polysporus squamosus (PSL) has an extended carbohydrate combining site, which exhibits a high specificity and affinity toward the NeuAc5alpha2,6Galbeta1,4Glc/GlcNAc trisaccharide sequence of asparagine-linked oligosaccharides. Therefore, PSL should be a superior reagent to the lectin from Sambucus nigra (SNA), which does not discriminate between alpha2,6-linked NeuAc5 present either in asparagine- or serine/threonine-linked oligosaccharides. We have prepared a digoxigenin-conjugated PSL and applied it for histochemistry and blotting. We observed a more restricted staining pattern by PSL as compared to SNA in paraffin sections from different rat organs. Pretreatment of sections with N-glycanase F abolished PSL staining indicating that it interacts only with asparagine-linked oligosaccharides. Furthermore, PSL staining was neuraminidase sensitive. In contrast, SNA staining was only partially sensitive to N-glycanase F pretreatment demonstrating that it was in part due to alpha2,6-linked NeuAc5 present in serine/threonine-linked oligosaccharides. The most striking observation in this regard was that PSL, in contrast to SNA, did not stain the mucus of sheep submandibular gland, which is extremely rich in serine/threonine-linked Neu5Acalpha2,6N-acetylgalactosamine. Furthermore, in some tissues neuraminidase pretreatment resulted in increased intensity of SNA staining probably due to binding to exposed terminal N-acetylgalactosamine residues. Collectively, these results indicate that PSL is a useful tool for the histochemical detection of alpha2,6-linked NeuAc5 in asparagine-linked oligosaccharides.

Carbohydrate binding properties of banana (Musa acuminata) lectin I. Novel recognition of internal alpha1,3-linked glucosyl residues.

Examination of lectins of banana (Musa acuminata) and the closely related plantain (Musa spp.) by the techniques of quantitative precipitation, hapten inhibition of precipitation, and isothermal titration calorimetry showed that they are mannose/glucose binding proteins with a preference for the alpha-anomeric form of these sugars. Both generate precipitin curves with branched chain alpha-mannans (yeast mannans) and alpha-glucans (glycogens, dextrans, and starches), but not with linear alpha-glucans containing only alpha1,4- and alpha1,6-glucosidic bonds (isolichenan and pullulan). The novel observation was made that banana and plantain lectins recognize internal alpha1,3-linked glucosyl residues, which occur in the linear polysaccharides elsinan and nigeran. Concanavalin A and lectins from pea and lentil, also mannose/glucose binding lectins, did not precipitate with any of these linear alpha-glucans. This is, the authors believe, the first report of the recognition of internal alpha1,3-glucosidic bonds by a plant lectin. It is possible that these lectins are present in the pulp of their respective fruit, complexed with starch.

Carbohydrate binding properties of banana (Musa acuminata) lectin II. Binding of laminaribiose oligosaccharides and beta-glucans containing beta1,6-glucosyl end groups.

This paper extends our knowledge of the rather bizarre carbohydrate binding poperties of the banana lectin (Musa acuminata). Although a glucose/mannose binding protein which recognizes alpha-linked gluco-and manno-pyranosyl groups of polysaccharide chain ends, the banana lectin was shown to bind to internal 3-O-alpha-D-glucopyranosyl units. Now we report that this lectin also binds to the reducing glucosyl groups of beta-1,3-linked glucosyl oligosaccharides (e.g. laminaribiose oligomers). Additionally, banana lectin also recognizes beta1,6-linked glucosyl end groups (gentiobiosyl groups) as occur in many fungal beta1,3/1,6-linked polysaccharides. This behavior clearly distinguishes the banana lectin from other mannose/glucose binding lectins, such as concanavalin A and the pea, lentil and Calystegia sepium lectins.

Purification and characterization of a Neu5Acalpha2-6Galbeta1-4Glc/GlcNAc-specific lectin from the fruiting body of the polypore mushroom Polyporus squamosus.

A lectin has been purified from the carpophores of the mushroom Polyporus squamosus by a combination of affinity chromatography on beta-D-galactosyl-Synsorb and ion-exchange chromatography on DEAE-Sephacel. Gel filtration chromatography, SDS-polyacrylamide gel electrophoresis, and N-terminal amino acid sequencing indicated that the native lectin, designated P. squamosus agglutinin, is composed of two identical 28-kDa subunits associated by noncovalent bonds. P. squamosus agglutinin agglutinated human A, B, and O and rabbit red blood cells but precipitated only with human alpha(2)-macroglobulin, of many glycoproteins and polysaccharides tested. The detailed carbohydrate binding properties of the purified lectin were elucidated using three different approaches, i.e. precipitation inhibition assay (in solution binding assay), fluorescence quenching studies, and glycolipid binding by lectin staining on high-performance thin layer chromatography (solid-phase binding assay). Based on the results obtained by these assays, we conclude that although the P. squamosus lectin binds beta-D-galactosides, it has an extended carbohydrate-combining site that exhibits highest specificity and affinity toward nonreducing terminal Neu5Acalpha2, 6Galbeta1,4Glc/GlcNAc (6'-sialylated type II chain) of N-glycans (2000-fold stronger than toward galactose). The strict specificity of the lectin for alpha2,6-linked sialic acid renders this lectin a valuable tool for glycobiological studies in biomedical and cancer research.

Xanthosoma sagittifolium tubers contain a lectin with two different types of carbohydrate-binding sites.

An unusual lectin possessing two distinctly different types of carbohydrate-combining sites was purified from tubers of Xanthosoma sagittifolium L. by consecutive passage through two affinity columns, i.e. asialofetuin-Sepharose and invertase-Sepharose. SDS-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and gel filtration chromatography of the purified lectin showed that the X. sagittifolium lectin is a heterotetrameric protein composed of four 12-kDa subunits (alpha(2)beta(2)) linked by noncovalent bonds. The results obtained by quantitative precipitation and hapten inhibition assays revealed that the lectin has two different types of carbohydrate-combining sites: one type for oligomannoses, which preferentially binds to a cluster of nonreducing terminal alpha1,3-linked mannosyl residues, and the other type for complex N-linked carbohydrates, which best accommodates a non-sialylated, triantennary oligosaccharide with N-acetyllactosamine (i.e. Galbeta1,4GlcNAc-) or lacto-N-biose (i.e. Galbeta1,3GlcNAc-) groups at its three nonreducing termini.

Sialic-acid-binding lectin from the slug Limax flavus--cloning, expression of the polypeptide, and tissue localization.

A cDNA library of Limax flavus was constructed and screened for sialic-acid-specific lectins. Complementary DNA clones were categorized into seven groups corresponding to closely related but different sequences. Group 1 clones contained an ORF encoding 199 amino acids including a sequence identical to the partial amino acid sequence obtained from the lectin protein. Within its 1074-bp 3' untranslated region, ten closely related 60-bp sequence repeats were found. Group 2 clones contained an ORF encoding a polypeptide chain of the same number of amino acid residues, with 89.1% overall identity to that of the group 1 and eight 60-bp repeat sequences in the 3' untranslated region. The remaining groups of clones contained ORF with highly similar full or partial sequences, with or without 60 bp repeats in the 3' untranslated region. The large number of closely related but different cDNA clones obtained indicated that the slug sialic-acid-specific lectin gene is a member of a multigene family. The lectin amino acid sequence showed significant similarity with the fibrinogen domain of human tenascin-C, with a human C-type serum lectin, and with pig ficolin. Immunostaining analysis of slug tissue for the lectin indicated that it is present primarily on the epidermal surface and in mucous glands. Recombinant slug lectin protein lacking the 20-amino-acid N-terminal signal sequence produced in a bacterial expression system from a group-1 clone accumulated as aggregates in inclusion bodies, suggesting that large-scale production of the active agglutinin may be possible.

Isolation of a novel plant lectin with an unusual specificity from Calystegia sepium.

A novel plant lectin has been isolated from the rhizomes of Calystegia sepium (hedge bindweed) and partially characterized. The lectin is a dimeric protein composed of two identical non-covalently linked subunits of 16 kDa. Hapten inhibition studies indicate that the novel lectin is best inhibited by maltose and mannose and hence exhibits a sugar binding specificity that differs in some respects from that of all previously isolated plant lectins. Mitogenicity tests have shown that the Calystegia lectin is a powerful T-cell mitogen. Affinity purification of human, plant and fungal glycoproteins on immobilized C. sepium lectin demonstrates that this novel lectin can be used for the isolation of glycoconjugates from various sources. Moreover, it can be expected that by virtue of its distinct specificity, the new lectin will become an important tool in glycobiology.

Molecular cloning of two different mannose-binding lectins from tulip bulbs.

Two lectins were isolated from the bulbs of Tulipa cv. Apeldoorn and their corresponding cDNA clones analyzed. The first, called TxLMII (second mannose-binding Tulipa hybrid lectin), is a novel mannose-binding tulip lectin. Based on its molecular structure, carbohydrate-binding specificity and amino acid sequence, TxLMII belongs to the superfamily of mannose-binding monocot lectins which are also found in representatives of the plant families Amaryllidaceae, Alliaceae, Orchidaceae and Araceae. Molecular cloning of the second lectin, called TxLCI (first Tulipa hybrid lectin with complex specificity), allowed determination unambiguously of the molecular structure of this previously described protein. In addition, evidence is presented that each TxLCI subunit possesses a mannose-binding site and an N-acetylgalactosamine-binding site, which act independently of each other. Both binding sites are located in a separate domain of the lectin polypeptide. Since the first domain of TxLCI shows sequence similarity to TxLMII, it is suggested that their genes evolved from a common ancestor.

Alpha-(2-->3)- and alpha-(2-->6)-sialyltransferase activities present in three variants of Ehrlich tumor cells: identification of the products derived from N-acetyllactosamine and beta-D-Gal-(1-->3)-alpha-D-GalNAc-(1-->O)-Bn.

We compared several sialytransferase activities related to synthesis of O-linked and N-linked sialyglycoproteins in Ehrlich ascites tumor cells that grow normally in murine ascites, but are not adherent nor grow in tissue culture (na-EAT cells), with those in cells that were selected to grow in tissue culture and adhere to extracellular matrices (a-EAT cells). Crude Golgi preparations from both cell types contained predominantly beta-D-Gal-(1-->3)-D-GalNAc alpha-(2-->3)-sialyltransferase activity. Sialylation of N-acetyllactosamine, lacto-N-tetraose, and benzyl alpha-D-GalNAc occurred at from 1 to 4% of that activity. Analysis, by ion-exchange HPLC at high pH, of sialylated N-acetyllactosamine showed that na-EAT cells sialylated beta-D-Gal-(1-->4)-D-GlcNAc mostly by alpha-(2-->3)-sialyltransferase, whereas beta-D-Gal-(1-->4)-D-GlcNAc alpha-(2-->6)-sialyltransferase activity was prominent in a-EAT cells. In addition, preparations from na-EAT cells formed significant quantities of an unknown tritiated product from CMP-[9-3H]sialic acid, suggesting at least one other difference in enzyme levels between the cell types. a-EAT cells reestablished in murine ascites for 11 passages retained the sialyltransferase levels characteristic of a-EAT cells. When viable cells were labeled with D-[3H]glucosamine, na-EAT cells formed larger amounts of sialic acid in O-linked glycoproteins than did a-EAT cells.

Glutamate uptake system in the presynaptic vesicle: glutamic acid analogs as inhibitors and alternate substrates.

A variety of naturally occurring amino acids, their isomers, and synthetic analogs were tested for their ability to inhibit uptake of [3H]glutamate into presynaptic vesicles from bovine cerebral cortex. Strongest inhibition (Ki < 1mM) was observed for trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) and erythro-4-methyl-L-glutamic acid (MGlu), while 4-methylene-L-glutamic acid (MeGlu) was only moderately inhibitory (Ki = approximately 3mM), indicating that the synaptic vesicle glutamate translocator has higher affinity for trans-ACPD and MGlu than for glutamate. A few other amino acids, e.g., 4-hydroxyglutamic acid, S-carboxyethyl cysteine, and 5-fluorotryptophan, were slightly inhibitory; all L- and DL-isomers of protein amino acids and longer chain acidic amino acids were without measurable inhibition. Potassium tetrathionate and S-sulfocysteine exhibited strong to moderate noncompetitive or irreversible inhibition. Inhibition by t-ACPD, MGlu, or MeGlu was competitive with glutamic acid. Each of these competitive inhibitors was also taken up by the vesicle preparation in an ATP-dependent manner, as indicated by their being recovered unchanged from filtered vesicles. Similar results were obtained with reconstituted vesicles, while glutamate uptake by partially purified rat synaptosomes was inhibited only by MGlu. These results indicate that the glutamate translocator of presynaptic vesicles has stringent structural requirements distinct from those of the plasma membrane translocator and the metabotropic type of postsynaptic glutamate receptor. They further suggest possible structural requirements of pharmacologically significant compounds that can substitute for glutamic acid in the presynaptic side of glutamatergic synapses, thus serving to moderate or control glutamate excitation and associated excitotoxic effects in these neurons.

4-methyleneglutamine amidohydrolase from peanut leaves : preparation, catalytic properties, and immunological responses of a highly purified form of the enzyme.

4-Methyleneglutamine amidohydrolase has been extracted and purified over 1000-fold from 14-day-old peanut (Arachis hypogaea) leaves by modification of methods described previously. The purified enzyme shows two bands of activity and three to four bands of protein after electrophoresis on nondenaturing gels. Each of the active bands is readily eluted from gel slices and migrates to its original position on subsequent electrophoresis. Although they are electrophoretically distinct, the two forms of the enzyme are immunologically identical by Ouchterlony double-diffusion techniques and have similar catalytic properties. Activity toward glutamine that has a threefold lower V(max) and a four-fold higher K(m) value copurifies with MeGln aminohydrolase activity. 4-Methyleneglutamine and 4-methyleneglutamic acid inhibit the hydrolysis of glutamine while glutamine inhibits 4-methyleneglutamine hydrolysis, further indicating the identity of the activity toward both substrates. Amidohydrolase activity is stimulated up to threefold by preincubation with either ionic or non-ionic detergents (0.1%) and also by added proteins (0.5% bovine serum albumin or whole rabbit serum); it is inhibited 50% by 1 millimolar borate or the glutamine analog, albizziin (10 millimolar). Rabbit antiserum to the purified peanut enzyme cross-reacts with one or more proteins in extracts of some plants but not others; in no instance, however, was 4-methyleneglutamine amidohydrolase activity detected in other species. Overall, the results support the hypothesis that 4-methyleneglutamine supplies N, via its hydrolysis by the amidohydrolase, to the growing shoots of peanut plants, whereas glutamine hydrolysis is prevented by the prepon-derance of the preferred substrate. Some results also suggest that this amidohydrolase activity may be regulated by metabolites and/or by association with other cellular components.

Specificity of aspartate aminotransferases from leguminous plants for 4-substituted glutamic acids.

Aspartate aminotransferase (glutamate-oxalacetate transaminase) was partially purified from extracts of germinating seeds of peanut (Arachis hypogaea), honey locust (Gleditsia triacanthos), soybean (Glycine max), and Sophora japonica. The ability of these enzyme preparations, as well as aspartate aminotransferase purified from pig heart cytosol, to use 4-substituted glutamic acids as amino group donors and their corresponding 2-oxo acids as amino group acceptors in the aminotransferase reaction was measured. All 4-substituted glutamic acid analogs tested were poorer substrates than was glutamate or 2-oxoglutarate. 2-Oxo-4-methyleneglutarate was least effective (lowest relative V(m)/K(m)) as a substrate for the enzyme from peanuts and honey locust, which are the two species studied that accumulate 4-methyleneglutamic acid and 4-methyleneglutamine. Of the different aminotransferases tested, the enzyme from honey locust was the least active with 2-oxo-4-hydroxy-4-methylglutarate, the corresponding amino acid of which also accumulates in that species. These results suggest that transamination of 2-oxo-4-substituted glutaric acids is not involved in the biosynthesis of the corresponding 4-substituted glutamic acids in these species. Rather, accumulation of certain 4-substituted glutamic acids in these instances may be, in part, the result of the inefficacy of their transamination by aspartate aminotransferase.

4-Methyleneglutamic acid and 4-methyleneglutamine: isolation from extracts of peanut seedlings and determination by high-performance liquid chromatography.

The non-protein amino acids, 4-methyleneglutamic acid and 4-methyleneglutamine, are isolated from aqueous extracts of peanut seedlings in good yield and high purity using a simple HCl-gradient elution from a column of cation-exchange resin followed, in some instances, by a gradient elution with acetic acid from a column of an anion-exchange resin. All of the 4-substituted glutamic acids commonly found in legume species are resolved by a combination of these two system. For analytical purposes, resolution of the acidic amino acids as their phenylthiocarbamoyl derivatives is achieved by HPLC but not by conventional ion-exchange amino acid analysis. Although 4-methyleneglutamine undergoes cyclic deamidation in acidic medium at a slower rate than glutamine, this reaction occurs to a significant extent at 22 degrees C but not a 4 degrees C during the cation-exchange chromatographic fractionation.

Purification and characterization of a novel 4-methyleneglutamine synthetase from germinated peanut cotyledons (Arachis hypogaea).

A newly detected amide synthetase, designated 4-methyleneglutamine synthetase, has been partially purified from extracts of 5- to 7-day germinated peanut cotyledons (Arachis hypogaea). Purification steps include fractionation with protamine sulfate and ammonium sulfate followed by column chromatography on Bio-Gel and DEAE-cellulose; synthetase purified over 300-fold is obtained. The enzyme has a molecular weight estimated to be approximately 250,000 and a broad pH optimum with maximal activity at approximately pH 7.5. Maximal rates of activity are obtained with NH+4 (Km = 3.7 mM) as the amide donor and the enzyme is highly specific for 4-methylene-L-glutamic acid (Km = 2.7 mM) as the amide acceptor. Product identification and stoichiometric studies establish the reaction catalyzed to be: 4-methyleneglutamic acid + NH4+ + ATP Mg2+----4-methyleneglutamine + AMP + PPi. PPi accumulates only when F- is added to inhibit pyrophosphatase activity present in synthetase preparations. This enzymatic activity is completely insensitive to the glutamine synthetase inhibitors, tabtoxinine-beta-lactam and F-, and is only partially inhibited by methionine sulfoximine. It is, however, inhibited by added pyrophosphate in the presence of F- as well as by certain divalent metal ions (other than Mg2+) including Hg2+, Ni2+, Mn2+, and Ca2+. All data obtained indicate that this newly detected synthetase is distinct from the well-known glutamine and asparagine synthetases.

Inhibition by auxins of 4-methyleneglutamic Acid synthesis in tissue cultures of peanut seeds.

Callus cultures of peanut (Arachis hypogaea L. cv Valencia Tennessee Red) cotyledons grown on Linsmaier and Skoog medium containing normal levels of auxin and cytokinin do not synthesize either 4-methyl-eneglutamic acid or 4-methyleneglutamine, which nonprotein amino acids are normally found in significant amounts in peanut plants. If mature peanut embryos (with cotyledons removed) are germinated and grown on a similar medium containing no added phytohormone, normal levels of these two amino acids accumulate. The addition of an auxin, however, prevents formation of 4-methyleneglutamic acid and 4-methyleneglutamine; typical levels of other free amino acids are seen and excised embryos so cultured develop into apparently otherwise normal plants. Kinetin addition to embryo cultures has little or no effect. 4-Methyleneglutamine is formed when 4-methyleneglutamic acid is added to embryo cultures maintained on auxin-containing medium, indicating that the phytohormone does not block amidation but rather the biosynthesis of 4-methyleneglutamic acid.

Dimerization occurs during the reversible acid inactivation of 2-oxo-4-hydroxyglutarate aldolase from Escherichia coli.

Exposure of Escherichia coli 2-oxo-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate glyoxylate-lyase, EC 4.1.3.16) (molecular weight = 63 000) to phosphoric acid at pH 1.6 for 10 min at 4 degrees C causes 95% or greater inactivation. No significant effect on the rate or extent of inactivation is caused by varied aldolase concentrations or the presence of exogenous proteins. Chloride ion (50-100 mM) or 10 mM 2-oxo-4-hydroxyglutarate markedly decreases both the rate and extent of inactivation; good protection is also afforded by 10 mM pyruvate, glyoxylate, glyoxal, 2-oxoglutarate or 2-oxobutyrate. Whereas native aldolase has two free and three buried sulfhydryl groups, all five are exposed in the acid-inactivated enzyme and the molecular weight of this species at pH 1.6 is 126 000. Ultraviolet absorbance difference spectra, circular dichroism spectra and ultracentrifugation studies establish that the inactivation process is characterized by an alteration of secondary and tertiary structure as well as an aggregation to a dimer of the native molecule. Reactivation of enzyme activity to 60-80% of the original level is seen within 20 min at pH 6 to 8; examination of inactivation/reactivation as a function of pH indicates that these two processes occur via kinetically distinct pathways. Native and reactivated enzymes are identical in molecular weight, sulfhydryl titer, Km and alpha-helix content.

4-methyleneglutamine synthetase: a new amide synthetase present in germinating peanuts.

Enzymatic activity which catalyzes the synthesis of 4-methyleneglutamine from 4-methyleneglutamic acid + ammonia was detected in and partially purified from cotyledons of peanut seeds germinated 5 to 7 days. This activity was separated from glutamine and asparagine synthetases by ammonium sulfate precipitation and DEAE-cellulose chromatography. The enzyme is distinct from these other amide synthetases in its substrate specificity, lack of amide/hydroxylamine exchange, and use of ammonium ion as amide donor together with formation of AMP from ATP. The activity is quite labile in solution, but is retained as a precipitate in ammonium sulfate or when frozen in 12.5% glycerol at -77 degrees C. This activity might be responsible for catalyzing the rapid synthesis of 4-methyleneglutamine which occurs in germinating peanuts.