Ferric chelate reduction by suspension culture cells and roots of soybean: A kinetic comparison

The abilities of suspension cultures and intact roots of soybean (Glycine max L. cv. Hawkeye) to reduce ferric chelate were compared. Ferric chelate was supplied as ferric hydroxyethylethylenediaminetriacetic acid (FeHEDTA) and reduction was measured spectrophotometrically using bathophenan-throlinedisulfonic acid (BPDS) as the ferrous scavenger. Ferric chelate reduction by cell suspension cultures showed typical saturation kinetics; however, no difference was observed between cells that had been continuously grown with Fe (+Fe) and those that had been grown for four days without added Fe (–Fe). Values for K_m and V_max, determined from a Lineweaver-Burk plot, were 57 M and nmoles mg^-1 dry weight for the +Fe cells and 50 M and 22 nmoles mg^-1 dry weight for the -Fe cells, respectively. Ferric chelate reduction by Fe-deficient roots also exhibited saturation kinetics, while roots grown with adequate Fe did not reduce ferric chelate. The K_m and V_max values for Fe-deficient roots were 45 M and 20 nmoles mg^-1 dry weight, respectively, and did not differ from values obtained for cells in culture. This study offers strong evidence that the mechanism responsible for the reduction of ferric chelate is the same for cultured cells and roots and that the process is controlled at the cellular level. We propose that suspension cultures can be used as an alternative to intact roots in the study of ferric chelate reduction.     

Biochemical properties of lipoxygenase from opium poppy chloroplasts

Lipoxygenase (LOX) from opium poppy (Papaver somniferum L.) chloroplasts was isolated and 126.1-fold purified to electrophoretic homogeneity by combination of ion-exchange chromatography on HA-Ultragel column and affinity chromatography on a linoleyl-aminopropyl agarose column. The relative molecular mass of the LOX determined by SDS-PAGE was 92 kDa. Kinetic properties of purified LOX were determined in spectrophotometric assay by using of linoleic acid (K_M = 1.78 mM and V_max = 11.4 μmol mg⁻¹ min⁻¹) and linolenic acid (K_M = 1.27 mM and V_max = 10.2 μmol mg⁻¹ min⁻¹). The optimum pH was 6.0 for both linoleic and linolenic acid dioxygenation catalyzed by LOX. HPLC analysis of the products revealed a dual positional specificity of linoleic acid dioxygenation at pH 6.0 with ratio of 9- and 13-hydroperoxide products being about 1:1. The activity of purified LOX was stimulated by Mg²⁺ and Ca²⁺.     

Uptake of iron byGeotrichum candidum,a non-siderophore producer

Summary Geotrichum candidum (isolate 1–9) pathogenic on citrus fruits, appears to lack siderophore production. Iron uptake byG. candidum is mediated by two distinct iron-regulated, energy-and temperature-dependent transport systems that require sulfhydryl groups. One system exhibits specificity for either ferric or ferrous iron, whereas the other exhibits specificity for ferrioxamine-B-mediated iron uptake and presumably other hydroxamate siderophores. Radioactive iron uptake from⁵⁹FeCl₃ showed an optimum at pH 6 and 35° C, and Michaelis-Menten kinetics (apparentK _m = 3 m,V _max = 0.054 nmol · mg^–1 · min^–1). The maximal rate of Fe²⁺ uptake was higher than Fe³⁺ (V _max = 0.25 nmol · mg^–1 · min^–1) but theK _m was identical. Reduction of ferric to ferrous iron prior to transport could not be detected. The ferrioxamine B system exhibits an optimum at pH 6 and 40° C and saturation kinetics (K _m = 2 M,V _max = 0.22 nmol · mg^–1 · min^–1). The two systems were distinguished as two separate entities by negative reciprocal competition, and on the basis of differential response to temperature and phenazine methosulfate. Mössbauer studies revealed that cells fed with either⁵⁷FeCl₃ or⁵⁷FeCl₂ accumulated unknown ferric and ferrous binding metabolites.     

Energetic basis of development of salt-tolerant transport in a moderately halophilic bacterium,Vibrio costicola

Active transport of -aminoisobutyric acid (AIB) in Vibrio costicola utilizes a system with affinity for glycine, alanine and, to some extent, methionine. AIB transport was more tolerant of high salt concentrations (3–4 M NaCl) in cells grown in the presence of 1.0 M NaCl than in those grown in the presence of 0.5 M NaCl. The former cells could also maintain much higher ATP contents than the latter in high salt concentrations.Transport kinetic studies performed with bacteria grown in 1.0 M NaCl revealed three effects of the Na⁺ ion: the first effect is to increase the apparent affinity (K _t) of the transport system for AIB at Na⁺ concentrations <0.2 M, the second to increase the maximum velocity (V _max) of transport (Na⁺ concentrations between 0.2 and 1.0 M), and the third to decrease the V _max without affectig K _t (Na⁺ concentrations >1.0 M). Cells grown in the presence of 0.5 M or 1.0 M NaCl had similar affinity for AIV. Thus, the differences in salt response of transport in these cells do not seem due to differences in AIB binding. Large, transport-inhibitory concentrations of NaCl resulted in efflux of AIB from cells preloaded in 0.5 M or 1.0 M NaCl, with most dramatic efflux occurring from the cells whose AIB transport was more salt-sensitive. Our results suggest that the degree to which high salt concentrations affect the transmembrane electrochemical energy source used for transport and ATP synthesis is an important determinant of salt tolerance.Abbreviations AIB -aminoisobutyric acid - pmf proton motive force     

Uptake of phosphate by symbiotic and free-living dinoflagellates

Uptake of phosphate in the light by Amphidinium carterae, Amphidinium klebsii, cultured and symbiotic Gymnodinium microadriaticum conformed to Michaelis-Menten type saturation kinetics with all organisms showing similar K _m values, namely 0.005 to 0.016 M phosphorus. V _max values were 0.009–0.32 nmol phosphorus · 10⁵ cells^-1 · 10 min^-1. Phosphate uptake by all the dinoflagellates was greater in the dark than in the light. The metabolic inhibitor 3-(3,4-dichlorophenyl) 1,1-dimethylurea stimulated phosphate uptake in the light by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. Carbonylcyanide 3-chlorophenylhydrazone (CCCP) inhibited phosphate uptake by A. carterae and A. klebsii under both light and dark conditions. Uptake of phosphate by cultured and symbiotic G. microadriaticum in the light, but not in the dark, was inhibited by CCCP. Low concentrations of arsenate (5 g As · l^-1) stimulated phosphate by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. High concentrations of arsenate (100 g As · l^-1) did not affect uptake of phosphate by A. carterae and A. klebsii.     

Selenite uptake and incorporation by Selenomonas ruminatium

Selenite uptake and incorporation in Selenomonas ruminantium was constitutive with an inducible component. It was distinct from sulphate or selenate transport, since sulphate and selenate did not inhbit uptake, nor could sulphate or selenate uptake be demonstrated. Selenite uptake had an apparent K _m of 1.28 mM and a V _max of 148 ng Se min^-1 mg^-1 protein. Uptake was sensitive to inhibition by 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), azide, iodoacetic acid (IAA) and N-ethylmaleimide (NEM), but not chloropromazine (CPZ), N,N-dicyclohexyl-carbodiimide (DCCD), quinine, arsenate, or fluoride. Treatment of cells accumulating ⁷⁵[Se]-Selenite with 2,4,DNP inhibited uptake, but did not cause efflux. Transport of selenite was inhibited by sulphite and nitrite, but not by nitrate, phosphate, sulphate of selenate. ⁷⁵[Se]-Selenite was incorporated into selenocystine, selenoethionine, selenohomocysteine, and selenomethionine and was also reduced to red elemental selenium.     

Characterization of uptake and release processes ford- andl-aspartate in primary cultures of astrocytes and cerebellar granule cells

The uptake ofl-andd-aspartate was studied in astrocytes cultured from prefrontal cortex and in granule cells cultured from cerebellum. A high affinity uptake system forl- andd-aspartate was found in both cell types, and the two stereoisomers exhibited essentially the sameK _m - andV _max -values in bouth astrocytes (l-aspartate:K _m 77 μM;V _max 11.8 nmol×min^?1×mg^?1;d-aspartate:K _m 83 μM;V _max 14.0 nmol×min^?1×mg^?1) and granule cells (l-aspartate:K _m 32 μM;V _max 2.8 nmol ×min^?1×mg^?1;d-aspartate:K _m 26 μM;V _max 3.0 nmol×min^?1×mg^?1). To investigate whetherl-glutamate,l-aspartate andd-aspartate use the same uptake system a detailed kenetic analysis was performed. The uptake kinetics of each one of the three amino acids was studied in the presence of the two other amino acids, and no essential differences between the uptake characteristics of the amino acids were found. In addition to the uptake studies the release ofD-aspartate from cerebellar granule cells was investigated and compared withl-glutamate release. A Ca²⁺-dependent, K⁺-induced release was found for both amino acids.     

Competition between sulfate-reducing and methanogenic bacteria for H2 under resting and growing conditions

The basis for the outcome of competition between sulfidogens and methanogens for H₂ was examined by comparing the kinetic parameters of representatives of each group separately and in co-culture. Michaelis-Menten parameters (V _max and K _m) for four methanogens and five sulfate-reducing bacteria were determined from H₂-depletion data. Further, Monod growth parameters (_max, K _s, Y _H2) for Desulfovibrio sp. G11 and Methanospirillum hungatei JF-1 were similarly estimated. H₂ K _m values for the methanogenic bacteria ranged from 2.5 M (Methanospirillum PM1) to 13 M for Methanosarcina barkeri MS; Methanospirillum hungatei JF-1 and Methanobacterium PM2 had intermediate H₂ K _m estimates of 5 M. Average H₂ K _m estimates for the five sulfidogens was 1.2 M. No consistent difference among the V _max estimates for the above sulfidogens (mean=100 nmol H₂ min^-1 mg^-1 protein) and methanogens (mean=110 nmol H₂ min^-1 mg^-1 protein) was found. A two-term Michaelis-Menten equation accurately predicted the apparent H₂ K _m values and the fate of H₂ by resting co-cultures of sulfate-reducers and methanogens. Half-saturation coefficients (K _s) for H₂-limited growth of Desulfovibrio sp. G11 (2–4 M) and Methanospirillum JF-1 (6–7 M) were comparable to H₂ K _m estimates obtained for these organisms. Maximum specific growth rates for Desulfovibrio sp. G11 (0.05 h^-1) were similar to those of Methanospirillum JF-1 (0.05–0.06 h^-1); whereas G11 had an average yield coefficient 4 x that of JF-1. Calculated _max and V _max/K _m values for the methanogens and sulfidogens studied predict that the latter bacterial group will process more H₂ whether these organisms are in a growing or resting state, when the H₂ concentration is in the first-order region.     

Uptake and metabolism of GABA in astrocytes cultured from dissociated mouse brain hemispheres

Uptake kinetics and contents of GABA in cultured, normal (i.e. nontransformed) glia cells obtained from the brain hemispheres of newborn mice were measured together with the activity of the GABA transaminase. During three weeks of culturing the activity of the transaminase rose from a low neonatal value toward the level in the adult brain. The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis-Menten kinetics. Both theK _m (40 M) and theV _max (0.350 nmol×min^–1×mg^–1 cell protein) were reasonably comparable to the corresponding values in brain slices, and theV _max was much higher than that reported for other glial preparations. The GABA content was low (<5 nmol/mg cell protein), which is in agreement with the high activity of the GABA transaminase.     

Ammonium uptake by cowpea Rhizobium strain MNF 2030 and Rhizobium trifolii MNF 1001

Free-living Rhizobium trifolii MNF 1001 and cowpea Rhizobium MNF 2030 grown in chemostat culture under nitrogen limitation had high activities of an ammonium permease. In phosphate-limited, nitrogen-excess conditions, strains MNF 1001 and MNF 2030 retained 20% and 50%, respectively, of the ammonium uptake activity found in nitrogen-limited cells. Uptake in both strains was sensitive to azide, cyanide, carbonyl cyanide m-chlorophenyl hydrazone and 2,4-dinitrophenol. A gradient of ammonium concentration greater than 150-fold developed across the membrane within 20 min in cells of strain MNF 1001 grown under ammonia limitation. The pH optimum for ammonium uptake by N-limited cells of both MNF 1001 and MNF 2030 was around pH 7. The apparent K _m values for the ammonium permease in strains MNF 2030 and MNF 1001 were 3.9±1.6 M and 2.0±1.6 M respectively, and the V _max was 47±2.6 nmol min^-1 (mg protein)^-1 for MNF 2030 and 101±5.1 nmol min^-1 (mg protein)^-1 for MNF 1001. Isolated snake bean bacteroids of strain MNF 2030 capable of transporting succinate and l-glutamate had no detectable ammonium uptake activity. It therefore appears that the ammonium permeases in cells of these two strains are not as tightly regulated as in R. leguminosarum MNF 3841.Abbreviations CCCP Carbonyl cyanide m-chlorophenyl hydrzone - HEPES N-Hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Multiple Effects of Lysophosphatidylcholine on the Activity of the Plasma Membrane H+-ATPase of Radish Seedlings*

We analyzed the effect of lysophosphatidylcholine (lysoPC) on the activity of the plasma membrane (PM) H⁺-AT-Pase measured at pH 6.3 or 7.5 in inside-out PM vesicles isolated from germinating radish seeds. LysoPC stimulated PM H⁺-ATPase at both pHs, but the dependence of the effect on lysoPC concentration was different: at pH 6.3 maximal stimulation was observed with 40 to 200 μg ml^?1 lysoPC, while at pH 7.5 a sharp peak of activation was observed at about 50 μg ml^?1 lysoPC, higher concentrations becoming dramatically inhibitory; this inhibitory effect was considerably reduced in the presence of 10% (v/v) glycerol. In trypsin-treared PM lysoPC stimulated the H⁺-ATPase activity assayed at pH 6.3, but only marginally that assayed at pH 7.5. LysoPC increased both V_max (from 190 to 280nmol min^?1 mg^?1 prot) and apparent K_M (from 0.15 to 0.3 mM) of the H⁺-ATPase at pH 6.3, while it increased V_max (from 120 to 230 nmol min^?1 mg^?1 prot) and decreased apparent K_m (from 0.8 to 0.4 mM) at pH 7.5. Low concentrations of Nacetylimidazole (10 to 50 mM), which modifies tyrosine residues, abolished the stimulation by lysoPC of the PM H⁺-ATPase activity at pH 7.5, but not that observed at pH 6.3. These results indicate that lysoPC influences the PM H⁺-ATPase through different mechanisms, and that its effect can only partly be ascribed to its ability to hamper the inhibitory interaction of the regulatory C-terminal domain with the catalytic site. N-acety-limidazole did not affect the stimulation of PM H⁺-ATPase by controlled trypsin treatment or by fusicoccin, indicating that the requirement for the tyrosine residue(s) modified by low Nacetylimidazole concentrations is specific for lysoPC-induced displacement of the C-terminal domain.     

Acetate oxidation to CO2 via a citric acid cycle involving an ATP-citrate lyase: a mechanism for the synthesis of ATP via substrate level phosphorylation in Desulfobacter postgatei growing on acetate and sulfate

Desulfobacter postgatei is an acetate-oxidizing, sulfate-reducing bacterium that metabolizes acetate via the citric acid cycle. The organism has been reported to contain a si-citrate synthase (EC 4.1.3.7) which is activated by AMP and inorganic phosphate. It is show now, that the enzyme mediating citrate formation is an ATP-citrate lyase (EC 4.1.3.8) rather than a citrate synthase. Cell extracts (160,000xg supernatant) catalyzed the conversion of oxaloacetate (apparent K _m=0.2 mM), acetyl-CoA (app. K _m=0.1 mM), ADP (app. K _m=0.06 mM) and phosphate (app. K _m=0.7 mM) to citrate, CoA and ATP with a specific activity of 0.3 mol·min^-1·mg^-1 protein. Per mol citrate formed 1 mol of ATP was generated. Cleavage of citrate (app. K _m=0.05 mM; V _max=1.2 mol · min^-1 · mg^-1 protein) was dependent on ATP (app. K _m=0.4 mM) and CoA (app. K _m=0.05 mM) and yielded oxaloacetate, acetyl-CoA, ADP, and phosphate as products in a stoichiometry of citrate:CoA:oxaloacetate:ADP=1:1:1:1. The use of an ATP-citrate lyase in the citric acid cycle enables D. postgatei to couple the oxidation of acetate to 2 CO₂ with the net synthesis of ATP via substrate level phosphorylation.     

Direct evidence for the presence of a rotenone-resistant NADH dehydrogenase on the inner surface of the inner membrane of plant mitochondria

Submitochondrial particles (SMP) were produced from Jerusalem artichoke (Helianthus tuberosus L.) mitochondria by sonication and differential centrifugation. The SMP were about 50% inside-out as measured by the access of reduced cytochrome c to cytochrome c oxidase. Uncoupled NADH oxidation (1 mM NADH) by the SMP was 120 nmol O₂ min^?1mg^?1, which was reduced to 98 nmol O₂ min^?1 (mg mitochondrial protein)^?1 in the presence of EGTA. In contrast, the oxidation of NADH by intact mitochondria was completely inhibited by EGTA (from 182 to 14 nmol O₂ min^?1mg^?1). The EGTA-resistant NADH oxidation by the SMP is ascribed to the NADH dehydrogenase(s) on the inside of the inner membrane and exposed to the medium in the inside-out SMP. In the presence of EGTA it could be shown that two NADH dehydrogenase activities were present in the SMP. One had an apparent K_m of 7 μM for NADH, a V_max of 80 nmol NADH min^?1mg^?1, and was rotenone-sensitive. This dehydrogenase is equivalent to the mammalian Complex I NADH dehydrogenase. The other dehydrogenase, which was rotenone-resistant, had a K_m of 80 μM and a V_max of 131 nmol NADH min^?1mg^?1; it is probably responsible for the rotenone-resistant oxidation of organic acids often observed in plant mitochondria. The redox poise of the pyridine nucleotides had only a small effect on the relative rates of the two internal dehydrogenases. Electron flow through these dehydrogenases appears, therefore, to be regulated mainly by the concentration of NADH in the matrix of the mitochondria.     

Multiphasic uptake of potassium by barley roots of low and high potassium content: Separate sites for uptake and transitions

In the range 10^?6M - 5 × 10^?2M uptake of K⁺ in excised roots of barley (Hordeum vulgare L. cv. Herta) with low and high K content could in both cases be represented by an isotherm with four phases. Uptake, especially in the range of the lower phases, was reduced in high K roots through decreases in V_max and increases in K_m. Similar data for other plants are also shown to be consistent with multiphasic kinetics. The concentrations at which transitions occurred were not affected by the K status, indicating the existence of separate uptake and transition sites. Uptake was markedly reduced in the presence of 10^?5M 2,4-dinitrophenol, especially at low K⁺ concentrations, but the isotherms remained multiphasic. This contraindicates major contributions from a non-carrier-mediated, passive flux. A tentative hypothesis for multiphasic ion uptake envisions a structure which changes conformation as a result of all-or-none changes in a separate transition site. The structure is “tight” at low external ion concentrations (low V_max. low K_m. active uptake, allosteric regulation) and “loose” at high concentrations (high V_max- high K_m- facilitated diffusion, no regulation).     

Characteristics of 3-O-methylfluorescein phosphate hydrolysis by the (Na+ + K+)-ATPase

With 3-O-methylfluorescein phosphate (3-OMFP) as substrate for the phosphatase reaction catalyzed by the (Na⁺ + K⁺)-ATPase, a number of properties of that reaction differ from those with the common substratep-nitrophenyl phosphate (NPP): theK _m is 2 orders of magnitude less and the V_max is two times greater, and dimethyl sulfoxide (Me₂SO) inhibits rather than stimulates. In addition, reducing the incubation pH decreases both theK _m and V_max for K⁺-activated 3-OMFP hydrolysis as well as theK _0.5 for K⁺ activation. However, reducing the incubation pH increases inhibition by P_i and the V_max for 3-OMFP hydrolysis in the absence of K⁺. When choline chloride is varied reciprocally with NaCl to maintain the ionic strength constant, NaCl inhibits K⁺-activated 3-OMFP hydrolysis modestly with 10 mM KCl, but stimulates (in the range 5–30 mM NaCl) with suboptimal (0.35 mM) KCl. In the absence of K⁺, however, NaCl stimulates increasingly over the range 5–100 mM when the ionic strength is held constant. These observations are interpreted in terms of (a) differential effects of the ligands on enzyme conformations; (b) alternative reaction pathways in the absence of Na⁺, with a faster, phosphorylating pathway more readily available to 3-OMFP than to NPP; and (c) a (Na⁺ + K⁺)-phosphatase pathway, most apparent at suboptimal K⁺ concentrations, that is also more readily available to 3-OMFP.Abbreviations Et₃N triethyl amine - FITC fluorescein isothiocyanate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - MES 2-(N-morpholino)ethanesulfonate - Me₂SO dimethyl sulfoxide - NPP p-nitrophenyl phosphate - 3-OMFP 3-O-methylfluorescein phosphate - TNP-ATP 2, (or 3)-O-(2,4,6-trinitrophenyl)-ATP     

毛白杨细胞质抗坏血酸过氧化物酶基因的克隆及表达分析

利用同源克隆技术得到1个毛白杨细胞质抗坏血酸过氧化物酶基因,命名为PcAPX。该基因编码249个氨基酸残基,预测分子量为33.01kD。采用原核表达技术在大肠杆菌中表达并纯化该蛋白并进行酶活性分析,结果表明:重组PcAPX蛋白对抗坏血酸(AsA)和过氧化氢(H2O2)有很高的活性,其对抗坏血酸的米氏常数(Km)和最大反应速度(Vmax)分别为(0.71±0.03)mmol·L-1和(0.41±0.02)mmol·L-1·min-1·mg-1;对过氧化氢的Km和Vmax分别为(0.60±0.21)mmol·L-1和(0.35±0.12)mmol·L-1·min-1·mg-1,表明PcAPX对AsA和H2O2拥有较高的催化底物的能力和催化效率。利用实时定量RT-PCR分析毛白杨PcAPX基因的表达模式,结果表明其在老叶中表达量高于新叶、韧皮部、形成层和根部。该研究结果将进一步促进毛白杨APX基因家族成员参与植物生长调控的研究。     

Transport and catabolism of proline betaine in salt-stressed Rhizobium meliloti

Exogenous proline betaine (N,N-dimethylproline or stachydrine) highly stimulated the growth rate of Rhizobium meliloti, in media of inhibitory concentration of NaCl whereas proline was ineffective. High levels of proline betaine uptake occurred in cells grown in media of elevated osmotic strength; on the contrary, only low activity was found in cells grown in minimal medium. The apparent K _m was 10 M with a maximal transport rate of 25 nmol min^-1 mg^-1 of protein in 0.3 M NaCl-grown cells. The concentrative transport was totally abolished by KCN (2 mM), 2,4-dinitrophenol (2 mM), and carbonyl cyanide-m-chlorophenyl hydrazone (CCCP 10 M) but was insensitive to arsenate (5 mM). Glycine betaine was a very potent inhibitor of proline betaine uptake while proline was not. Proline betaine transport was not reduced in osmotically shocked cells and no proline betaine binding activity was detected in the crude periplasmic shock fluid. In the absence of salt stress, Rhizobium meliloti actively catabolized proline betaine but this catabolism was blocked by increasing the osmotic strength of the medium. The osmolarity in the growth medium regulates the use of proline betaine either as a carbon and nitrogen source or as an osmoprotectant.Abbreviations LAS lactate-aspartate-salts - MSY mannitol-salts-yeast - CCCP carbonyl cyanide-m-chlorophenyl hydrazone - DCCD dicyclohexylcarbodiimide - KCN potassium cyanide - Hepes 4-(2-hydroxyethyl)-1-piperzine-ethanesulphonic acid     

A novel isolate of Desulfovibrio sp. with enhanced ability to reduce Cr(VI)

Kinetic analysis of the reduction of Cr(VI) by resting cell suspensions of Desulfovibrio vulgaris ATCC 29579 and a new isolate, Desulfovibrio sp. (`Oz7') was studied using lactate as the electron donor at 30 °C. The apparent K <sub>m</sub> (K <sub>m app</sub>) and V <sub>max</sub> with respect to Cr(VI) reduction was compared for both strains. Desulfovibio sp. `Oz7' had a K _{m app} of 90 M (threefold lower than that of D. vulgaris ATCC 29579) and a V _max of 120 nmol h^–1 mg^–1 biomass dry wt (approx. 30% lower than for the reference strain). The potential of the new isolate for bioremediation of Cr(VI) wastewaters is discussed.     

Isolation and characterization of glutathione-S-transferase isozyme Q3 from the northern quahog,Mercinaria mercinaria

Three glutathione-S-transferase (GST) isozymes (Q1, Q2, and Q3) from the northern quahog (Mercinaria mercinaria) were purified and separated with a combination of affinity and ion exchange chromatography. SDS-PAGE analysis of the separated quahog GSTs indicated there are four distinct subunits of the enzyme with molecular masses ranging between 23 and 27 kDa. The electrophoretic analysis in combination with GST information from literature indicates that among the quahog GST isozymes, there is a single homodimer and two heterodimers. Enzymatic kinetic analysis of the homodimeric quahog GST (Q3) using 1-chloro-2,4-dinitrobenzene and glutathione as reactants resulted in V _max and K _m values of 33.2 mol min^–1 mg^–1 and 0.40 mM, respectively. A pH profile analysis of the Q3 GST indicates that the optimum catalytic pH is 7.6. The Q3 isozyme composes about 28% of the ion exchange purified GSTs but accounts for only 9% of the total GST enzymatic activity (25 mol min^–1 mg^–1). An analysis investigating the dependence of the Q3 GST activity on temperature resulted in a retention of enzymatic activity (50–30% at temperature extremes from –13°C to 100°C), suggesting a unconventional role for the Q3 GST in quahog metabolism.     

Ammonium-stimulated,sodium-dependent uptake of glutamine in Bacillus pasteurii

The uptake of glutamine was studied in Bacillus pasteurii DSM 33. Only one uptake system was detected in the concentration range studied (between 1 and 100 M glutamine) which exhibited Michaelis-Menten saturation kinetics, with an apparent K _t of 10.7 (±3.5) M glutamine. The uptake was sodium-dependent (apparent K _t=0.2 mM Na⁺); none of several monovalent cations tested was able to replace sodium in the uptake reaction. Ionophores interfering with proton, sodium or potassium gradients across membranes strongly inhibited uptake of glutamine. Low uptake rates correlating with low potassium content and an acidic cytoplasm were measured in cells grown at high ammonium¹ concentrations. Ammonium and other permeant amines as well as potassium stimulated the uptake reaction in these cells, leading to an increase of up to 100-fold in V _max without affecting the affinity of the uptake system. In cells grown at low concentrations of ammonium, an alkaline cytoplasm and both high glutamine uptake activities and potassium content were measured; the uptake reaction was not further stimulated by permeant amines or potassium in such cells. Growth of the strain was inhibited by Tris at high concentrations; this inhibition was relieved by the addition of increasing amounts of ammonium.Abbreviations CCCP carbonylcyanide-m-chlorphenylhydrazone - DCCD dicyclohexylcarbodiimide This work is dedicated to Prof. Dr. H. Kaltwasser on the occasion of his 60th birthday