Magnesium ions alleviate the negative effect of manganese on Glomus claroideum BEG23

The phytotoxicity of excessive levels of manganese (Mn), an essential micronutrient, can be alleviated significantly by a high supply of magnesium (Mg) ions to plants. A similar interaction of these two elements in the development of arbuscular mycorrhizal (AM) fungi has been verified in two experimental systems. In in vitro experiments, an outgrowth of hyphae from excised, surface-disinfected root segments colonised with Glomus claroideum BEG23 was measured after 5 days incubation in liquid media. When only Mn ions were present in the media at higher concentrations (>0.05 mM), the growth of hyphae from root segments was reduced significantly. Addition of magnesium sulphate to the incubation solution reduced the inhibitory effects of Mn on hyphal growth. Alleviation of Mn toxicity by Mg ions observed in in vitro experiments was verified also for the symbiotic association between G. claroideum and maize as a host plant in a hydroponics sand culture experiment.     

Ribonucleotide reductase of Brevibacterium ammoniagenes is a manganese enzyme

Ribonucleotide reduction and not DNA replication is the site for the specific manganese requirement of DNA synthesis and cell growth in the coryneform bacterium Brevibacterium ammoniagenes. To characterize the metal effect we have isolated and purified ribonucleoside-diphosphate reductase from overproducing bacteria that were first deprived of and then reactivated by manganese ions. Purification on columns of Sephacryl S400, DEAE-cellulose and hydroxyapatite provided an apparently homogeneous enzyme consisting of two protein subunits. These were characterized by affinity chromatography on 2',5'-ADP-Sepharose as nucleotide-binding protein B1 (Mr = 80,000) and catalytic protein B2 (Mr = 100,000, composed of two Mr = 50,000 polypeptides), which were both necessary for activity. In vitro the purified enzyme does not require added metal ions except for an unspecific, twofold activity increase observed in the presence of Mg2+ and other divalent cations. Enzyme activity is inhibited by hydroxyurea (I50 = 2.5 mM). The electronic spectrum with maxima around 455 nm and 485 nm closely resembles that of manganese(III)-containing pseudocatalase and of oxo-bridged binuclear Mn(III) model complexes. Denaturation of the enzyme in trichloroacetic acid liberated an equimolar amount of Mn(II) which was detected by EPR spectroscopy. It was not possible to remove and reintroduce metal ions without loss of enzyme activity. Manganese-deficient cell cultures were also grown in the presence of 54MnCl2. Ribonucleotide reductase activity and radioactivity cochromatographed in several systems. Non-denaturing polyacrylamide gel electrophoresis showed that protein subunit B2 was specifically 54Mn-labeled. All these properties suggest that the ribonucleotide reductase of B. ammoniagenes is a manganese-containing analog of the non-heme-iron-containing reductases of Escherichia coli and eukaryotes.     

Effects of polyamines and methylglyoxal bis(guanylhydrazone) on Escherichia coli ribonucleic acid polymerase and the template activity of hepatic cell nuclei in vitro

Escherichia coli RNA polymerase was assayed with 4 mM Mg2+ and 1 mM Mn2+ using native DNA, heat-denatured DNA, histone-nucleate and isolated rat liver nuclei as the template source. With purified DNA and either or both divalent metal ions, 0.1--5 mM amine stimulated enzyme activity. Spermidine resulted in the greatest stimulation (1.7-fold at 5 mM); whereas, spermine or methylglyoxal bis(guanylhydrazone) first stimulated, then above 3 mM inhibited, the reaction. The addition of unfractionated histone to purified DNA inhibited the reaction by 90%. The subsequent addition of amines resulted in a slight stimulation in incorporation (1.5-fold) in the range of 1--3 mM amine. Alternatively, when enzyme was combined with DNA before histone, only a 20% inhibition was observed and this could be completely prevented by 3 mM spermidine. The addition of amines to isolated nuclei resulted in marked alterations in ultrastructure and Mg2+ content; however, relatively small effects on RNA polymerase activity were observed. With the E. coli enzyme, 0.1--1.0 mM amine stimulated RNA synthesis (1.5-fold) whereas, none of the amines stimulated endogeneous activity in the absence or presence of 300 mM (NH4)2SO4.     

Kinetic effects of ATP, divalent metal ions and pH on chicken liver mevalonate 5-diphosphate decarboxylase

The activity of chicken liver mevalonate 5-diphosphate decarboxylase was measured over a wide range of Mg2+ and ATP concentrations. It was found that free ATP activated the enzyme, whereas free Mg2+ had no effect on the enzyme activity. Computed analyses of free species concentrations and pH studies indicated that MgATP2- is the true substrate. The relative efficiencies of Mg2+, Mn2+, Cd2+, and Zn2+ as activating metal ions were evaluated in terms of V/Km for the corresponding (metal-ATP)2- complexes, and the relative ratios were: Mn2+ 100, Cd2+ 37, Mg2+ 14, Zn2+ 1.7. Inhibitory effects were demonstrated for all free divalent cations tested, except for Mg2+, and were in the order Zn2+ greater than Cd2+ greater than Mn2+.     

Temperature and the regulation of enzyme activity in poikilotherms. Properties of rainbow-trout fructose diphosphatase

1. The properties of fructose diphosphatase from the liver of rainbow trout (Salmo gairdnerii) were examined over the physiological temperature range of the organism. 2. Saturation curves for substrate (fructose 1,6-diphosphate) and a cofactor (Mg(2+)) are sigmoidal, and Hill plots of the results suggest a minimum of two interacting fructose 1,6-diphosphate sites and two interacting Mg(2+) sites per molecule of enzyme. 3. Mn(2+)-saturation curves are hyperbolic, and the K(a) for Mn(2+), which inhibits the enzyme at high concentrations, is 50-100-fold lower than the K(a) for Mg(2+). 4. Fructose diphosphatase is inhibited by low concentrations of AMP; this inhibition appears to be decreased and reversed by increasing the concentrations of Mg(2+) and Mn(2+). Higher concentrations of AMP are required to inhibit the trout fructose diphosphatase in the presence of Mn(2+). 5. The affinities of fructose diphosphatase for fructose diphosphate and Mn(2+) appear to be temperature-independent, whereas the affinities for Mg(2+) and AMP are highly temperature-dependent. 6. The pH optimum of the enzyme depends on the concentrations of Mg(2+) and Mn(2+). In addition, pH determines the K(a) for Mg(2+); at high pH, K(a) for Mg(2+) is lowered. 7. The enzyme is inhibited by Ca(2+) and Zn(2+), and the inhibition is competitive with respect to both cations. 8. The possible roles of these ions and AMP in the modulation of fructose diphosphatase and gluconeogenic activity are discussed in relation to temperature adaptation.     

Binding of manganese ions to the Na+/K+-ATPase during phosphorylation by ATP

The aim of the present work was to study the Mg2+-Na+/K+-ATPase interaction that was proposed to lead to the formation of a stable Mg-enzyme complex during phosphorylation from ATP. Instead of Mg we used Mn, which can replace Mg as essential activator of Na+/K+-ATPase activity. The amounts of steady-state Mn bound to the enzyme were estimated at 0 degree C on the basis of the 54Mn remaining in the effluent after passing the reaction mixture through a cation exchange resin column. As a function of the MnCl2 concentration, the amount of Mn retained by the enzyme in the absence and presence of ATP showed a saturable and a linear component; the slope of the linear component was the same in both instances (0.016 nmol/mg per microM). The ATP-dependent Mn binding could be adjusted to a hyperbolic function with a Km of 0.76 microM. The ratio [ATP-dependent E-Mn]/[E-P] measured at 5 microM MnCl2 and 5 microM ATP was not different from 1.0, both in native (Mn-E2-P) as well as in a chymotrypsin treated enzyme (Mn-E1-P). When the Mn.E-P complex was allowed to react with KCl (E2-P form) or ADP (E1-P form), the enzyme was dephosphorylated and simultaneously lost the strongly bound Mn in such a way that the ratio [ATP-dependent E-Mn]/[E-P] remained 1:1. These results show the existence of strongly bound Mn ions to Na+/K+-ATPase during phosphorylation by ATP. That binding is (i) of high affinity for Mn, (ii) probably on a single site, and (iii) with a stoichiometry Mn-Pi of 1:1.     

Purification and characterization of chromatin-bound DNA-dependent RNA polymerase I from parsley (Petroselinum crispum). Influence of nucleoside triphosphates.

The isolation and purification of DNA-dependent RNA polymerase I (EC 2.7.7.6) from parsley (Petroselinum crispum) callus cells grown in suspension culture is described. The enzyme was solubilized from isolated chromatin. Purification was achieved by using DEAE- and phospho-cellulose in batches, followed by column chromatography on DEAE- and phospho-cellulose (two columns) and density-gradient centrifugation. The highly purified enzyme was stable over several months. The properties of purified parsley RNA polymerase I were investigated. Optimum concentration for Mn2+ was 1 mM, and for Mg2+ 4-6 mM, Mn2+ was slightly more stimulatory than Mg2+. The enzyme was most active at low ionic strengths [10-20 mM-(NH4)SO4]. The influence of various phosphates was tested: pyrophosphate inhibited RNA polymerase at low concentrations, whereas orthophosphate had no effect on the enzyme activity. ADP was slightly inhibitory, and AMP had no effect on the enzyme reaction. Nucleoside triphosphates and bivalent cations in equimolar concentrations in the range 4-11 mM did not influence the RNA synthesis in vitro. Free nucleoside triphosphates in excess of this 1:1 ratio inhibited the enzyme activity, unlike free bivalent cations, which stimulated RNA polymerase I.     

Physiologically relevant metal cofactor for methionine aminopeptidase-2 is manganese

The identity of the physiological metal cofactor for human methionine aminopeptidase-2 (MetAP2) has not been established. To examine this question, we first investigated the effect of eight divalent metal ions, including Ca(2+), Co(2+), Cu(2+), Fe(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+), on recombinant human methionine aminopeptidase apoenzymes in releasing N-terminal methionine from three peptide substrates: MAS, MGAQFSKT, and (3)H-MASK(biotin)G. The activity of MetAP2 on either MAS or MGAQFSKT was enhanced 15-25-fold by Co(2+) or Mn(2+) metal ions in a broad concentration range (1-1000 microM). In the presence of reduced glutathione to mimic the cellular environment, Co(2+) and Mn(2+) were also the best stimulators (approximately 30-fold) for MetAP2 enzyme activity. To determine which metal ion is physiologically relevant, we then tested inhibition of intracellular MetAP2 with synthetic inhibitors selective for MetAP2 with different metal cofactors. A-310840 below 10 microM did not inhibit the activity of MetAP2-Mn(2+) but was very potent against MetAP2 with other metal ions including Co(2+), Fe(2+), Ni(2+), and Zn(2+) in the in vitro enzyme assays. In contrast, A-311263 inhibited MetAP2 with Mn(2+), as well as Co(2+), Fe(2+), Ni(2+), and Zn(2+). In cell culture assays, A-310840 did not inhibit intracellular MetAP2 enzyme activity and did not inhibit cell proliferation despite its ability to permeate and accumulate in cytosol, while A-311263 inhibited both intracellular MetAP2 and proliferation in a similar concentration range, indicating cellular MetAP2 is functioning as a manganese enzyme but not as a cobalt, zinc, iron, or nickel enzyme. We conclude that MetAP2 is a manganese enzyme and that therapeutic MetAP2 inhibitors should inhibit MetAP2-Mn(2+).     

Effect of metal ions on the activity of the catalytic domain of calcineurin

Calcineurin (CN) is a heterodimer, composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). There are four functional domains present in CNA, which are catalytic domain (CNa), CNB-binding domain (BBH), CaM-binding domain (CBH) and autoinhibitory domain (AI). It has been shown previously that the in vitro activity of calcineurin is relied primarily on the binding of metal ions. Mn2+ and Ni2+ are the most crucial cation-activators for this enzyme. In order to determine which domain(s) in CN is functionally regulated by metal ions, the rat CNA alpha subunit and its catalytic domain (CNa) were cloned and expressed in E. coli. The effects of Mn2+, Ni2+ and Mg2+ on the catalytic activity of these purified proteins were examined. Our results demonstrate that all the metal ions tested in this study activated either CNA or CNa. However, the activation degree of CNa by the metal ions was much higher than that of CNA. In term of different metal ions, the activating extents to CNA and CNa were different. To CNA, the activating order from high to low was Mg2+ > > Ni2+ > Mn2+, but Mn2+ > Ni2+ > > Mg2+ to CNa. No effect of CaM/Ca2+ and CNB/Ca2+ on the activity of CNa was observed in our experiments. Moreover, a weak interaction (or untight coordination binding) between metal ions and the enzyme molecule was also identified. These results suggest that the activation of these enzymes by the exogenous metal ions might be via both regulating fragment of CNA (including BBH, CBH and AI) and catalytic domain (CNa), and mainly via regulating fragment to CNA and mainly via catalytic domain to CNa. The activating extents of metal ions via catalytic domain were higher than that via regulating fragment. The results obtained in this study should be very useful for understanding the molecular mechanism underlying the interaction between calcineurin and metal ions, especially Mn2+, Ni2+ and Mg2+.     

Differences between manganese and magnesium ions with regard to fatty acid biosynthesis, acetyl-coenzyme A carboxylase activity and malonyl-coenzyme A decarboxylation

Fatty acid-biosynthetic activity in rat liver cytosol fractions is much greater when the bivalent cation in the assay system is Mn(2+) than when it is Mg(2+). This difference between bivalent cations can be abolished if the cytosol fractions are preincubated with isocitrate and the bivalent cation for 30min before assay of fatty acid-biosynthetic activity. In a search for the biochemical basis of this phenomenon, the following differences between Mg(2+) and Mn(2+) were established: (1) Mn(2+) promotes acetyl-CoA carboxylase activity of the protomeric form of the enzyme under conditions in which Mg(2+) does not; (2) Mn(2+)+ATP have little inhibitory effect on the polymerization of acetyl-CoA carboxylase whereas Mg(2+)+ATP are markedly inhibitory; (3) under conditions in which utilization of malonyl-CoA in condensation reactions is prevented, the steady-state concentration of malonyl-CoA formed by a cytosol fraction is much greater with Mn(2+) than with Mg(2+). The role that each of these specific differences between Mn(2+) and Mg(2+) might play in causing liver cytosol preparations to have greater fatty acid-biosynthetic activity in the presence of Mn(2+) is discussed.     

Regulation and properties of glutamine synthetase purified from Bacillus cereus

The glutamine synthetase from Bacillus cereus IFO 3131 was purified to homogeneity. The enzyme is a dodecamer with a molecular weight of approximately 600,000, and its subunit molecular weight is 50,000. Both Mg2+ and Mn2+ activated the enzyme as to the biosynthesis of L-glutamine, but, unlike in the case of the E. coli enzyme, the Mg2+-dependent activity was stimulated by the addition of Mn2+. The highest activity was obtained when 20 mM Mg2+ and 0.5 mM Mn2+ were added to the assay mixture. For each set of optimal assay conditions, the apparent Km values for glutamate, ammonia and a divalent cation X ATP complex were 1.03, 0.34, and 0.40 mM (Mn2+: ATP = 1: 1); 14.0, 0.47, and 0.91 mM (Mg2+: ATP = 4: 1); and 9.09, 0.45, and 0.77 mM (Mg2+: Mn2+: ATP = 4: 0.2: 1), respectively. At each optimum pH, the Vmax values for these reactions were 6.1 (Mn2+-dependent), 7.4 (Mg2+-dependent), and 12.9 (Mg2+ plus Mn2+-dependent) mumoles per min per mg protein, respectively. Mg2+-dependent glutamine synthetase activity was inhibited by the addition of AMP or glutamine; however, this inhibitory effect was suppressed in the case of the Mg2+ plus Mn2+-dependent reaction. These results suggest that the activity of the B. cereus glutamine synthetase is regulated by both the intracellular concentration and the ratio of Mn2+/Mg2+ in vivo. Also in the present investigation, a potent glutamine synthetase inhibitor(s) was detected in crude extracts from B. cereus.     

Properties of cholera toxin- and NaF-stimulated adenylate cyclase from mouse thymocytes.

Kinetic parameters of mouse thymocyte adenylate cyclase activity were determined. NaF and cholera toxin stimulated adenylate cyclase. Stimulation by either agent did not change the pH or Mg2+ optima relative to control (unstimulated cyclase). The Km value for ATP of adenylate cyclase stimulated by NaF was significantly reduced from control. By contrast, cholera toxin treatment did not change the Km relative to control. Adenylate cyclase, when stimulated by NaF, had an optimum for Mn2+ alone, or Mn2+ in combination with Mg2+, at least twice that of control. In contrast, cyclase activity prepared from cells treated with cholera toxin remained unchanged with regard to these divalent cations when compared to control. Addition of NaF to adenylate cyclase prepared from cells treated with cholera toxin resulted in a significant reduction (30%) in activity suggesting that both NaF and cholera toxin were acting on the same cyclase. NaF inhibition of cholera toxin-stimulated activity was shown to be a direct interaction of fluoride on the stimulated cyclase enzyme. This inhibition appeared to be immediate and independent on pH, Mg2+ or ATP concentrations. Although NaF inhibition was lost when Mn2+ was present in the reaction mixture, the activity expressed by addition of NaF to cyclase prepared from cholera toxin-treated cells was much less than by addition of NaF to control. As observed with cholera toxin stimulation alone, activity expressed by the inhibited enzyme (cholera toxin treated + NaF) exhibited a Km for ATP and an optimum for Mn2+ alone or in combination with Mg2+ similar to control.     

Effect of inhibitors and cations on the proteolytic activity of Bacillus mesentericus

The effect of some inhibitors and bivalent metal cations (Mn2+, Ca2+, Fe2+, Zn2+, Mg2+, Co2+ and Cu2+) on the proteolytic activity of two Bacillus mesentericus strains (strain 8 and strain 64 M-variant) was comparatively studied. The both enzymes were shown to be serine proteinases, but the proteinase of strain 64 was also a metal-dependent enzyme. Metal ions exerted no essential effect on the proteinase of strain 8. Ca2+ and Mg2+ ions stimulated the proteinase activity of strain 64 whereas Fe2+ and Zn2+ ions inhibited it in the case of three substrates. Therefore, the two proteinases are different.     

Studies on the pathogenesis of liver necrosis by α-amanitin. Effect of α-amanitin on ribonucleic acid synthesis and on ribonucleic acid polymerase in mouse liver nuclei

1. Injection of alpha-amanitin to mice causes a decreased incorporation of [6-(14)C]-orotic acid into liver RNA in vivo. 2. The activity of RNA polymerase activated by Mn(2+) and ammonium sulphate is greatly impaired in liver nuclei isolated from mice poisoned with alpha-amanitin, and is inhibited by the addition of the same toxin in vitro. 3. The activity of the Mg(2+)-activated RNA polymerase is only slightly affected by alpha-amanitin either administered to mice or added in vitro.     

Effect of micromolar concentrations of manganese ions on calcium-ion cycling in rat liver mitochondria.

The effects of micromolar concentrations of Mn2+ on the rat liver mitochondrial Ca2+ cycle were investigated. It was found that the addition of Mn2+ to mitochondria which were cycling 45Ca2+ led to a rapid dose dependent decrease in the concentration of extramitochondrial 45Ca2+ of about 1 nmol/mg of protein. The effect was complete within 30 s, was half maximal with 10 microM Mn2+ and was observed in the presence of 3 mM Mg2+ and 1 mM ATP. It occurred over a broad range of incubation temperatures, pH and mitochondrial Ca2+ loads. It was not observed when either Mg2+ or phosphate was absent from the incubation medium, or in the presence of Ruthenium Red. These findings indicate that micromolar concentrations of Mn2+ stimulate the uptake of Ca2+ by rat liver mitochondria, and provide evidence for an interaction between Mg2+ and Mn2+ in the control of mitochondrial Ca2+ cycling.     

Temperature and the regulation of enzyme activity in poikilotherms. Properties of lungfish fructose diphosphatase

1. The properties of fructose diphosphatase from liver of South American lungfish (Lepidosiren paradoxa) were examined. 2. Saturation curves for substrate (fructose diphosphate) and both cofactors (Mn(2+) and Mg(2+)) are sigmoidal and Hill plots of these results suggest about 2 interacting substrate and cofactor sites/molecule of enzyme. 3. Mn(2+) is an efficient positive modulator of the enzyme and K(a) for Mn(2+) is about 20-30-fold lower than the K(a) for Mg(2+). 4. Lungfish fructose diphosphatase is inhibited by low concentrations of AMP, and the affinity of the enzyme for AMP is insensitive to temperature. 5. The affinities of fructose diphosphatase for fructose diphosphate and Mn(2+) appear to be dependent on temperature, whereas affinity for Mg(2+) is temperature-independent. 6. The pH optimum of the enzyme depends on the presence of the particular cofactor. As pH increases, the K(a) values of both cations are lowered, maximum velocities are increased and the saturation curves for cofactor become hyperbolic. 7. The possible roles of these ions, pH and substrate in the modulation of fructose diphosphatase and gluconeogenic activity in the lungfish are discussed in relation to aestivation and temperature adaptation.     

Some aspects of the kinetics of rat liver pyruvate carboxylase

1. The kinetics of rat liver pyruvate carboxylase were examined and the effect of various agents as activators or inhibitors determined. 2. Essentially similar results were obtained in comparisons of kinetics determined by a radioactivity method involving extracts of acetone-dried powders from whole livers and with a spectrophotometric assay using partially purified enzyme from the mitochondrial fraction. Activity per g of liver from fed or starved rats assayed under optimum substrate and activator conditions was 3 or 6 mumol of oxaloacetate formed/min at 30 degrees C, respectively. 3. The enzyme exhibited cold-lability and lost activity on standing, even in 1.5m-sucrose. 4. The K(m) towards pyruvate was about 0.33mm and towards bicarbonate 4.2mm. K(m) towards MgATP(2-) was 0.14mm. Mg(2+) ions activated the enzyme, in addition to their role in MgATP(2-) formation. From calculations of likely concentrations of free Mg(2+) in the assay medium a K(a) towards Mg(2+) of about 0.25mm was deduced. Mn(2+) also activated the enzyme as well as Mg(2+), but at much lower concentrations. It appeared to be inhibitory when concentrations of free Mn(2+) as low as 0.1mm were present. 5. Excess of ATP is inhibitory, and this appears at least in part independent of the trapping of Mg(2+). 6. Both Co(2+) and Zn(2+) were inhibitory; 2mol of the latter appeared to be bound even in the presence of excess of Mg(2+) and the inhibition was time-dependent. 7. Ca(2+) inhibited by competition with Mg(2+) (K(i) about 0.38mm). The inhibition due to Ca(2+) was less pronounced when activation was with Mn(2+). Inhibition by Ca(2+) and ATP appeared to be additive. 8. Hill plots suggested that no interactions occurred between ATP-binding sites. Although similar plots for total Mg(2+) gave n=3.6, no conclusions could be drawn due to the chelation of the cation with other components of the assay. Similar difficulties arose in assessing the values for Ca(2+). 9. The enzyme was inactive in the absence of acetyl-CoA and showed a sigmoidal response in its presence. K(a) was about 0.1mm with possibly up to four binding sites. Malonyl-CoA was a competitive inhibitor, with K(i) 0.01mm. 10. There was no apparent inhibition by glucose, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-diphosphate, acetoacetate, beta-hydroxybutyrate, malate, aspartate, pyruvate, palmitoylcarnitine, octanoate, glutathione, butacaine, triethyltin or potassium chloride under the conditions used. Inhibition was found with citrate (possibly by chelation) and adenosine, and also by phosphoenolpyruvate, AMP, ADP and cyclic AMP, K(i) towards the last four being 0.55, 0.76, 0.25 and 1.4mm respectively.     

Purification and properties of UDP-GlcNAc:dolichyl-phosphate GlcNAc-1-phosphate transferase. Activation and inhibition of the enzyme

The GlcNAc-1-P transferase was solubilized from pig aorta microsomal fractions using 0.5% Nonidet P-40. The activity of the solubilized enzyme was stimulated by exogeneously added phospholipids in the order phosphatidylglycerol greater than phosphatidylinositol greater than phosphatidylserine. When the enzyme was stored in 20% glycerol containing 20 micrograms of phosphatidylglycerol/mg of protein, more than 80% of the activity remained after storage for 6 days at 0-4 degrees C. On the other hand, in the absence of the stabilizers, the enzyme lost most of its activity within 24 h. The transferase was purified about 68-fold using ammonium sulfate and DEAE-cellulose fractionation. The DEAE-cellulose chromatography separated a heat-stable factor from the enzyme, which when added back to the partially purified enzyme stimulated about 5-fold. With this partially purified enzyme, the Km for UDP-GlcNAc was found to be 1 X 10(-7) M, and that for dolichyl-P about 1 X 10(-6) M. The stimulatory factor increased the Vmax for both UDP-GlcNAc and dolichyl-P 5-10-fold, but the Km values remained the same. The pH optimum for the enzyme was between 7.4 and 7.6, and either Mn2+ (1 mM) or Mg2+ (10 mM) was required for optimum activity. The GlcNAc-1-P transferase was also stimulated by the addition of GDP-mannose (or other purine sugar nucleotides) or dolichyl-phosphoryl-mannose to the incubation mixtures. These two compounds acted in different ways on the enzyme since their stimulatory effects were additive. The effect of GDP-mannose was found to be due to protection of the substrate, UDP-GlcNAc, from degradation, but the effect of dolichyl-P-mannose remains to be established. In addition, the stimulations shown by phosphatidylglycerol, GDP-mannose, and factor, or phosphatidylglycerol, dolichyl-P-mannose, and factor, were all additive, indicating that they were acting at different sites on the enzyme. The transferase was quite sensitive to the action of sulfhydryl reagents such as N-ethylmaleimide or p-chloromercuribenzene sulfonate, and was rapidly inactivated in their presence. The enzyme could be protected to the extent of about 50% when all of the substrates (UDP-GlcNAc, dolichyl-P, Mn2+) were added before the addition of the sulfhydryl reagents.     

Degradation of benzo[a]pyrene by the litter-decomposing basidiomycete Stropharia coronilla: role of manganese peroxidase

The litter-decomposing basidiomycete Stropharia coronilla, which preferably colonizes grasslands, was found to be capable of metabolizing and mineralizing benzo[a]pyrene (BaP) in liquid culture. Manganese(II) ions (Mn(2+)) supplied at a concentration of 200 micro M stimulated considerably both the conversion and the mineralization of BaP; the fungus metabolized and mineralized about four and twelve times, respectively, more of the BaP in the presence of supplemental Mn(2+) than in the basal medium. This stimulating effect could be attributed to the ligninolytic enzyme manganese peroxidase (MnP), whose activity increased after the addition of Mn(2+). Crude and purified MnP from S. coronilla oxidized BaP efficiently in a cell-free reaction mixture (in vitro), a process which was enhanced by the surfactant Tween 80. Thus, 100 mg of BaP liter(-1) was converted in an in vitro reaction solution containing 1 U of MnP ml(-1) within 24 h. A clear indication was found that BaP-1,6-quinone was formed as a transient metabolite, which disappeared over the further course of the reaction. The treatment of a mixture of 16 different polycyclic aromatic hydrocarbons (PAHs) selected by the U.S. Environmental Protection Agency as model standards for PAH analysis (total concentration, 320 mg liter(-1)) with MnP resulted in concentration decreases of 10 to 100% for the individual compounds, and again the stimulating effect of Tween 80 was observed. Probably due to their lower ionization potentials, poorly bioavailable, high-molecular-mass PAHs such as BaP, benzo(g,h,i)perylene, and indeno(1,2,3-c,d)pyrene were converted to larger extents than low-molecular-mass ones (e.g., phenanthrene and fluoranthene).