Exogenous Mn2 Research Articles

Microcalorimetric studies on the non-competitive inhibition of bovine liver arginase

Two methods, e.g. initial rate method and thermokinetic reduced extent method were presented for studies on non-competitive inhibition. Arginase-catalyzed the hydrolysis of L-arginine toL-ornithine and urea and the inhibition of this reaction by sodium fluoride were studied in the absence and presence of exogenous of Mn2+at 37°C in 40 mM sodium barbiturate-hydrochloric acid buffer solution (pH 7.4). Both methods were successively used to determine the values of K1. The advances and disadvantages of each method were compared in this paper. Exogenous Mn2+ could result in more sensitivity of arginase to F-1. Since the inhibition of arginase by F-1 depends on the pH values of the reaction system and behave as a non-competitive inhibition, it probably due to its small volume and high electronic density allow it access to the activity site of the enzyme and replaces of µ-OH2 (or µ-OH) as the bridge ligand with Mn(II, II) cluster. However, further studies are necessary to determine the modes of interaction of F-1 with bovine liver arginase.

Photoinhibition and recovery of photosynthesis in leaves of Vitis berlandieri and Vitis rupestris

Photoinhibition of photosynthesis was studied in Vitis berlandieri and Vitis rupestris leaves under controlled conditions (irradiation of detached leaves to about 1900 micromol m(-2) s(-1)). The degree of photoinhibition was determined by means of the ratio of variable to maximum chlorophyll (Chl) fluorescence (Fv/Fm) and electron transport measurements. The potential efficiency of PS2, Fv/Fm declined, Fo increased significantly in leaves of V. berlandieri, while Fo decreased in V. rupestris. In isolated thylakoids, the rate of whole chain and PS2 activity markedly decreased in high light irradiated more in leaves of V. berlandieri than in leaves of V. rupestris. A smaller inhibition of PS1 activity was also observed in both leaves. In the subsequent dark incubation, fast recovery was observed in both leaves and reached maximum PS2 efficiencies similar to those observed in non-photoinhibited leaves. The artificial exogenous electron donors DPC, NH2OH and Mn2+ failed to restore the high light induced loss of PS2 activity in V. berlandieri leaves, while DPC and NH2OH significantly restored in V. rupestris leaves. It is concluded that high light inactivates on the donor side of PS2 and acceptor side of PS2 in V. rupestris and V. berlandieri leaves, respectively. Quantification of the PS2 reaction center protein D1 and 33 kDa protein of water splitting complex following high light exposure of leaves showed pronounced differences between V. berlandieri and V. rupestris leaves. The marked loss of PS2 activity in high light irradiated leaves was due to the marked loss of D1 protein and 33 kDa protein in V. berlandieri and V. rupestris leaves, respectively.

Isoniazid Activation Defects in Recombinant Mycobacterium tuberculosis Catalase-Peroxidase (KatG) Mutants Evident in InhA Inhibitor Production

Mycobacterium tuberculosis KatG catalyzes the activation of the antitubercular agent isoniazid to yield an inhibitor targeting enoyl reductase (InhA). However, no firm biochemical link between many KatG variants and isoniazid resistance has been established. In the present study, six distinct KatG variants identified in clinical Mycobacterium tuberculosis isolates resistant to isoniazid were generated by site-directed mutagenesis, and the recombinant mutant proteins (KatG(A110V), KatG(A139P), KatG(S315N), KatG(L619P), KatG(L634F), and KatG(D735A)) were purified and characterized with respect to their catalase-peroxidase activities (in terms of k(cat)/K(m)), rates of free-radical formation from isoniazid oxidation, and, moreover, abilities to activate isoniazid. The A110V amino acid replacement did not result in significant alteration of KatG activities except that the peroxidase activity was enhanced. The other mutations, however, resulted in modestly reduced catalase and peroxidase catalytic efficiencies and, for the four mutants tested, significantly lower activities to oxidize isoniazid. Compared to the wild-type enzyme, the ability of the KatG(L634F), KatG(A139P), and KatG(D735A) variants to activate isoniazid decreased by 36%, 76%, and 73%, respectively, whereas the KatG(S315N) and KatG(L619P) variants completely lost their abilities to convert isoniazid into the InhA inhibitor. In addition, the inclusion of exogenous Mn(2+) to the isoniazid activation reaction mix significantly improved the ability of wild-type and KatG mutants to produce the InhA inhibitor.

Activation of calcineurin by the trivalent metal terbium.

As a possible probe for metal activation of calcineurin, Tb3+ was tested for effects on calcineurin activity. Calcineurin was activated by Tb3+ with the following kinetic parameters estimated: k(cat) = 0.78 +/- 0.02 sec(-1), Km(pNPP) = 32.6 +/- 1.8 mM, and K(act)(Tb3+) = 0.08 +/- 0.03 mM. Terbium luminescence was demonstrated in the presence of the heterodimer of calcineurin and exploited to localize the binding of exogenous metal to the enzyme active site. Exogenous Mn2+ reduced luminescence, although the affinity of calcineurin for Tb3+ seemed to be greater. Putative active-site ligands, such as para-nitrophenol and a synthetic peptide from the autoinhibitory region, reduced the luminescence of terbium. Collectively, these data suggested that Tb3+ was binding directly at the active site of calcineurin, with the corollary that exogenous activating metal (Mn2+) binds at the active site of the enzyme. These data support the hypothesis that activating, exogenous divalent metal participates directly in catalysis.

Selective Activation of Calcineurin by Dipicolinic Acid

Calcineurin was activated at 30°C by incubation with dipicolinic acid, a metal chelator, in the absence of activating, exogenous Mn2+. The activation reached a plateau after 90 min with 8- to 12-fold higher activity. Inclusion of the activating metal Mn2+(1.0 mm) in the incubation mixture slightly lessened the activation induced by dipicolinic acid. The chelator 1,10-phenanthroline had no effect on the activity of calcineurin in concurrent experiments. Activation by dipicolinic acid was reversed by the addition of Zn2+or Fe3+. The reversal occurred within 30 min after the addition of either metal and returned the activity of calcineurin to its initial level. Atomic absorption spectrometry analysis showed no loss of iron or zinc from calcineurin after activation (2 h) by dipicolinic acid. Because there seemed to be no interaction between dipicolinic acid and exogenous metal, the effect of dipicolinic acid was concluded to result from masking of at least one intrinsic metal. Calcineurin incubated with 1.0 mmMn2+(saturating levels) also did not show any loss of intrinsic metal by atomic absorption analysis. The consequences of these data concerning the role(s) of intrinsic metals in calcineurin catalysis are discussed.

Electron donation to photosystem II by diphenylcarbazide is inhibited both by the endogenous manganese complex and by exogenous manganese ions.

Diphenylcarbazide (DPC) is an efficient electron donor to the inactive oxygen-evolving complex of photosystem II (PSII). We investigated the role of manganese on the rate of electron donation from DPC to PSII in both Mn-depleted (Tris washed) and Mn-retaining (NaCl washed) PSII preparations. The rate of electron donation from DPC to PSII was significantly higher in Mn-depleted than in Mn-retaining preparations, indicating a negative role of native Mn complex on DPC electron donation. The apparent Km values for DPC were found to be 0.11 and 0.17 mM for Mn-depleted and Mn-retaining PSII preparations, respectively. This difference in the Km values also indicates an antagonistic effect of endogenous Mn cluster on electron donation from DPC, which was markedly inhibited by exogenous Mn2+. However, the magnitude of inhibition was greater in Mn-depleted than in Mn-retaining PSII preparations. This indicates a higher accessibility to DPC to PSII in the absence of native Mn complex. Our results suggest (i) that Mn, either endogenous or added, acts as an accessibility barrier for DPC to donate electrons to PSII and (ii) that the native Mn complex not only functions as an accumulator of oxidizing equivalents but may also protect PSII from exogenous reductants.

Purification and characterization of the imidazoleglycerol-phosphate dehydratase of Saccharomyces cerevisiae from recombinant Escherichia coli.

The HIS3+ gene of Saccharomyces cerevisiae was overexpressed in Escherichia coli and the recombinant imidazoleglycerol-phosphate dehydratase (IGPD) purified to homogeneity. Laser-desorption and electrospray m.s. indicated a molecular ion within 2 units of that expected (23833.3) on the basis of the protein sequence, with about half of the polypeptide lacking the N-terminal formylmethionine residue. IGPD initially purified as an apoprotein was catalytically inactive and mainly a trimer of M(r) 70,000. Addition of Mn2+ (but not Mg2+) caused this to assemble to an active (40 units/mg) enzyme (Mn-IGPD) comprising of 24 subunits (M(r) 573,000) and containing 1.35 +/- 0.1 Mn atoms/polypeptide subunit. An enzyme with an identical activity and metal content was also obtained when the fermenter growth medium of recombinant Escherichia coli was supplemented with MnCl2, and IGPD was purified through as Mn-IGPD rather than as the apoenzyme and assembled in vitro. Inhibition by EDTA indicated that the intrinsic Mn2+ was essential for activity. The retention of activity over time after dilution to very low concentrations of enzyme (< 20 nM) indicated that the metal remained in tight association with the protein. A novel continuous assay method was developed to facilitate the kinetic characterization of Mn-IGPD. At pH 7.0, the Km for IGP was 0.10 +/- 0.02 mM and the Ki value for inhibition by 1,2,4-triazole, 0.12 +/- 0.02 mM. In contrast with other reports, thiols had no influence on catalytic activity. The activity of Mn-IGPD varied with enzyme concentration in such a way as to suggest that it dissociates to a less active form at very low concentrations. Significant inhibition by the product, imidazole acetol phosphate, was inferred from the shape of the progress curve. Titration with, the potent competitive inhibitor, 2-hydroxy-3-(1,2,4-triazol-1-yl)propyl phosphonate indicated that Mn-IGPD contained 0.9 +/- 0.1 catalytic sites/protomer. The activity nearly doubled in the presence of high concentrations of Mn2+; the apparent Ks for stimulation was 20 microM. The basis of this effect was obscure, since there was no corresponding increase in the titre of active sites. Neither was there a discernable shift in the values of Km or Ki (above), although exogenous Mn2+ did reduce the optimum pH for kcat, from 7.2 to 6.8. On the basis of a single site/subunit, the maximum rate of catalytic turnover at 30 degrees C was 32 s-1.

The rate of reduction of oxidized redox-active tyrosine, Z+, by exogenous Mn2+ is slowed in a site-directed mutant, at aspartate 170 of polypeptide D1 of photosystem II, inactive for photosynthetic oxygen evolution

Eleven different site-directed mutants at Asp-170 of Photosystem II polypeptide D1 ofSynechocystis PCC 6803 show varying degrees of activity for light-driven water oxidation depending on the nature of the substitution (Nixon, P.J. and Diner, B.A. (1992) Biochemistry 31, 942–948). These range from the most active, glutamate, with near wild-type rates of oxygen evolution to the totally inactive serine and alanine. Photosystem II core complexes lacking the tetranuclear Mn cluster responsible for water oxidation and isolated from wild-type and D1-Asp170Ser, were compared with respect to the kinetics of reduction by Mn2+ of the photooxidized redox-active tyrosine, Z+. The dependence of the rate of reduction of Z+ on the Mn2+ concentration shows mixed first and second-order behavior in the case of the wild-type and second-order behavior alone in the case of the mutant. The second-order rate constants for the wild-type and mutant complexes were 3.1 · 107 M−1 s−1 and 5.5 · 105 M−1 s−1, respectively. The ratio of these rate constants is consistent with theKm values determined earlier for the blockage of charge recombination by Mn2+. D1-Aspartate 170 is therefore implicated in a facilitated pathway for the oxidation of Mn2+, which most likely includes participation in a high-affinity metal binding site (Mn2+ and/or Mn3+) in the Photosystem II reaction center.

Electron-transfer events leading to reconstitution of oxygen-evolution activity in manganese-depleted photosystem II membranes

O2-evolution activity and the Mn complex can be reconstituted in photosystem II by a process called photoactivation. We have studied the elementary steps in photoactivation by using electron paramagnetic resonance spectroscopy to probe electron transport in Mn-depleted photosystem II membranes. The electron donation reactions in Mn-depleted photosystem II were found to be identical with those in untreated photosystem II, except that electron donation from the Mn complex was absent and could be replaced by slower electron donation from exogenous Mn2+. Mn2+ photooxidation by Mn-depleted photosystem II membranes correlates with reconstitution of O2-evolution activity. However, photooxidation of Mn2+ occurs in competition with photooxidation of the tyrosine residue YD, and cytochrome b-559. Thus, these two species are excluded from direct participation in the initial steps in the assembly of the Mn complex. Because photooxidation of Mn2+ is slower than photooxidation of the competing electron donors, cytochrome b-559 and chlorophyll, as well as recombination of the charge-separated states chlorophyll+QA- or YZ+QA-, these other reactions dominate in a single photochemical turnover reaction. This provides a molecular basis for both the low yield and low quantum yield of photoactivation. The first photochemical step in the assembly of the Mn complex results in photooxidation of one Mn2+ ion. Therefore, the first intermediate in assembly of the Mn complex contains Mn3+. On the basis of these results and previous kinetic studies [Miller, A.-F., & Brudvig, G. W. (1989) Biochemistry 28, 8181], we conclude that the second intermediate of Mn complex assembly contains Mn2+Mn3+, which is photooxidized to Mn3+2.

EFFECT OF EDTA ON ENDOGENOUS MANGANESE DURING INDOLEACETIC ACID‐INDUCED GROWTH OF A VENA COLEOPTILE SEGMENTS

SummaryThe effect of ethylenediamine tetracetic acid (EDTA) on endogenous Mn2+ was studied by electron spin resonance (ESR) spectroscopy during indoleacetic acid (IAA)‐induced growth of Avena coleoptile segments. An enhancement in the concentration of endogenous Mn2+ was recorded in coleoptile segments treated with low concentrations (1 × 10−6 M) of EDTA or ethyleneglycol‐bis‐(β‐aminoethylether) N, N″‐tetracetic acid (EGTA ‐ 1 × 10−6 to 1 × 10−5 M), while at higher concentrations both EDTA and EGTA appeared to chelate Mn2+. An intermediate concentration of EDTA (5 × 10−5 M) interacted with IAA in enhancing the growth, while both lower (1 × 10−6M) and higher (5 × 10−6M) concentrations inhibited IAA‐induced growth. Inhibition of IAA‐induced growth at higher concentrations of EDTA was reversed by exogenous Mn2+. Concentration of endogenous Mn2+ declined further when IAA was present along with different concentrations of EDTA, except in coleoptile segments treated with 5 × 10−4 M EDTA. The data suggest that endogenous Mn2+ is essential for IAA‐induced growth and that a precise balance of free to bound/complexed Mn2+ may be involved in IAA action.

Regulation of phosphoglycerate phosphomutase in developing forespores and dormant and germinated spores of Bacillus megaterium by the level of free manganous ions

The large depot of phosphoglyceric acid (PGA) which is accumulated within spores of Bacillus megaterium is greater than 99% 3-phosphoglyceric acid (3-PGA). The 3-PGA depot is stable in forespores and dormant spores, but is utilized rapidly during spore germination. When spores were germinated in KBr plus NaF, the PGA depot was not utilized, but 13% of the 3-PGA was converted to 2-PGA. These data suggest phosphoglycerate phosphomutase as the enzyme which is regulated to allow 3-PGA accumulation during sporulation. Young isolated forespores, in which 3-PGA was normally stable, utilized their 3-PGA rapidly when incubated with Mn2+ plus the divalent cation ionophore X-537A; Mn2+ or ionophore alone or Mg2+ or Ca2+ plus ionophore was without effect. Young forespores contained significant amounts of Mn2+. However, forespore Mn2+ exchanged slowly with exogenous Mn2+ and was removed poorly by toluene treatment. This suggests that much of the forespore Mn2+ is tightly bound to some forespore component. Since phosphoglycerate phosphomutase from B. megaterium has an absolute and specific requirement for Mn2+, these data suggest that the activity of this enzyme in vivo may be regulated to a large degree by the level of free Mn2+. Indeed, the activity of this enzyme in forespore or dormant spore extracts was stimulated greater than 25-fold by Mn2+, whereas comparable extracts from cells or germinated spores were stimulated only two- to fourfold.

Effect of cyanide on NADPH oxidation by granules from human polymorphonuclear leukocytes

Cyanide has been shown to stimulate both oxygen uptake and hexose monophosphate shunt activity in phagocytizing human polymorphonuclear leukocytes. It also stimulates the oxidation of NADPH by a particulate fraction derived from phagocytizing cells. This stimulation of NADPH oxidase is not observed in the presence of exogenous Mn2+. Studies with purified enzymes have shown that CN- also stimulates NADPH oxidation by horseradish peroxidase or lactoperoxidase, suggesting that the respiratory burst might be initiated by activation of a peroxidase-like enzyme in the human polymorphonuclear leukocyte. Based on studies of others, however, it does not appear as though the enzyme is identical to myeloperoxidase. The mechanism of the CN- stimulation appears to involve an oxidatic chain reaction, since it stimulates markedly NADPH oxidation in the presence of an artificial superoxide-generating system.

Effect of Cyanide on NADPH Oxidation by Granules From Human Polymorphonuclear Leukocytes

Cyanide has been shown to stimulate both oxygen uptake and hexose monophosphate shunt activity in phagocytizing human polymorphonuclear leukocytes. It also stimulates the oxidation of NADPH by a particulate fraction derived from phagocytizing cells. This stimulation of NADPH oxidase is not observed in the presence of exogenous Mn2+. Studies with purified enzymes have shown that CN- also stimulates NADPH oxidation by horseradish peroxidase or lactoperoxidase, suggesting that the respiratory burst might be initiated by activation of a peroxidase-like enzyme in the human polymorphonuclear leukocyte. Based on studies of others, however, it does not appear as though the enzyme is identical to myeloperoxidase. The mechanism of the CN- stimulation appears to involve an oxidatic chain reaction, since it stimulates markedly NADPH oxidation in the presence of an artificial superoxide-generating system.