Purification and Properties of Manganese Superoxide Dismutase from Norway Spruce (Picea abies L. Karst)

A manganese-containing superoxide dismutase (SOD; EC 1.15.1.1 [EC] )was purified to electrophoretic homogeneity from seeds of Norwayspruce (Picea abies L.). The apparent molecular mass of thepurified enzyme was 86 kDa, as determined by gel filtration.The subunit molecular mass, estimated by SDS-polyacrylamidegel electrophoresis, was 22 kDa both in the presence and inthe absence of 2-mercaptoethanol. Thus, the native enzyme isa homotetramer with subunits that were not linked by disulfidebonds. The isoelectric point of this Mn-SOD was 5.5. The specificactivity of the Mn-SOD was strongly pH-dependent and was 400units per nmol SOD at pH 7.8 and 30 units per nmol SOD at pH10.4. The first 25 amino acid residues in the amino terminalregion of spruce Mn-SOD exhibited a high degree of sequencehomology to those of Mn-SODs from other organisms. In Mn-deficientneedles the activity of Mn-SOD was only half of that in non-deficientneedles, whereas the activity of CuZn-SOD was doubled. (Received May 20, 1994; Accepted October 31, 1994)     

Crystal structure of the phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with myo-inositol.

Phosphatidylinositol (PI), once regarded as an obscure component of membranes, is now recognized as an important reservoir of second messenger precursors and as an anchor for membrane enzymes. PI-specific phospholipase C (PI-PLC) is the enzyme that cleaves PI, invoking numerous cellular responses. The crystal structure of PI-PLC from Bacillus cereus (EC 3.1.4.10) has been solved at 2.6 A resolution and refined to a crystallographic R factor of 18.7%. The structure consists of an imperfect (beta alpha)8-barrel similar to that first observed for triose phosphate isomerase and does not resemble any other known phospholipase structure. The active site of the enzyme has been identified by determining the structure of PI-PLC in complex with its inhibitor, myo-inositol, at 2.6 A resolution (R factor = 19.5%). This substrate-like inhibitor interacts with a number of residues highly conserved among prokaryotic PI-PLCs. Residues His32 and His82, which are also conserved between prokaryotic and eukaryotic PI-PLCs, most likely act as general base and acid respectively in a catalytic mechanism analogous to that observed for ribonucleases.     

The rhodium(III) trisacetonitrile complexes: a re-investigation of the synthesis of fac- and mer- [RhCl3(CH3CN)3], their characterization by 2D NMR spectroscopy and the X-ray crystal structure of mer-[RhCl3(CH3CN)3] · CH3CN

It is shown that the reaction of RhCl₃·3H₂O with acetonitrile normally produces mixtures of mer- and fac-[RhCl₃(CH₃CN)₃] (1a and 1b, respectively). The IR and ¹H NMR spectra of these isomers were re-investigated. Their two-dimensional (¹⁰³Rh,¹H) NMR spectra were also recorded. Equilibrium and exchange studies of 1a and 1b in CD₃C were performed. It was found that in 1a the exchange rate of the nitrile molecule trans to Cl is much faster than those of mutually trans nitriles. Also the nitrile molecules in 1b underwent fast exchange in CD₃CN; however, their rate was slightly faster than that of the more labile CH₃CN in 1a. The X-ray crystal structure of mer-[RhCl₃(CH₃CN)₃]·CH₃CN (1c) was determined. Crystal data: triclinic space group

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Application of H(N)CA,CO-E.COSY experiments for calibrating the φ angular dependences of vicinal couplings J(C′i−1,Hi α), J(C′i−1,Ci β) and J(C′i−1,C′i) in proteins

A triple-resonance NMR technique suitable for the determination ofcarbonyl-related couplings in polypeptide systems is introduced. Theapplication of three novel pulse sequences to uniformly¹³C/¹⁵N-enriched proteins yields E.COSY-likemultiplet patterns exhibiting either one of the³J(C_i–1,H_i ), ³J(C_i–1,C_i ) and³J(C_i–1,C_i)coupling constants in the indirectly detected ¹³Cdimension, depending on the passive spin selected. The experiments aredemonstrated with oxidized flavodoxin from Desulfovibrio vulgaris. On thebasis of the J-values measured and the backbone -angles derived from ahigh-resolution X-ray structure of the protein, the three associated Karplusequations were reparametrized. The root-mean-square differences between theexperimental coupling constants and those predicted by the optimized Karpluscurves are 0.41, 0.33 and 0.32 Hz for³J(C_i–1,H_i ),³J(C_i–1,C_i ) and³J(C_i–1,C_i),respectively. The results are compared with the Karplus parameters previouslypublished for the same couplings.     

Interactions of NH4+ and L-glutamate with NO3- transport processes of non-mycorrhizal Fagus sylvatica roots

The processes of NO₃^– uptake and transport and the effectsof NH₄⁺ or L-glutamate on these processes were investigatedwith excised non-mycorrhizal beech (Fagus sylvatica L.) roots.NO₃^– net uptake followed uniphasic Michaelis-Menten kineticsin a concentration range of 10µM to 1 mM with an apparentK_m of 9.2 µM and a V_max of 366 nmol g^–1 FW h^–1.NH₄⁺, when present in excess to NO₃^–, or 10 mM L-glutamateinhibited the net uptake of NO₃^– Apparently, part of NO₃^–taken up was loaded into the xylem. Relative xylem loading ofNO₃^– ranged from 3.21.6 to 6.45.1% of NO₃^– netuptake. It was not affected by treatment with NH₄⁺ or L-glutamate.¹⁶N/¹³N double labelling experiments showed that NO₃^–efflux from roots increased with increasing influx of NO₃^–and, therefore, declined if influx was reduced by NH₄⁺ or L-glutamateexposure. From these results it is concluded that NO₃^–net uptake by non-mycorrhizal beech roots is reduced by NH₄⁺or L-glutamate at the level of influx and not at the level ofefflux. Key words: Nitrate transport, net uptake, influx, efflux, ammonium, Fagus, Fagaceae     

Crystallization of the complex of human IFN-γ and the extracellular domain of the IFN-γ receptor

A complex of human interferon-γ (IFN- γ) with the soluble extracellular domain of the IFN- γ receptor α-chain (IFN-γ-R) has been crystallised. Crystals of the complex were grown using PEG 4000 as the precipitating agent in the presence of β-octyl glucoside. The receptor-ligand complex crystallizes in a monoclinic space group and diffracts to about 3.0 Å resolution. Isomorphous crystals have been obtained with complex containing selenomethionine and cysteine mutants of IFN-γ, which may facilitate the ongoing X-ray structure determination. © 1995 Wiley-Liss, Inc.