1064 nm Nd:YAG laser light affects transmembrane mitochondria respiratory chain complexes.

Photobiomodulation (PBM) is a non-plant-cell manipulation through a transfer of energy by means of light sources at the non-ablative or thermal intensity. Authors showed that cytochrome-c-oxidase (complex IV) is the specific chromophore's target of PBM at the red (600-700 nm) and NIR (760-900 nm) wavelength regions. Recently, it was suggested that the infrared region of the spectrum could influence other chromospheres, despite the interaction by wavelengths higher than 900 nm with mitochondrial chromophores was not clearly demonstrated. We characterized the interaction between mitochondria respiratory chain, malate dehydrogenase, a key enzyme of Krebs cycle, and 3-hydroxyacyl-CoA dehydrogenase, an enzyme involved in the ß-oxidation (two mitochondrial matrix enzymes) with the 1064 nm Nd:YAG (100mps and 10 Hz frequency mode) irradiated at the average power density of 0.50, 0.75, 1.00, 1.25 and 1.50 W/cm2 to generate the respective fluences of 30, 45, 60, 75 and 90 J/cm2 . Our results show the effect of laser light on the transmembrane mitochondrial complexes I, III, IV and V (adenosine triphosphate synthase) (window effects), but not on the extrinsic mitochondrial membrane complex II and mitochondria matrix enzymes. The effect is not due to macroscopical thermal change. An interaction of this wavelength with the Fe-S proteins and Cu-centers of respiratory complexes and with the water molecules could be supposed.

Activity Based Protein Profiling Leads to Identification of Novel Protein Targets for Nerve Agent VX.

Organophosphorus (OP) nerve agents continue to be a threat at home and abroad during the war against terrorism. Human exposure to nerve agents such as VX results in a cascade of toxic effects relative to the exposure level including ocular miosis, excessive secretions, convulsions, seizures, and death. The primary mechanism behind these overt symptoms is the disruption of cholinergic pathways. While much is known about the primary toxicity mechanisms of nerve agents, there remains a paucity of information regarding impacts on other pathways and systemic effects. These are important for establishing a comprehensive understanding of the toxic mechanisms of OP nerve agents. To identify novel proteins that interact with VX, and that may give insight into these other mechanisms, we used activity-based protein profiling (ABPP) employing a novel VX-probe on lysates from rat heart, liver, kidney, diaphragm, and brain tissue. By making use of a biotin linked VX-probe, proteins covalently bound by the probe were isolated and enriched using streptavidin beads. The proteins were then digested, labeled with isobarically distinct tandem mass tag (TMT) labels, and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Quantitative analysis identified 132 bound proteins, with many proteins found in multiple tissues. As with previously published ABPP OP work, monoacylglycerol lipase associated proteins and fatty acid amide hydrolase (FAAH) were shown to be targets of VX. In addition to these two and other predicted neurotransmitter-related proteins, a number of proteins involved with energy metabolism were identified. Four of these enzymes, mitochondrial isocitrate dehydrogenase 2 (IDH2), isocitrate dehydrogenase 3 (IDH3), malate dehydrogenase (MDH), and succinyl CoA (SCS) ligase, were assayed for VX inhibition. Only IDH2 NADP+ activity was shown to be inhibited directly. This result is consistent with other work reporting animals exposed to OP compounds exhibit reduced IDH activity. Though clearly a secondary mechanism for toxicity, this is the first time VX has been shown to directly interfere with energy metabolism. Taken together, the ABPP work described here suggests the discovery of novel protein-agent interactions, which could be useful for the development of novel diagnostics or potential adjuvant therapeutics.

Maize cytosolic NADP-malic enzyme (ZmCytNADP-ME): a phylogenetically distant isoform specifically expressed in embryo and emerging roots.

Two maize plastidic NADP-malic enzyme isoforms have been characterized: the bundle sheath-located photosynthetic isoform (ZmC(4)-NADP-ME) and a constitutively expressed one (Zm-nonC(4)-NADP-ME). In this work, the characterization of the first maize cytosolic NADP-ME (ZmCytNADP-ME) is presented, which transcript is exclusively found in embryo and emerging roots. ZmCytNADP-ME expression in roots decreases with development, while Zm-nonC ( 4 ) -NADP-ME increases concomitantly. On the other hand, ZmCytNADP-ME accumulation is differentially modulated by several stress conditions and shows coordination with that of Zm-nonC ( 4 ) -NADP-ME in maize young roots. Recombinant ZmCytNADP-ME displays clearly distinct kinetic parameters and metabolic regulation than the plastidic isoforms. The particular properties and the specific-expression pattern of this novel isoform suggest that it may be involved in the control of cytosolic malate levels in emerging roots, e.g. during hypoxia. ZmCytNADP-ME is phylogenetically related to other cytosolic mono and dicot NADP-MEs, and data indicate that it belongs to an ancestral unique group among plant NADP-MEs.

Genomic diversity of Erwinia carotovora subsp. carotovora and its correlation with virulence.

We used genetic and biochemical methods to examine the genomic diversity of the enterobacterial plant pathogen Erwinia carotovora subsp. carotovora. The results obtained with each method showed that E. carotovora subsp. carotovora strains isolated from one ecological niche, potato plants, are surprisingly diverse compared to related pathogens. A comparison of 23 partial mdh sequences revealed a maximum pairwise difference of 10.49% and an average pairwise difference of 2.13%, values which are much greater than the maximum variation (1.81%) and average variation (0.75%) previously reported for Escherichia coli. Pulsed-field gel electrophoresis analysis of I-CeuI-digested genomic DNA revealed seven rrn operons in all E. carotovora subsp. carotovora strains examined except strain WPP17, which had only six copies. We identified 26 I-CeuI restriction fragment length polymorphism patterns and observed significant polymorphism in fragment sizes ranging from 100 to 450 kb for all strains. We detected large plasmids in two strains, including the model strain E. carotovora subsp. carotovora 71. The two least virulent strains had an unusual chromosomal structure, suggesting that a particular pulsotype is correlated with virulence. To compare chromosomal organization of multiple enterobacterial genomes, several genes were mapped onto I-CeuI fragments. We identified portions of the genome that appear to be conserved across enterobacteria and portions that have undergone genome rearrangements. We found that the least virulent strain, WPP17, failed to oxidize cellobiose and was missing several hrp and hrc genes. The unexpected variability among isolates obtained from clonal hosts in one region and in one season suggests that factors other than the host plant, potato, drive the evolution of this common environmental bacterium and key plant pathogen.

Solvent interactions of halophilic malate dehydrogenase.

Malate dehydrogenase from the extreme halophilic Haloarcula marismortui (Hm MalDH) is an acidic protein that is unstable below molar salt concentrations. The solvated folded protein was studied by small-angle neutron scattering in solvents containing salt: NaCl, NaCH(3)CO(2), KF, NH(4)Cl, NH(4)CH(3)CO(2), (NH(4))(2)SO(4), MgCl(2), and MgSO(4). It was found that the global solvent interactions depend mainly on the nature of the cation. Complementary mass density measurements in MgCl(2), NaCl, NaCH(3)CO(2), and (NH(4))(2)SO(4) allowed determining the partial molal volumes of the protein, which were found to increase slightly with the salt, and the preferential salt binding parameters for each solvent condition. These are strongly dependent on the cation type and salt concentration. Hm MalDH can be modeled as an invariant particle binding 4100 water molecules in MgCl(2) and 2000 +/- 200 in NaCl, NaCH(3)CO(2), or (NH(4))(2)SO(4). The number of salt molecules associated to the particle decreases from about 85 to 0 in the order MgCl(2) > NaCl = NaCH(3)CO(2) > (NH(4))(2)SO(4). Alternatively, we considered exchangeable sites for water and salt with the effects of solvent nonideality. It does not change the description of the solvent interactions. Solvent anions act on Hm MalDH stability through a limited number of strong binding sites, as those seen at the interfaces of Hm MalDH by crystallography. Cations would act through some strong and numerous weak binding sites defined on the folded protein, in possible addition to nonspecific hydration effects.

The cytotoxic lipid peroxidation product, 4-hydroxy-2-nonenal, specifically inhibits decarboxylating dehydrogenases in the matrix of plant mitochondria.

4-Hydroxy-2-nonenal (HNE), a cytotoxic product of lipid peroxidation, inhibits O(2) consumption by potato tuber mitochondria. 2-Oxoglutarate dehydrogenase (OGDC), pyruvate dehydrogenase complex (PDC) (both 80% inhibited) and NAD-malic enzyme (50% inhibited) are its major targets. Mitochondrial proteins identified by reaction with antibodies raised to lipoic acid lost this antigenicity following HNE treatment. These proteins were identified as acetyltransferases of PDC (78 kDa and 55 kDa), succinyltransferases of OGDC (50 kDa and 48 kDa) and glycine decarboxylase H protein (17 kDa). The significance of the effect of these inhibitions on the impact of lipid peroxidation and plant respiratory functions is discussed.

Mechanism of light modulation: identification of potential redox-sensitive cysteines distal to catalytic site in light-activated chloroplast enzymes.

Light-dependent reduction of target disulfides on certain chloroplast enzymes results in a change in activity. We have modeled the tertiary structure of four of these enzymes, namely NADP-linked glyceraldehyde-3-P dehydrogenase, NADP-linked malate dehydrogenase, sedoheptulose bisphosphatase, and fructose bisphosphatase. Models are based on x-ray crystal structures from non-plant species. Each of these enzymes consists of two domains connected by a hinge. Modeling suggests that oxidation of two crucial cysteines to cystine would restrict motion around the hinge in the two dehydrogenases and influence the conformation of the active site. The cysteine residues in the two phosphatases are located in a region known to be sensitive to allosteric modifiers and to be involved in mediating structural changes in mammalian and microbial fructose bisphosphatases. Apparently, the same region is involved in covalent modification of phosphatase activity in the chloroplast.

Cloning, site-specific mutagenesis, expression and characterization of full-length chloroplast NADP-malate dehydrogenase from Pisum sativum.

Chloroplast NADP-dependent malate dehydrogenase is regulated by a dithiol redox reaction. The assignment of the groups involved, requires the primary structure of the enzyme to be known. Using the polymerase chain reaction and the cDNA library of Pisum sativum, the sequence of the enzyme and its targeting signal was determined. The gene was cloned in Escherichia coli JM83 and expressed in E. coli JM83 and E. coli B at high yield. The determination of the physical properties of the gene product proves the recombinant protein to be indistinguishable from the enzyme purified from the plant. This holds true, in spite of the fact that the plant enzyme lacks 11 N-terminal residues. The lengths of the complete polypeptide chain of the recombinant enzyme and its transit peptide are 388 and 53 residues, respectively. The comparison of the sequences of the mature enzyme with those of known chloroplast NADP-MDH shows 83-95% identity, but with mitochondrial or bacterial MDH only approximately 20%. Reduction of the (inactive) oxidized enzyme with dithiothreitol allows mimicking of the in vivo activation. The reaction follows a consecutive second-order-kinetics mechanism. Guanidinium chloride (GdmCl) at concentrations below 0.4 M leads to a significant activation of the oxidized form of the enzyme. At [GdmCl] = 0.4-0.46 M, both oxidized and reduced NADP-MDH show highly cooperative changes in the hydrodynamic and spectral properties, indicating the synchronous breakdown of the quaternary, tertiary and secondary structures. Site-directed mutations C23A and C28A do not quench the regulatory properties of the enzyme; additional substitution of alanine for Cys206 and Cys376 renders the enzyme equally active in both the reduced and the oxidized state. Therefore, one can consider these residues, either alone or in combination with Cys23 and Cys28, as responsible for enzyme activation.

Effects of N-terminal truncations upon chloroplast NADP-malate dehydrogenases from pea and spinach.

Using the purification procedure of Fickenscher and Scheibe (Biochim. Biophys. Acta 749 (1983), 249-254) and a modification of the method, we produced a series of NADP-MDH forms from spinach and pea-leaf extracts that were characterized by a stepwise shortening of the N-terminal sequences. Limited proteolysis of the enzymes resulted in the generation of even shorter forms. Immunoprecipitation of the NADP-MDH from crude extracts revealed that the sequences of the intact enzymes from pea, spinach and maize started at a position (Ser) identical with that established for the Sorghum enzyme (Crétin, C., et al. (1990) Eur. J. Biochem. 192, 299-303). Spinach NADP-MDH isolated by conventional methods was shown to represent the intact form. Thus, the kinetic, regulatory and structural properties of the various truncated forms could be compared with those of an intact form. Removal of 5 or 11 amino acids, as occurred during isolation of the pea NADP-MDH, was without any significant effect. The enzymes were all dimeric and still exhibited the characteristic redox-regulatory properties. However, removal of 31 and 37 amino acids using aminopeptidase K resulted in the formation of active monomers characterized by only slightly lowered affinities towards the substrates, a shift of their pH optimum from 8 to 7, the loss of oxaloacetate inhibition and an increased maximal velocity. Although these forms lacked most or all of the N-terminal extra-peptide, including the 2 cysteines involved in redox-modification, they were still sensitive to the redox-potential. However, the low concentration of thiol required for immediate and complete restoration of any lost activity (40 mM beta-mercaptoethanol) suggested that this reaction might not be relevant for redox-regulation in vivo.

Limited proteolysis of NADP-malate dehydrogenase from pea chloroplast by aminopeptidase K yields monomers. Evidence of proteolytic degradation of NADP-malate dehydrogenase during purification from pea.

NADP-malate dehydrogenase (L-malate: NADP oxidoreductase, EC 1.1.1.82) from leaves of Pisum sativum has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis. In the crude leaf extract and in the absence of protease inhibitors in the isolation medium, the N-terminus of NADP-MDH was found to be highly susceptible to proteolysis. Evidence of proteolysis during purification includes observations of reduced subunit size on SDS-PAGE and reduced specific activity. Experiments were carried out to investigate the function of the N-terminal amino acid sequence extension of NADP-MDH. Limited proteolysis of highly active (600 units/mg protein) NADP-MDH using aminopeptidase K yielded catalytically active monomers of 34.7 kDa. The results support the conclusions that the N-terminal region is located at the surface of the protein, and that for maintenance of the native NADP-MDH dimer an N-terminal amino acid sequence is important.

Primary structure and analysis of the location of the regulatory disulfide bond of pea chloroplast NADP-malate dehydrogenase.

Purified pea chloroplast NADP-malate dehydrogenase (S)-malate: NADP+ oxidoreductase, EC 1.1.1.82) was digested with trypsin and the resulting peptides were separated by HPLC and sequenced. Together with the information from earlier work (Fickenscher, K. et al. (1987) Eur. J. Biochem. 168, 653-658) the total sequence is not known to an extent of 78%. Comparison with the sequence of the corn NADP-malate dehydrogenase deduced from its cDNA (Metzler, M.C. et al. (1989) Plant Mol. Biol. 12, 713-722) showed 84% agreement; however, the 11 N-terminal residues exhibit only 27% similarity. The N- and C-terminal extrapeptides of the pea NADP-malate dehydrogenase when aligned with non-regulatory NAD-malate dehydrogenases from bacteria or mammals consist of 30 and 17 amino acids, respectively. Since all cysteine-containing peptides were sequenced, the number of eight cysteines per subunit of the pea enzyme was established. The native, oxidized enzyme is characterized by an extremely slow reactivity of two thiols. Titration of the thiols of the denatured, oxidized enzyme both with DTNB and with pCMB resulted in six thiols not involved in disulfide formation. Therefore, one disulfide bridge must be present per 38.9 kDa subunit. Analysis of disulfide bonds by urea gel electrophoresis confirmed this finding. Using digestion products of NADP-malate dehydrogenase with aminopeptidase K, the location of the single disulfide bridge was established to be on the N-terminal arm (Cys-12 and Cys-17) of the polypeptide chain.