Studies on dilution inactivation of sheep liver pyruvate carboxylase

When sheep liver pyruvate carboxylase was diluted below 4 EU/ml, it underwent inactivation involving two kinetically distinct processes, i.e., a rapid initial burst followed by a slower second phase. The catalytic activity of the diluted enzyme eventually approached zero, suggesting the occurrence of an irreversible process. Analysis of the quaternary structure of the enzyme by gel filtration chromatography and electron microscopy showed that most of the enzyme molecules occur as tetramers at high enzyme concentrations. However, dilution of the enzyme below 4 EU/ml led to the appearance of dimers and monomers which were essentially inactive under the conditions of the assay system used. The presence of acetyl-CoA during dilution prevented inactivation from occurring and preserved the tetrameric structure. When added after dilution, acetyl-CoA prevented further inactivation from occurring but did not reactivate the enzyme. However, acetyl-CoA did cause a relatively rapid reassociation of the inactive monomers and dimers to form inactive tetramers.     

Electron microscopic localization of hydrogenase genes on the megaplasmid pHG 1 in Alcaligenes eutrophus

Summary Plasmids carrying hydrogenase genes in Alcaligenes eutrophus wild type H 16 and in two transposon Tn5 —induced mutants have been investigated by electron microscopy. Besides the pHG1 megaplasmid (458±27 kb) carrying genes coding for structural and regulatory properties of hydrogenases, small plasmids of unknown significance have been detected. The sizes of EcoRI fragments obtained from pHG1 were measured from electron micrographs. They were significantly different from sizes determined previously by agarose gel electrophoresis.Plasmid pHG1 isolated from the wild type H 16 was shown to contain two inverted repeats (IR 16-1 and IR 16-2) with sizes similar to known transposons.From electron microscopic hybridization studies, it was deduced that the sites of insertion of Tn5 into a regulation gene on pHG1 for both soluble and membrane-bound hydrogenase, and of Tn5-Mob into the gene coding for structural properties of the soluble hydrogenase, are about 67.2 kb apart. One of the inverted repeats (IR 16-1) was localized in between these sites.     

Structural analysis of EcoRI-DNA complexes as revealed by electron microscopy

Electron microscopy of negatively stained isolated restriction enzyme EcoRI revealed particle projections with triangular or square outlines, indicating that the enzyme, in its tetrameric state, is tetrahedron-like. The two dimers making up the tetramer appear to be arranged in two planes orthogonal to each other. Complexes formed by EcoRI with the plasmids pBR322 or pGW10 were investigated by electron microscopic spreading techniques. In the presence of Mg²⁺, EcoRI was bound to the DNA molecules to form pearl necklace-like aggregates. The number of bound EcoRI particles was much higher as the sum of EcoRI-and 5..AATT..3 sites (with exceptions, the 5..AATT..3 sites may function as one type of EcoRI^* sites) along the DNAs, indicating unspecific binding. In the absence of Mg²⁺, EcoRI was bound to the DNA only at the recognition site for EcoRI and the sites where the tetranucleotide sequence 5..AATT..3 was present. A direct correlation of the local concentrations of the bases A and T within the flanking sequences of the binding sites with the frequency of EcoRI to the DNA was observed. Dimers and tetramers of the enzyme was found to bind to the DNA. Tetramers occasionally exhibited two binding sites for DNA as indicated by the observation of DNA loops originating at the sites of bound tetrameric EcoRI particles.Abbreviations BAC Benzyldimethylalkylammoniumchloride - bp base pairs - Kb kilobases - SDS sodium dodecylsulfate Enzymes (EC 3.1.23.13) Restrictionendonuclease EcoRI - (EC 3.1.23.21) Restrictionendonuclease HindIII - (EC 3.1.23.37) Restrictionendonuclease SalGI Dedicated Professor H. G. Schlegel on occasion of this 60th birthday     

Two-photon imaging of glutathione levels in intact brain indicates enhanced redox buffering in developing neurons and cells at the cerebrospinal fluid and blood-brain interface

Glutathione is the major cellular thiol present in mammalian cells and is critical for maintenance of redox homeostasis. However, current assay systems for glutathione lack application to intact animal tissues. To map the levels of glutathione in intact brain with cellular resolution (acute tissue slices and live animals), we have used two-photon imaging of monochlorobimane fluorescence, a selective enzyme-mediated marker for reduced glutathione. Previously, in vitro experiments using purified components and cultured glial cells attributed cellular monochlorobimane fluorescence to a glutathione S-transferase-dependent reaction with GSH. Our results indicate that cells at the cerebrospinal fluid or blood-brain interface, such as lateral ventricle ependymal cells (2.73 +/- 0.56 mm; glutathione), meningeal cells (1.45 +/- 0.09 mm), and astroglia (0.91 +/- 0.08 mm), contain high levels of glutathione. In comparison, layer II cortical neurons contained 20% (0.21 +/- 0.02 mm) the glutathione content of nearby astrocytes. Neuronal glutathione labeling increased 250% by the addition of the cell-permeable glutathione precursor N-acetylcysteine indicating that the monochlorobimane level or glutathione S-transferase activity within neurons was not limiting. Regional mapping showed that glutathione was highest in cells lining the lateral ventricles, specifically ependymal cells and the subventricular zone, suggesting a possible function for glutathione in oxidant homeostasis of developing neuronal progenitors. Consistently, developing neurons in the subgranular zone of dentate gyrus contained 3-fold more glutathione than older neurons found in the neighboring granular layer. In conclusion, mapping of glutathione levels in intact brain demonstrates a unique role for enhanced redox potential in developing neurons and cells at the cerebrospinal fluid and blood-brain interface.     

Structural aspects of the soluble NAD-dependent hydrogenase isolated from Alcaligenes eutrophus H16 and from Nocardia opaca 1b

The soluble NAD-dependent hydrogenase (hydrogen-NAD oxidoreductase, EC 1.12.1.2), consisting of four non-identical subunits, was isolated from Alcaligenes eutrophus H16 and from Nocardia opaca 1b and analyzed by a HPLC gel permeation technique and electron microscopy. The tetrameric enzyme particles from both origins, as determined from negatively stained electron microscopic samples, were found to be elongated and very similar in shape and size. The A. eutrophus enzyme was measured in more detail. It exhibited dimensions of 12.7 nm by 5.5 nm (axial ratio 2.3:1). Dissociation into smaller particles and unspecific aggregation combined with partial inactivation were observed in the presence of the inhibitor NADH. Kept in buffer without added nickel, the enzyme was partially dissociated. Reassociation of tetramers without restored enzyme activity was achieved by addition of 0.5 mM NiCl₂. A working model for the structural organization of the tetrameric enzyme particle is presented.     

Electron microscopy studies of DNA complexes with restriction endonuclease SalGI

The binding properties of the type II restriction endonuclease SalGI to the plasmid DNA pGW 10 has been investigated by electron microscopic studies. Samples were spread by the BAC technique. In the presence of magnesium, SalGI binds as dimers and tetramers to the specific recognition site 5'-G-T-C-G-A-C-3' and with lower rate to the sequence 5'-G-T-C-A-A-C-3', which represents the recognition site of the restriction endonucleases Hind II and Hinc II.     

SIGNR3‐dependent immune regulation by Lactobacillus acidophilus surface layer protein A in colitis

Intestinal immune regulatory signals govern gut homeostasis. Breakdown of such regulatory mechanisms may result in inflammatory bowel disease (IBD). Lactobacillus acidophilus contains unique surface layer proteins (Slps), including SlpA, SlpB, SlpX, and lipoteichoic acid (LTA), which interact with pattern recognition receptors to mobilize immune responses. Here, to elucidate the role of SlpA in protective immune regulation, the NCK2187 strain, which solely expresses SlpA, was generated. NCK2187 and its purified SlpA bind to the C-type lectin SIGNR3 to exert regulatory signals that result in mitigation of colitis, maintenance of healthy gastrointestinal microbiota, and protected gut mucosal barrier function. However, such protection was not observed in Signr3^−/− mice, suggesting that the SlpA/SIGNR3 interaction plays a key regulatory role in colitis. Our work presents critical insights into SlpA/SIGNR3-induced responses that are integral to the potential development of novel biological therapies for autoinflammatory diseases, including IBD.     

Pyruvate carboxylase from Pseudomonas citronellolis: shape of the enzyme, and localization of its prosthetic biotin group by electron microscopic affinity labeling

Pseudomonas citronellolis is known to contain a pyruvate carboxylase with an alpha 4 beta 4 composition. All the other pyruvate carboxylases investigated so far are made up of four seemingly identical subunits. Nevertheless, this exceptional pyruvate carboxylase exhibits a size and overall shape similar to other pyruvate carboxylases. Electron microscopic affinity labeling with avidin revealed that the prosthetic biotin groups (one per alpha beta unit, i.e. four per enzyme particle) are located close to the inter-unit junctions of pairs of alpha beta units making up the enzyme. This position of the prosthetic biotin groups is very similar to the location of the biotin in the other carboxylases.     

Nitrate reductase from yeast: cultivation,partial purification and characterization

Summary Several yeast strains were assayed for occurence of nitrate reductase after growth in a defined medium with nitrate as the sole nitrogen source, Candida boidinii DSM 70026, showing the highest specific activity, was further investigated. The procedures for yeast fermentation and nitrate reductase purfication are described in detail. Nitrate reductase from this yeast was characterized as NAD(P)H: nitrate oxidoreductase (E.C.1.6.6.2). The enzyme activity with NADH (NADPH) was highest at pH 7.0 (7.1) and 30° C (25° C). The values of K _m determinations with NADH/NADPH were both 4 × 10^–4 mol/l; values for the substrate inhibition constant (K _i) were 6 × 10^–4 mol/l. The molecular mass of the native enzyme was estimated by gel permeation chromatography to be approximately 350 kDa. Offprint requests to: R. Gromes