Carboxylate and aromatic active‐site residues are determinants of high‐affinity binding of ω‐aminoaldehydes to plant aminoaldehyde dehydrogenases

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87000/1/j.1742-4658.2011.08239.x.pd

Biomolecular Profiling by MALDI-TOF Mass Spectrometry in Food and Beverage Analyses

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has frequently been applied to the analysis of biomolecules. Its strength resides not only in compound identification but particularly in acquiring molecular profiles providing a high discriminating power. The main advantages include its speed, simplicity, versatility, minimum sample preparation needs, and a relatively high tolerance to salts. Other benefits are represented by the possibility of automation, high throughput, sensitivity, accuracy, and good reproducibility, allowing quantitative studies. This review deals with the prominent use of MALDI-TOF MS profiling in food and beverage analysis ranging from the simple detection of sample constituents to quantifications of marker compounds, quality control, and assessment of product authenticity. This review summarizes relevant discoveries that have been obtained with milk and milk products, edible oils, wine, beer, flour, meat, honey, and other alimentary products. Marker molecules are specified: proteins and peptides for milk, cheeses, flour, meat, wine and beer; triacylglycerols and phospholipids for oils; and low-molecular-weight metabolites for wine, beer and chocolate. Special attention is paid to sample preparation techniques and the combination of spectral profiling and statistical evaluation methods, which is powerful for the differentiation of samples and the sensitive detection of frauds and adulterations

Pseudotrypsin: A Little-Known Trypsin Proteoform

Trypsin is the protease of choice for protein sample digestion in proteomics. The most typical active forms are the single-chain β-trypsin and the two-chain α-trypsin, which is produced by a limited autolysis of β-trypsin. An additional intra-chain split leads to pseudotrypsin (ψ-trypsin) with three chains interconnected by disulfide bonds, which can be isolated from the autolyzate by ion-exchange chromatography. Based on experimental data with artificial substrates, peptides, and protein standards, ψ-trypsin shows altered kinetic properties, thermodynamic stability and cleavage site preference (and partly also cleavage specificity) compared to the above-mentioned proteoforms. In our laboratory, we have analyzed the performance of bovine ψ-trypsin in the digestion of protein samples with a different complexity. It cleaves predominantly at the characteristic trypsin cleavage sites. However, in a comparison with common tryptic digestion, non-specific cleavages occur more frequently (mostly after the aromatic residues of Tyr and Phe) and more missed cleavages are generated. Because of the preferential cleavages after the basic residues and more developed side specificity, which is not expected to occur for the major trypsin forms (but may appear anyway because of their autolysis), ψ-trypsin produces valuable information, which is complementary in part to data based on a strictly specific trypsin digestion and thus can be unnoticed following common proteomics protocols

The Influence of Metabolic Inhibitors, Antibiotics, and Microgravity on Intact Cell MALDI-TOF Mass Spectra of the Cyanobacterium Synechococcus Sp. UPOC S4

The aim and novelty of this paper are found in assessing the influence of inhibitors and antibiotics on intact cell MALDI-TOF mass spectra of the cyanobacterium Synechococcus sp. UPOC S4 and to check the impact on reliability of identification. Defining the limits of this method is important for its use in biology and applied science. The compounds included inhibitors of respiration, glycolysis, citrate cycle, and proteosynthesis. They were used at 1–10 μM concentrations and different periods of up to 3 weeks. Cells were also grown without inhibitors in a microgravity because of expected strong effects. Mass spectra were evaluated using controls and interpreted in terms of differential peaks and their assignment to protein sequences by mass. Antibiotics, azide, and bromopyruvate had the greatest impact. The spectral patterns were markedly altered after a prolonged incubation at higher concentrations, which precluded identification in the database of reference spectra. The incubation in microgravity showed a similar effect. These differences were evident in dendrograms constructed from the spectral data. Enzyme inhibitors affected the spectra to a smaller extent. This study shows that only a long-term presence of antibiotics and strong metabolic inhibitors in the medium at 10−5 M concentrations hinders the correct identification of cyanobacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF)

Purification of Maize Nucleotide Pyrophosphatase/Phosphodiesterase Casts Doubt on the Existence of Zeatin Cis–Trans Isomerase in Plants

Almost 25 years ago, an enzyme named zeatin cis–trans isomerase from common bean has been described by Bassil et al. (1993). The partially purified enzyme required an external addition of FAD and dithiothreitol for the conversion of cis-zeatin to its trans- isomer that occurred only under light. Although an existence of this important enzyme involved in the metabolism of plant hormones cytokinins was generally accepted by plant biologists, the corresponding protein and encoding gene have not been identified to date. Based on the original paper, we purified and identified an enzyme from maize, which shows the described zeatin cis–trans isomerase activity. The enzyme belongs to nucleotide pyrophosphatase/phosphodiesterase family, which is well characterized in mammals, but less known in plants. Further experiments with the recombinant maize enzyme obtained from yeast expression system showed that rather than the catalytic activity of the enzyme itself, a non-enzymatic flavin induced photoisomerization is responsible for the observed zeatin cis–trans interconversion in vitro. An overexpression of the maize nucleotide pyrophosphatase/phosphodiesterase gene led to decreased FAD and increased FMN and riboflavin contents in transgenic Arabidopsis plants. However, neither contents nor the ratio of zeatin isomers was altered suggesting that the enzyme is unlikely to catalyze the interconversion of zeatin isomers in vivo. Using enhanced expression of a homologous gene, functional nucleotide pyrophosphatase/phosphodiesterase was also identified in rice

Identification of cisplatin-binding sites on the large cytoplasmic loop of the Na+/K+-ATPase

Cisplatin is the most widely used chemotherapeutic drug for the treatment of various types of cancer; however, its administration brings also numerous side effects. It was demonstrated that cisplatin can inhibit the Na+/K+-ATPase (NKA), which can explain a large part of the adverse effects. In this study, we have identified five cysteinyl residues (C452, C456, C457, C577, and C656) as the cisplatin binding sites on the cytoplasmic loop connecting transmembrane helices 4 and 5 (C45), using site-directed mutagenesis and mass spectrometry experiments. The identified residues are known to be susceptible to glutathionylation indicating their involvement in a common regulatory mechanism

Heteroleptic Copper(II) Complexes Containing 2′-Hydroxy-4-(Dimethylamino)Chalcone Show Strong Antiproliferative Activity

A series of six heteroleptic copper(II) complexes with 2′-hydroxy-4-(dimethylamino)chalcone (HL) with the composition [Cu(N-N)(L)]NO3 (1–6), where N-N stands for dmbpy = 5,5′-dimethyl-2,2′-bipyridine (1), bphen = 4,7-diphenyl-1,10-phenanthroline (2), dbbpy = 4,4′-di-tert-butyl-2,2′-bipyridine (3), nphen = 5-nitro-1,10-phenanthroline (4), bpy = 2,2′-bipyridine, (5), and dpa = 2,2′-dipyridylamine (6), was prepared and thoroughly characterized. The in vitro cytotoxicity screening on eight human cancer cell lines identified complex 2, containing the bulkiest N-donor ligands (bphen) as highly cytotoxic against cancer cells, with IC50 values ranking from 1.0 to 2.3 μM, with good selectivity and low toxicity against healthy human fetal lung fibroblasts MRC-5. The cell-based assays, involving the most effective complex 2 in A2780 cancer cells, revealed its strong pro-apoptotic effects based on the effective activation of caspases 3/7, ROS overproduction, and autophagy in the A2780 cells while not impeding the cell cycle and mitochondrial membrane functions. The cellular uptake studies in A2780 and 22Rv1 cells uncovered no intracellular transport of the cationic complex 2, supporting the hypothesis that the in vitro anticancer effects of complex 2 are based on the combined extrinsic activation of apoptosis and autophagy induction

Heteroleptic Copper(II) Complexes Containing 2′-Hydroxy-4-(Dimethylamino)Chalcone Show Strong Antiproliferative Activity

A series of six heteroleptic copper(II) complexes with 2′-hydroxy-4-(dimethylamino)chalcone (HL) with the composition [Cu(N-N)(L)]NO3 (1–6), where N-N stands for dmbpy = 5,5′-dimethyl-2,2′-bipyridine (1), bphen = 4,7-diphenyl-1,10-phenanthroline (2), dbbpy = 4,4′-di-tert-butyl-2,2′-bipyridine (3), nphen = 5-nitro-1,10-phenanthroline (4), bpy = 2,2′-bipyridine, (5), and dpa = 2,2′-dipyridylamine (6), was prepared and thoroughly characterized. The in vitro cytotoxicity screening on eight human cancer cell lines identified complex 2, containing the bulkiest N-donor ligands (bphen) as highly cytotoxic against cancer cells, with IC50 values ranking from 1.0 to 2.3 μM, with good selectivity and low toxicity against healthy human fetal lung fibroblasts MRC-5. The cell-based assays, involving the most effective complex 2 in A2780 cancer cells, revealed its strong pro-apoptotic effects based on the effective activation of caspases 3/7, ROS overproduction, and autophagy in the A2780 cells while not impeding the cell cycle and mitochondrial membrane functions. The cellular uptake studies in A2780 and 22Rv1 cells uncovered no intracellular transport of the cationic complex 2, supporting the hypothesis that the in vitro anticancer effects of complex 2 are based on the combined extrinsic activation of apoptosis and autophagy induction