Sławomir Orzechowski

A novel, simple, and sensitive colorimetric method to determine aromatic amino acid aminotransferase activity using the Salkowski reagent

This study describes the development of a new colorimetric assay to determine aromatic amino acid aminotransferase (ArAT) activity. The assay is based on the transamination of l-tryptophan in the presence of 2-oxoglutarate, which yields indole-3-pyruvate (IPyA). The amount of IPyA formed was quantified by reaction with the Salkowski reagent. Optimized assay conditions are presented for ArAT isozymes isolated from Pseudomonas putida. For comparative purposes, ArAT activity was also determined by high-performance liquid chromatography. ArAT activity staining in polyacrylamide gels with the Salkowski reagent is also presented.

Starch phosphorylation: insights and perspectives

During starch metabolism, the phosphorylation of glucosyl residues of starch, to be more precise of amylopectin, is a repeatedly observed process. This phosphorylation is mediated by dikinases, the glucan, water dikinase (GWD) and the phosphoglucan, water dikinase (PWD). The starch-related dikinases utilize ATP as dual phosphate donor transferring the terminal γ-phosphate group to water and the β-phosphate group selectively to either C6 position or C3 position of a glucosyl residue within amylopectin. By the collaborative action of both enzymes, the initiation of a transition of α-glucans from highly ordered, water-insoluble state to a less order state is realized and thus the initial process of starch degradation. Consequently, mutants lacking either GWD or PWD reveal a starch excess phenotype as well as growth retardation. In this review, we focus on the increased knowledge collected over the last years related to enzymatic properties, the precise definition of the substrates, the physiological implications, and discuss ongoing questions.

Analysis of the expression, subcellular and tissue localisation of phosphoglucan, water dikinase (PWD/GWD3) in Solanum tuberosum L.: a bioinformatics approach for the comparative analysis of two α-glucan, water dikinases (GWDs) from Solanum tuberosum L.

There are several important factors affecting the rate of starch decomposition in plants, including the circadian clock, the regulation of gene expression, the regulation of enzyme activities and starch phosphorylation by glucan, water dikinase activities (GWDs). One isoform of glucan, water dikinase named GWD3 or PWD (EC 2.7.9.5) was isolated for the first time from Arabidopsis thaliana, and now we report its isolation and identification in Solanum tuberosum L. leaves and tubers. We compare StGWD3 sequence to the other GWDs sequences using bioinformatics tools and propose also structural models for the starch-binding domains in StGWD3 and StGWD1. The StGWD3 gene expression and protein were localised in different heterotrophic and autotrophic potato tissues and organs using in situ RT-PCR and immunolocalisation methods, respectively. Diurnal changes in the transcript abundance of StGWD3 in leaves were analysed using quantitative real-time PCR and they appeared to be typical for most genes involved in starch degradation in chloroplasts.

Reduction of the plastidial phosphorylase in potato (Solanum tuberosum L.) reveals impact on storage starch structure during growth at low temperature.

Tubers of potato (Solanum tuberosum L.), one of the most important crops, are a prominent example for an efficient production of storage starch. Nevertheless, the synthesis of this storage starch is not completely understood. The plastidial phosphorylase (Pho1; EC 2.4.1.1) catalyzes the reversible transfer of glucosyl residues from glucose-1-phosphate to the non-reducing end of α-glucans with the release of orthophosphate. Thus, the enzyme is in principle able to act during starch synthesis. However, so far under normal growth conditions no alterations in tuber starch metabolism were observed. Based on analyses of other species and also from in vitro experiments with potato tuber slices it was supposed, that Pho1 has a stronger impact on starch metabolism, when plants grow under low temperature conditions. Therefore, we analyzed the starch content, granule size, as well as the internal structure of starch granules isolated from potato plants grown under low temperatures. Besides wild type, transgenic potato plants with a strong reduction in the Pho1 activity were analyzed. No significant alterations in starch content and granule size were detected. In contrast, when plants were cultivated at low temperatures the chain length distributions of the starch granules were altered. Thus, the granules contained more short glucan chains. That was not observed in the transgenic plants, revealing that Pho1 in wild type is involved in the formation of the short glucan chains, at least at low temperatures.

A triticale water-deficit-inducible phytocystatin inhibits endogenous cysteine proteinases in vitro

Water-deficit is accompanied by an increase in proteolysis. Phytocystatins are plant inhibitors of cysteine proteinases that belong to the papain and legumain family. A cDNA encoding the protein inhibitor TrcC-8 was identified in the vegetative organs of triticale. In response to water-deficit, increases in the mRNA levels of TrcC-8 were observed in leaf and root tissues. Immunoblot analysis indicated that accumulation of the TrcC-8 protein occurred after 72h of water-deficit in the seedlings. Using recombinant protein, inhibitory activity of TrcC-8 against cysteine proteases from triticale and wheat tissues was analyzed. Under water-deficit conditions, there are increases in cysteine proteinase activities in both plant tissues. The cysteine proteinase activities were inhibited by addition of the recombinant TrcC-8 protein. These results suggest a potential role for the triticale phytocystatin in modulating cysteine proteinase activities during water-deficit conditions.

Analysis of expression and inhibitory activity of a TrcC-6 phytocystatin present in developing and germinating seeds of triticale (×Triticosecale Wittm.).

Storage proteins of cereal seeds are processed during accumulation and degraded during germination primarily by cysteine proteinases. One of the mechanisms controlling the activity of these enzymes is the synthesis of specific inhibitors named phytocystatins. Here we present the complete gene sequence of a triticale ( × Triticosecale Wittm.) phytocystatin, TrcC-6, which encodes a 152-amino acid protein with a putative 25-amino acid signal peptide. This protein has a calculated molecular mass of 16.2 kDa, and was assigned to phylogenetic group B of phytocystatins. Because TrcC-6 transcripts are present in triticale seeds, we hypothesized that this phytocystatin regulates storage protein accumulation and degradation. Therefore, changes in gene expression during the entire period of seed development and germination were examined. TrcC-6 transcripts and TrcC-6 protein levels increased during the maturation of seeds and remained high during the first hours of germination. This enabled us to conclude that TrcC-6 likely regulates seed germination by the regulation of storage protein hydrolysis. For the analysis of TrcC-6 inhibitory activity, recombinant protein was expressed in Escherichia coli BL21 (DE3) and purified. Recombinant TrcC-6 proved to be a potent inhibitor of cysteine proteinases. It inhibited the in vitro activity of papain (EC 3.4.22.2) and ficin (EC 3.4.22.3). Furthermore, native PAGE analysis revealed that recombinant TrcC-6 inhibits the activity of endogenous cysteine proteinases present in germinating seeds of triticale. Based on these results, TrcC-6 is likely one of the important factors that regulate cysteine proteinase activity during the accumulation and mobilization of storage proteins.

FUNCTIONAL ANALYSIS OF GENES

Summary The aim of this article is to present the current literature concerning the expression analysis and methods of functional characteristics of genes. The progress in the analysis of gene expression within cells or whole tissues is undisputed and leads to a constant improvement of our understanding of the function of particular gene. The traditional methods of the functional characteristics of genes such as homology, inactivation and overexpression are more frequently being replaced by microarray and DNA chip analysis, which are extensively supported by bioinformatics tools. Knowledge of the functions and changes in gene expression has applications in medical diagnostics, the pharmaceutical industry and in plant and animal biotechnology.

Is quantity of protein in barley forms determined by proteins localized in the subaleurone layer

Barley biotypes from the world collection differ in their storage protein content even till 200 %. This is the first report including results of the research, in which the structure of grains containing different amount of protein was tested to explain this difference. The endosperm was investigated using scanning electron microscopy. The structure of the aleurone layer, storing large quantities of protein, did not differ between the high- and low-protein forms of barley. It has been proven that the large quantities of kernel protein may be stored in some cells of the zone adjacent to the aleurone layer, defined as the subaleurone cells. It has been shown that morphologically uniform kernels of the same plant and even of the same ear can vary greatly with respect to the number of these subaleurone cells. The purpose of the study was an examination of variation in protein structure in single kernels of a fodder, a brewery and in an extra high-protein form of barley as well. Moreover the studies were aimed to detect qualitative differences in the subaleurone protein. Application of mass spectrometry made possible the identification of several kinds of proteins which were present in subaleurne layer of kernels. In the granule-bound protein fraction isolated from the subaleurone type kernels, a much stronger representation of some protein was found, with the molecular mass between 29 and 45 kDa, in comparison with the low-protein kernels. It is supposed, that these protein are isoforms of z-type serpin and B3-hordein.

Subcellular distribution of alanine aminotransferase activity in maize (Zea mays L.) leaves

The intracellular distribution of alanine aminotransferase (AlaAT, EC 2.6.1.2) activity with L-alanine and 2-oxoglutarate as a substrates in maize whole leaf extract and bundle sheath cells was studied. After isolation of the mitochondrial-peroxisomal fraction, mitochondria and peroxisomes were separated by centrifugation on a linear 40–52 % (w/w) sucrose gradient. L-Alanine-2-oxoglutarate transaminating activity of whole leaf extract showed two peaks: first distinctly higher associated with mitochondria and second lower with peroxisomes. In bundle sheath cells only one peak of this activity was found. It corresponded to the mitochondrial region of the gradient. It is proposed that mitochondrial L-alanine — 2-oxoglutarate activity was brought about by AlaAT. Glycine aminotransferase (EC 2.6.1.4) could be responsible for the same activity in peroxisomes.

Purification and properties of alanine aminotransferase from maize [Zea mays L.] leaves

Alanine aminotransferase (AlaAT, EC 2.6.1.2) from leaves of 14-day-old maize seedlings was purified over 1600-fold to electrophoretical homogeneity. Specific activity of the purified enzyme measured with L-alanine and 2-oxoglutarate as substrates was 2125 nkat·(mg protein)−1 at 30 °C. The molecular weights of the native and sodium dodecyl sulfate — denatured AlaAT protein were 95 kDa and 50 kDa respectively, indicating that the native enzyme is probably a homodimer. AlaAT almost exclusively catalyzed amino group transfer from L-alanine to 2-oxoglutarate and the reverse reaction. The inhibitory experiments showed that pirydoxal phosphate is directly involved in the enzymatic catalysis and the enzyme molecule contains essential SH groups. The use of phenylglyoxal demonstrated the presence of arginine residue as anionic binding site in the active centre of AlaAT.