Comparative Analysis of Catalase Activity in Plants: Spectrophotometry and Native PAGE Approaches.

Catalase, a pivotal enzyme in plant antioxidative defense mechanisms, plays a crucial role in detoxifying hydrogen peroxide, a reactive oxygen species (ROS). In this chapter, a comparative analysis of catalase activity was conducted using two distinct methodologies: spectrophotometry and non-denaturing polyacrylamide gel electrophoresis (PAGE). The spectrophotometric approach allowed the quantification of catalase activity by measuring the breakdown rate of hydrogen peroxide, while native PAGE enabled the separation and visualization of catalase isozymes, based on their native molecular weight and charge characteristics, and specific staining assay. Both methods provide valuable insights into catalase activity, offering complementary information on the enzyme's functional diversity and distribution within different plant tissues. This study integrates different techniques, previously described, to comprehensively elucidate the role of catalase in plant metabolism. Furthermore, it provides the possibility of obtaining a holistic understanding of antioxidant defense mechanisms by considering both total activity and isoenzyme distribution of catalase enzyme.

An efficient protocol for extracting thylakoid membranes and total leaf proteins from Posidonia oceanica and other polyphenol-rich plants.

BACKGROUND: The extraction of thylakoids is an essential step in studying the structure of photosynthetic complexes and several other aspects of the photosynthetic process in plants. Conventional protocols have been developed for selected land plants grown in controlled conditions. Plants accumulate defensive chemical compounds such as polyphenols to cope with environmental stresses. When the polyphenol levels are high, their oxidation and cross-linking properties prevent thylakoid extraction. RESULTS: In this study, we developed a method to counteract the hindering effects of polyphenols by modifying the grinding buffer with the addition of both vitamin C (VitC) and polyethylene glycol (PEG4000). This protocol was first applied to the marine plant Posidonia oceanica and then extended to other plants synthesizing substantial amounts of polyphenols, such as Quercus pubescens (oak) and Vitis vinifera (grapevine). Native gel analysis showed that photosynthetic complexes (PSII, PSI, and LHCII) can be extracted from purified membranes and fractionated comparably to those extracted from the model plant Arabidopsis thaliana. Moreover, total protein extraction from frozen P. oceanica leaves was also efficiently carried out using a denaturing buffer containing PEG and VitC. CONCLUSIONS: Our work shows that the use of PEG and VitC significantly improves the isolation of native thylakoids, native photosynthetic complexes, and total proteins from plants containing high amounts of polyphenols and thus enables studies on photosynthesis in various plant species grown in natural conditions.

Investigation of Foreign Amylase Adulteration in Honey Distributed in Japan by Rapid and Improved Native PAGE Activity Staining Method.

Foreign amylase addition to honey in an effort to disguise diastase activity has become a widespread form of food fraud. However, since there is no report on the investigation in Japan, we investigated foreign amylases in 67 commercial honeys in Japan. First, the α-glucosidase and diastase activities of honeys were measured, which revealed that only α-glucosidase activity was significantly low in several samples. As both enzymes are secreted from honeybee glands, it is unlikely that only one enzyme was inactivated during processing. Therefore, we suspected the presence of foreign amylase. α-Amylase in honey were assigned using protein analysis software based on LC-QTOF-MS. As a result, α-amylases from Aspergillus and Geobacillus were detected in 13 and 6 out of 67 honeys, respectively. To detect foreign amylases easily, we developed a cost-effective method using native PAGE. Conventional native PAGE failed to separate the α-amylase derived from honeybee and Geobacillus. However, when native PAGE was performed using a gel containing 1 % maltodextrin, the α-amylase from honeybee did not migrated in the gel and the α-amylase could be separated from the other two α-amylases. The results from this method were consistent with those of LC-QTOF-MS method, suggesting that the novel native PAGE method can be used to detect foreign amylases.

Purification and characterization of extracellular lipase from a thermotolerant strain: Bacillus subtilis TTP-06.

In current study, lipase from a thermotolerant Bacillus subtilis TTP-06 was purified in a stepwise manner by using ammonium sulfate precipitation and column chromatography. Thenceforth, it was subjected to sodium dodecyl sulfate- and native-polyacrylamide gel electrophoresis to check the homogeneity of the purified enzyme. The ideal substrate concentration, pH, temperature, reaction duration and lipase specificity were identified. With a yield of 11.02%, purified lipase displayed activity of 8.51 U/mg. Thenceforward, the homogeneously purified enzyme was considered to be a homo-dimer of 30 kDa subunits. Enzyme had Km and Vmax value of 9.498 mM and 19.92 mol mg-1 min-1, respectively. Additionally, the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method was used to investigate the purified lipase and estimate its 3-D structure, which revealed a catalytic triad of serine, aspartate and histidine.

Conformational change in an engineered biliverdin-binding cyanobacteriochrome during the photoconversion process.

Cyanobacteriochromes (CBCRs) derived from cyanobacteria are linear-tetrapyrrole-binding photoreceptors related to the canonical red/far-red reversible phytochrome photoreceptors. CBCRs contain chromophore-binding cGMP-specific phosphodiesterase/adenylate cyclase/FhlA (GAF) domains that are highly diverse in their primary sequences and are categorized into many subfamilies. Among this repertoire, the biliverdin (BV)-binding CBCR GAF domains receive considerable attention for their in vivo optogenetic and bioimaging applications because BV is a mammalian intrinsic chromophore and can absorb far-red light that penetrates deep into the mammalian body. The typical BV-binding CBCR GAF domain exhibits reversible photoconversion between far-red-absorbing dark-adapted and orange-absorbing photoproduct states. Herein, we applied various biochemical and spectral studies to identify the details of the conformational change during this photoconversion process. No oligomeric state change was observed, whereas the surface charge would change with a modification of the α-helix structures during the photoconversion process. Combinatorial analysis using partial protease digestion and mass spectrometry identified the region where the conformational change occurred. These results provide clues for the future development of optogenetic tools.

Use of Native-PAGE for the Identification of Epichaperomes in Cell Lines.

Epichaperomes are disease-associated pathologic scaffolds, composed of tightly bound chaperones, co-chaperones, and other factors. They mediate anomalous protein-protein interactions inside cells, which aberrantly affects the function of protein networks, and in turn, cellular phenotypes. Epichaperome study necessitates the implementation of methods that retain these protein complexes in their native cellular states for analysis. Here we describe a protocol for detection and composition analysis of epichaperomes in cell homogenates through native polyacrylamide gel electrophoresis.

Iron Accumulation in Ferritin.

Understanding the iron accumulation process in ferritin protein nanocages has remained a centerpiece in the field of iron biochemistry/biomineralization, which ultimately has implications in health and diseases. Although mechanistic differences of iron acquisition and mineralization exist in the superfamily of ferritins, we describe the techniques that can be used to investigate the accumulation of iron in all the ferritin proteins by in vitro iron mineralization process. In this chapter, we report that the non-denaturing polyacrylamide gel electrophoresis coupled with Prussian blue staining (in-gel assay) can be useful to investigate the iron-loading efficiency in ferritin protein nanocage, by estimating the relative amount of iron incorporated inside it. Similarly, the absolute size of the iron mineral core and the amount of total iron accumulated inside its nanocavity can be determined by using transmission electron microscopy and spectrophotometry, respectively.

Isolation of Functional Mitochondria and Pure mtDNA from Murine Tissues.

Detailed analysis of mitochondrial function cannot be achieved without good quality preparations of isolated mitochondria. Ideally, the isolation protocol should be quick, while producing a reasonably pure pool of mitochondria that are still intact and coupled. Here, we describe a fast and simple method for the purification of mammalian mitochondria relying on isopycnic density gradient centrifugation. We describe specific steps that should be taken into consideration when functional mitochondria from different tissues should be isolated. This protocol is suitable for the analysis of many aspects of the organelle's structure and function.

Structural Characterization of Murine Phosphodiesterase 5 Isoforms and Involvement of Cysteine Residues in Supramolecular Assembly.

Phosphodiesterases (PDEs) are a superfamily of evolutionarily conserved cyclic nucleotide (cAMP/cGMP)-hydrolyzing enzymes, components of transduction pathways regulating crucial aspects of cell life. Within this family, the cGMP-dependent PDE5 is the major hydrolyzing enzyme in many mammalian tissues, where it regulates a number of cellular and tissular processes. Using Kluyveromyces lactis as a model organism, the murine PDE5A1, A2 and A3 isoforms were successfully expressed and studied, evidencing, for the first time, a distinct role of each isoform in the control, modulation and maintenance of the cellular redox metabolism. Moreover, we demonstrated that the short N-terminal peptide is responsible for the tetrameric assembly of MmPDE5A1 and for the mitochondrial localization of MmPDE5A2. We also analyzed MmPDE5A1, A2 and A3 using small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), structural mass spectrometry (MS) and polyacrylamide gel electrophoresis in their native conditions (native-PAGE) and in the presence of redox agents. These analyses pointed towards the role of a few specific cysteines in the isoforms' oligomeric assembly and the loss of enzymatic activity when modified.

Effector-dependent activation and oligomerization of plant NRC class helper NLRs by sensor NLR immune receptors Rpi-amr3 and Rpi-amr1.

Plant pathogens compromise crop yields. Plants have evolved robust innate immunity that depends in part on intracellular Nucleotide-binding, Leucine rich-Repeat (NLR) immune receptors that activate defense responses upon detection of pathogen-derived effectors. Most "sensor" NLRs that detect effectors require the activity of "helper" NLRs, but how helper NLRs support sensor NLR function is poorly understood. Many Solanaceae NLRs require NRC (NLR-Required for Cell death) class of helper NLRs. We show here that Rpi-amr3, a sensor NLR from Solanum americanum, detects AVRamr3 from the potato late blight pathogen, Phytophthora infestans, and activates oligomerization of helper NLRs NRC2 and NRC4 into high-molecular-weight resistosomes. In contrast, recognition of P. infestans effector AVRamr1 by another sensor NLR Rpi-amr1 induces formation of only the NRC2 resistosome. The activated NRC2 oligomer becomes enriched in membrane fractions. ATP-binding motifs of both Rpi-amr3 and NRC2 are required for NRC2 resistosome formation, but not for the interaction of Rpi-amr3 with its cognate effector. NRC2 resistosome can be activated by Rpi-amr3 upon detection of AVRamr3 homologs from other Phytophthora species. Mechanistic understanding of NRC resistosome formation will underpin engineering crops with durable disease resistance.

In-Plate and In-Gel Assays for the Assessment of Proteasome Activity in Caenorhabditis elegans.

This chapter describes two methods for the study of proteasome function in Caenorhabditis elegans (C. elegans). The first method, referred to as "in-plate activities," provides a quantitative measurement of proteasome activities in C. elegans lysates by means of a kinetic reaction in a 96-well plate. The second one, referred to as "in-gel activities," involves the separation of C. elegans protein lysates in a native polyacrylamide gel and the assessment of the activity of each proteasome form. Downstream immunoblotting also allows the semi-quantitative assessment of proteasome assembly. This chapter outlines two detailed protocols along with helpful schematics and representative results that will facilitate researchers to replicate both protocols accurately and reproducibly.

Predicting the bioremediation potential of earthworms of different ecotypes through a multi-biomarker approach.

Earthworms are attracting the attention of bioremediation research because of their short-term impact on pollutant fate. However, earthworm-assisted bioremediation largely depends on the earthworm sensitivity to target pollutants and its metabolic capacity to break down contaminants. The most studied species in soil bioremediation has been Eisenia fetida, which inhabits the soil surface feeding on decomposing organic residues. Therefore, its bioremediation potential may be limited to organic matter-rich topsoil. We compared the detoxification potential against organophosphate (OP) pesticides of three earthworm species representative of the main ecotypes: epigeic, anecic, and endogeic. Selected biomarkers of pesticide detoxification (esterases, cytochrome P450-dependent monooxygenase, and glutathione S-transferase) and oxidative homeostasis (total antioxidant capacity, glutathione levels, and glutathione reductase [GR] and catalase activities) were measured in the muscle wall and gastrointestinal tract of E. fetida (epigeic), Lumbricus terrestris (anecic) and Aporrectodea caliginosa (endogeic). Our results show that L. terrestris was the most suitable species to bioremediate OP-contaminated soil for the following reasons: 1) Gut carboxylesterase (CbE) activity of L. terrestris was higher than that of E. fetida, whereas muscle CbE activity was more sensitivity to OP inhibition than that of E. fetida, which means a high capacity to inactivate the toxic oxon metabolites of OPs. 2) Muscle and gut phosphotriesterase activities were significantly higher in L. terrestris than in the other species. 3) Enzymatic (catalase and GR) and molecular mechanisms of free radical inactivation (glutathione) were 3- to 4-fold higher in L. terrestris concerning E. fetida and A. caliginosa, which reveals a higher potential to keep the cellular oxidative homeostasis against reactive metabolites formed during OP metabolism. Together with biological and ecological traits, these toxicological traits suggest L. terrestris a better candidate for soil bioremediation than epigeic earthworms.

Gamma (γ)-radiation stress response of the cyanobacterium Anabaena sp. PCC7120: Regulatory role of LexA and photophysiological changes.

High radioresistance of the cyanobacterium, Anabaena sp. PCC7120 has been attributed to efficient DNA repair, protein recycling, and oxidative stress management. However, the regulatory network involved in these batteries of responses remains unexplored. In the present study, the role of a global regulator, LexA in modulating gamma (γ)-radiation stress response of Anabaena was investigated. Comparison of the cytosolic proteome profiles upon γ-radiation in recombinant Anabaena strains, AnpAM (vector-control) and AnlexA+ (LexA-overexpressing), revealed 41 differentially accumulated proteins, corresponding to 29 distinct proteins. LexA was found to be involved in the regulation of 27 of the corresponding genes based on the presence of AnLexA-Box, EMSA, and/or qRT-PCR studies. The majority of the regulated genes were found to be involved in C-assimilation either through photosynthesis or C-catabolism and oxidative stress alleviation. Photosynthesis, measured in terms of PSII photophysiological parameters and thylakoid membrane proteome was found to be affected by γ-radiation in both AnpAM and AnlexA+ cells, with LexA affecting them even under control growth conditions. Thus, LexA functioned as one of the transcriptional regulators involved in modulating γ-radiation stress response in Anabaena. This study could pave the way for a deeper understanding of the regulation of γ-radiation-responsive genes in cyanobacteria at large.

Native DIGE for Quantitative and Functional Analysis of Protein Interactomes.

Protein-protein interactions and multiprotein assemblies of water-soluble and membrane proteins are inherent features of the proteome, which also impart functional heterogeneity. One needs to consider this aspect while studying changes in abundance and activities of proteins in response to any physiological stimulus. Abundance changes in the components of a given proteome can be best visualized and efficiently quantified using electrophoresis-based approaches. Here, we describe the method of Blue Native Difference Gel Electrophoresis to quantify changes in abundance and activity of proteins in the context of protein-protein interactions. This method confers an additional advantage to monitor quantitative changes in membrane proteins, which otherwise is a difficult task.

Zymography assisted quick purification, characterization and inhibition analysis of K. pneumoniae alkaline phosphatase by mercury and thiohydroxyal compounds.

In-gel hydrolysis of para-nitrophenyl phosphate (p-NPP) to yellow colored para-nitrophenol was used to locate precisely the K. pneumoniae alkaline phosphatase (Kp-ALKP) on 7% native PAGE. Subsequent removal of the yellow-stained band and electroelution yielded a 54 kDa, Kp-ALKP with Km, Vmax and kcat values of (0.7 ± 0.02) mM, (80 ± 4.5) µmol min-1 and (39.2 ± 2.2) × 104 s-1 respectively for p-NPP. Kp-ALKP was optimally active at 70 °C and pH 7.2 that was activated by Mg2+, Ca2+, Co2+ and inhibited by EDTA, PO4, Pb2+, Cu2+ and Hg2+. The enzyme was trypsin resistant and retained 75% activity in presence of 10 mM PO4 and 65% activity at 3 mM Hg2+ showing it's PO43- irrepressibility and Hg2+-tolerance. Molecular dynamics simulation revealed increased structural stability of Kp-ALKP at 70 °C that accounts for it's optimal temperature. Zymography revealed that both DTT and ß-mercaptoethanol induced activity loss accompanied by mobility retardation of Kp-ALKP on 7% native PAGE. These results and in Silico analysis shows that both DTT and ßME reduce the C308-C358 disulfide bond, leading to an open conformation of the enzyme. However, Hg2+ had negligible effect on the in-gel mobility of Kp-ALKP indicating it's plausible non-covalent interaction with surface-accessible amino-acids without significant conformational change. For the first time our study reveals the zymography as an easy, inexpensive and convenient tool for quick purification, characterization and conformational analysis of K. pneumoniae alkaline phosphatase.

The effect of radiofrequency heat treatment on trypsin inhibitor activity and in vitro digestibility of soybean varieties (Glycine max. (L.) Merr.).

Kunitz (KTI) and Bowman-Birk (BBI) trypsin inhibitors were characterized in soybean seeds. Cultivars having KTI/BBI (Pannónia Kincse, PK) or lacking KTI (Aries; Hilario; Bahia) were assessed with well-characterized soybean varieties having Ti-a or ti types of KTI mobility. The TIA values of Pannónia Kincse (9.8 ± 0.48 mg/g) were not significantly different (p ≤ 0.05) from Ti-a samples (10.07 ± 1.86 mg/g), while of Aires, Bahia, Hilario (6.19 ± 1.89) were identical (p ≤ 0.05) with ti samples (6.63 ± 1.99). Radiofrequency heat treatment (RF) decreased TIA values (p ≤ 0.05) at ≥ 100 °C. However, in the traditional soybean variety, the RF at 110 °C was more effective in eliminating the residual KTI activity. The remaining or the disapperaing bioactive form of trypsin inhibitors were succesfully characterized by the means of a standardized in vitro digestion model. It showed that residual BBI-originated trypsin inhibitor activity was in the stomach even after RF at 110 °C, whereas its chymotrypsin inhibitor activity was not detectable at all. Although PK and KTI null types of soybean seeds still required an energy-saving, gentle heat treatment to inactive the trypsin inhibitors before using them as food or feed, the physicochemical properties and processing quality of soybean products were protected, improved.

Morphological and structural characterization of chitin as a substrate for the screening, production, and molecular characterization of chitinase by Bacillus velezensis.

The processing of shellfishery industrial wastes is gaining much interest in recent times due to the presence of valuable components. Chitin is one of the valuable components and is insoluble in most common solvents including water. In this study, a novel gram-positive bacterial strain capable of solubilizing chitin was screened from a prawn shell dumping yard. The chitinolytic activity of the isolated strain was observed through the zone of hydrolysis plate assay. The hyper-producing isolate was identified as Bacillus velezensis through the 16S rRNA sequencing technique. The structural and morphological characterization of raw and colloidal chitin preparation was carried out using FTIR, XRD, and SEM analysis. The residual protein and mineral content, degree of polymerization, and degree of acetylation were reported for both raw and colloidal chitin preparations. There was a linear increase in the chitinase activity with an increase in the colloidal chitin concentration. The maximum activity of chitinase was observed as 38.98 U/mL for the initial colloidal chitin concentration of 1.5%. Supplement of additional carbon sources, viz., glucose and maltose, did not improve the production of chitinase and resulted in a diauxic growth pattern. The maximum chitinase activity was observed to be 33.10 and 30.28 U/mL in the colloidal chitin-containing medium with and without glucose as a secondary carbon source, respectively. Interestingly, the addition of complex nitrogen sources has increased the production of chitinase. A 1.95- and 2.14-fold increase in the enzyme activity was observed with peptone and yeast extract, respectively. The chitinase was confirmed using SDS-PAGE, native PAGE, and zymograms. The optimum pH and temperature for chitinase enzyme activity were found to be 7.0 and 44 °C, respectively.

Oligomeric state of the aspartate:alanine transporter from Tetragenococcus halophilus.

The aspartate:alanine exchanger family of membrane transporters includes industrially important transporters such as succinate exporter and glutamate exporter. No high-resolution structure is available from this family so far, and the transport mechanism of these transporters also remains unclear. In the present study, we focus on the oligomeric status of the aspartate:alanine antiporter (AspT) of Tetragenococcus halophilus, which is the prototype of this family. To investigate the oligomeric structure of AspT, we established a system that produces high yields of highly purified AspT and determined the oligomeric structure of AspT by analysis with size exclusion chromatography coupled with multi-angle light scattering and blue native PAGE and by comparison of the wild-type AspT with a single-cysteine mutant that forms spontaneous inter-molecular thiol crosslinking. All the results consistently support the notion that AspT is a homodimer in solutions and in membranes.

A screening method for binding synthetic metallo-complexes to haem proteins.

The introduction of a second coordination sphere, in the form of a protein scaffold, to synthetic catalysts can be beneficial for their reactivity and substrate selectivity. Here we present semi-native polyacrylamide gel electrophoresis (semi-native PAGE) as a rapid screening method for studying metal complex-protein interactions. Such a screening is generally performed using electron spray ionization mass spectrometry (ESI-MS) and/or UV-Vis spectroscopy. Semi-native PAGE analysis has the advantage that it does not rely on spectral changes of the metal complex upon protein interaction and can be applied for high-throughput screening and optimization of complex binding. In semi-native PAGE non-denatured protein samples are loaded on a gel containing sodium dodecyl sulphate (SDS), leading to separation based on differences in structural stability. Semi-native PAGE gel runs of catalyst-protein mixtures were compared to gel runs obtained with native and denaturing PAGE. ESI-MS was additionally realised to confirm protein-complex binding. The general applicability of semi-native PAGE was investigated by screening the binding of various cobalt- and ruthenium-based compounds to three types of haem proteins.

Effect of Senna plant on the mitochondrial activity of Hymenolepis diminuta.

The peculiarity of energy metabolism in helminths is the ability to undergo transition from aerobic to anaerobic under low oxygen tension. during its adult stage. Fumarate reductase and succinate dehydrogenase of mitochondria are the two enzymes responsible during this transition and adaptation to this hypoxic environment. Earlier we had reported that three species of Senna plant, S. alata, S. alexandrina and S. occidentalis altered the morphology, ionic concentration and neurotransmission of the cestode parasite Hymenolepis diminuta. The present study aimed at exploring the mechanism of leaf extracts of the three plant species of Senna on the mitochondrial activity of the parasite that chiefly involve the NADH-fumarate reductase system which is the terminal step in phosphoenolpyruvate carboxykinase succinate pathway. The structure of mitochondria was observed through electron microsopy and its density was detected through confocal microscopy, spectroflourimetry and spectrophotometry, while enzyme activities were assayed through native gel and spectrophotometric assays. Praziquantel was tested on the parasites as a reference drug to compare its effects with that of the plant extracts. The mitochondria architecture was altered, and enzymes activity decraeased by 60% in all three plant species of Senna treated parasites which suggested that these three Senna species posses potent chemotherapeutic properties. Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-021-01415-9.