Philip L.R. Bonner

Factors influencing the free amino acid content of potato (Solanum tuberosum L) tubers during prolonged storage

Tubers of the potato cultivars Pentland Dell and Record were stored for a period of up to 40 weeks at 5°C and 10°C over four consecutive storage seasons (1989-1990 to 1992-1993). Reconditioning treatments at 20°C were also performed at early, mid and late stages in storage. To assess the turnover of proteins during storage, tubers were analysed for free amino acid and soluble protein content. The net direction of nitrogen flow was dependent on the state of dormancy of the tuber and hence storage duration. An increase in free amino acid content commonly occurred during the latter part of storage, caused by an upturn of proteinase activity on the break of dormancy. This increased enzyme activity was probably due to de novo synthesis of proteinases, in particular of a 47 kDa aspartic proteinase which was purified to homogeneity and shown to have a marked substrate-specificity for endogenous tuber proteins such as patatin. The rate of protein turnover increased with storage temperature, although the net direction of nitrogen flow was temperature independent. However, when nitrogen flow exhibited a distinct direction, such as the protein breakdown in Pentland Dell during late storage, this was enhanced by higher temperatures. The accumulation of free amino acids in Pentland Dell at 10°C corresponded with a deterioration of processing quality that could not be accounted for by an upturn in reducing sugar content. Despite an excess of free amino acids with respect to reducing sugars, amino acids had a probable synergistic influence on fry colour over the later stages of storage resulting in a darker colour per unit reducing sugar than in early storage. Reconditioning treatments were ineffective as a means of lowering the free amino acid pool size, these treatments operating solely through the decrease in reducing sugar content.

Effects of post-translational modifications catalysed by pollen transglutaminase on the functional properties of microtubules and actin filaments.

TGases (transglutaminases) are a class of calcium-dependent enzymes that catalyse the interactions between acyl acceptor glutamyl residues and amine donors, potentially making cross-links between proteins. To assess the activity of apple (Malus domestica) pollen TGase on the functional properties of actin and tubulin, TGase was prepared from apple pollen by hydrophobic- interaction chromatography and assayed on actin and tubulin purified from the same cell type. The enzyme catalysed the incorporation of putrescine into the cytoskeleton monomers. When tested on actin filaments, pollen TGase induced the formation of high-molecular-mass aggregates of actin. Use of fluorescein-cadaverine showed that the labelled polyamine was incorporated into actin by pollen TGase, similar to with guinea pig liver TGase. The pollen TGase also reduced the enzyme activity and the binding of myosin to TGase-treated actin filaments. Polymerization of tubulin in the presence of pollen TGase also yielded the formation of high-molecular-mass aggregates. Furthermore, the pollen TGase also affected the binding of kinesin to microtubules and reduced the motility of microtubules along kinesin-coated slides. These results indicate that the pollen TGase can control different properties of the pollen tube cytoskeleton (including the ability of actin and tubulin to assemble and their interaction with motor proteins) and consequently regulate the development of pollen tubes.

An extracellular transglutaminase is required for apple pollen tube growth

An extracellular form of the calcium-dependent protein-cross-linking enzyme TGase (transglutaminase) was demonstrated to be involved in the apical growth of Malus domestica pollen tube. Apple pollen TGase and its substrates were co-localized within aggregates on the pollen tube surface, as determined by indirect immunofluorescence staining and the in situ cross-linking of fluorescently labelled substrates. TGase-specific inhibitors and an anti-TGase monoclonal antibody blocked pollen tube growth, whereas incorporation of a recombinant fluorescent mammalian TGase substrate (histidine-tagged green fluorescent protein: His6-Xpr-GFP) into the growing tube wall enhanced tube length and germination, consistent with a role of TGase as a modulator of cell wall building and strengthening. The secreted pollen TGase catalysed the cross-linking of both PAs (polyamines) into proteins (released by the pollen tube) and His6-Xpr-GFP into endogenous or exogenously added substrates. A similar distribution of TGase activity was observed in planta on pollen tubes germinating inside the style, consistent with a possible additional role for TGase in the interaction between the pollen tube and the style during fertilization.

Aspects of amino acid metabolism in stored potato tubers (cv. Pentland Dell)

Tubers of the cv. Pentland Dell were stored at 5 and 10°C for up to 33 weeks and analysed for soluble protein and free amino acids. In addition, glutamine synthetase, NADH-GOGAT and acid proteinase activities were measured over the 33-week storage period. An accumulation of asparagine and glutamine occurred during late storage which coincided with an upturn in proteinase activity. The amide content of the major tuber storage proteins suggested that amidation of free amino acids must account for the high proportion of free amides. The activities of glutamine synthetase and NADH-GOGAT further indicated amidation of free amino acids throughout storage, independent of storage temperature. An increase of total soluble protein during storage suggested that much of the observed increase in amides resulted from translocation into the tuber core tissue, perhaps as a result of the chemical suppression of sprout growth.

Estimation of peptide concentration by a modified bicinchoninic acid assay

Although biuret based protein assays are theoretically applicable to peptide measurement, there is a high level of interpeptide variation, determined largely by peptide hydrophobicity. This variation in peptide reactivity can be significantly reduced by heat-denaturation of peptides at 95 degrees C for 5 min in the presence of 0.1 M NaOH containing 1% (w/v) SDS, prior to incubation for 30 min at 37 degrees C in BCA standard working reagent. This modification to the standard bicinchoninic acid (BCA) assay protocol allows for an accurate, rapid, and economical estimation of the peptide concentration within an unknown sample.

Assays for the measurement of tissue transglutaminase (type II) mediated protein crosslinking via ϵ-(γ-glutamyl) lysine and N′,N′-bis (γ-glutamyl) polyamine linkages using biotin labelled casein

Abstract Two colorimetric assays for tissue transglutaminase (type II) activity involving the crosslinking of proteins have been developed. In one assay, biotin labelled casein is crosslinked into chemically modified casein bound to a microtiter plate by tissue transglutaminase and the biotin labelled reaction product is detected by conjugation to Extravidin peroxidase. The assay can detect activity in 10 ng of commercially available purified guinea pig liver transglutaminase and in the crude homogenate derived from 400 human endothelial cells (cell line ECV 304). A correlation ( r 2 =0.977) was shown between this assay and the radiolabeled putrescine incorporation assay for the detection of transglutaminase activity. This assay measures the protein crosslinking activity of tissue transglutaminase as opposed to polyamine incorporation and offers a rapid, non-radiometric method for screening large sample numbers. Typical inter-assay variability is 13.9±1.5% ( n =8). In a second assay, the ability of tissue transglutaminase to catalyze the formation of N′,N′ -bis ( γ -glutamyl) polyamine bridges is measured. N′,N′ -dimethylcasein is bound to a microtiter plate and modified enzymatically using commercially available purified guinea pig liver transglutaminase to incorporate polyamines into glutamine residues. Biotin labelled casein is then crosslinked into the immobilized polyamines by tissue transglutaminase resulting in the formation of N′,N′- bis ( γ -glutamyl) polyamine linkages.

A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival

The regulation of tissue transglutaminase (TG2) activity by the GPCR family is poorly understood. In this study, we investigated the modulation of TG2 activity by the A1 adenosine receptor in cardiomyocyte-like H9c2 cells. H9c2 cells were lysed following stimulation with the A1 adenosine receptor agonist N(6)-cyclopentyladenosine (CPA). Transglutaminase activity was determined using an amine incorporating and a protein cross linking assay. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1 adenosine receptor-induced cytoprotection was investigated by monitoring hypoxia-induced cell death. CPA induced time and concentration-dependent increases in amine incorporating and protein crosslinking activity of TG2. CPA-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA were blocked by PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) inhibitors and by removal of extracellular Ca(2+). CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Fluorescence microscopy revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (Histone H4) and novel (Hexokinase 1) protein substrates for TG2. CPA pre-treatment reversed hypoxia-induced LDH release and decreases in MTT reduction. TG2 inhibitors R283 and Z-DON attenuated A1 adenosine receptor-induced cytoprotection. TG2 activity was stimulated by the A1 adenosine receptor in H9c2 cells via a multi protein kinase dependent pathway. These results suggest a role for TG2 in A1 adenosine receptor-induced cytoprotection.

Cardioprotective and cardiotoxic effects of quercetin and two of its in vivo metabolites on differentiated h9c2 cardiomyocytes.

Whilst mitotic rat embryonic cardiomyoblast‐derived H9c2 cells have been widely used as a model system to study the protective mechanisms associated with flavonoids, they are not fully differentiated cardiac cells. Hence, the aim of this study was to investigate the cardioprotective and cardiotoxic actions of quercetin and two of its major in vivo metabolites, quercetin 3‐glucuronide and 3′‐O‐methyl quercetin, using differentiated H9c2 cells. The differentiated cardiomyocyte‐like phenotype was confirmed by monitoring expression of cardiac troponin 1 after 7 days of culture in reduced serum medium containing 10 nM all‐trans retinoic acid. Quercetin‐induced cardiotoxicity was assessed by monitoring MTT reduction, lactate dehydrogenase (LDH) release, caspase 3 activity and reactive oxygen species production after prolonged flavonoid exposure (72 hr). Cardiotoxicity was observed with quercetin and 3′‐O‐methyl quercetin, but not quercetin 3‐glucuronide. Cardioprotection was assessed by pre‐treating differentiated H9c2 cells with quercetin or its metabolites for 24 hr prior to 2‐hr exposure to 600 μM H2O2, after which oxidative stress‐induced cell damage was assessed by measuring MTT reduction and LDH release. Cardioprotection was observed with quercetin and 3′‐O‐methyl quercetin, but not with quercetin 3‐glucuronide. Quercetin attenuated H2O2‐induced activation of ERK1/2, PKB, p38 MAPK and JNK, but inhibitors of these kinases did not modulate quercetin‐induced protection or H2O2‐induced cell death. In summary, quercetin triggers cardioprotection against oxidative stress‐induced cell death and cardiotoxicity after prolonged exposure. Further studies are required to investigate the complex interplay between the numerous signalling pathways that are modulated by quercetin and which may contribute to the cardioprotective and cardiotoxic effects of this important flavonoid.

Modulation of transglutaminase 2 activity in H9c2 cells by PKC and PKA signalling: a role for transglutaminase 2 in cytoprotection.

Tissue transglutaminase (TG2) has been shown to mediate cell survival in many cell types. In this study, we investigated whether the role of TG2 in cytoprotection was mediated by the activation of PKA and PKC in cardiomyocyte‐like H9c2 cells.

Nano-electrospray and microbore liquid chromatography-ion trap mass spectrometry studies of copper complexation with MHC restricted peptides

The formation of copper/peptide complex ions by nano-electrospray and microbore HPLC-electrospray mass spectrometry has been investigated for major histocompatibility complex (MHC) class I and class II restricted peptides. Post-column addition of copper(II) acetate following microbore HPLC-MS separation was carried out using a mixing T-piece or via the sheath flow inlet of the electrospray source. Optimal analytical conditions for copper complex ion formation were determined by variation of copper concentration, pH, nebulization gas supply and spray voltage. Tandem mass spectrometry of copper/peptide complex ions provides peptide sequence information and insight into the peptide chelation sites. Copper associated y fragment ions dominate the product ion spectrum for non-histidine containing peptides, but both b and y copper complex ions were observed for the histidine containing MHC class I associated peptide gp70.