Spermine delays leaf senescence in Lactuca sativa and prevents the decay of chloroplast photosystems

Aliphatic polyamines (PAs) are involved in the delay or prevention of plant senescence, but the molecular mechanism is not clarified. The hypothesis is put forward that one of the mechanisms by which PAs modulate leaf senescence and chlorophyll stabilisation could be due to their modification of chlorophyll-bound proteins, catalysed by transglutaminase (TGase, R-glutaminylpeptide-amine γ-glutamyltransferase; E.C. 2.3.2.13). The retardation of leaf senescence of Lactuca sativa L. by spermine (Spm) was examined during induced cell death using leaf discs, or during the normal developmental senescence of leaves. Over 3 days, in leaf discs, Spm caused a delay of chlorophyll (Chl) decay, an increase of endogenous TGase activity, and a three-fold increase in chlorophyll content when supplied together with exogenous TGase. Spm was conjugated, via TGase, mainly to 22–30 kDa proteins. Long-term experiments over 5 days showed a general decrease in all three parameters with or without Spm. When leaves remained on the plants, Spm-sprayed leaves showed an increase in free Spm 1 h after spraying, mainly in the young leaves, whereas over longer periods (15 days) there was an increase in perchloric acid-soluble and -insoluble Spm metabolites. In senescing leaves, Spm prevented degradation of chlorophyll b and some proteins, and increased TGase activity, producing more PA-protein conjugates. Spm was translocated to chloroplasts and bound mainly onto fractions enriched in PSII, but also those enriched in PSI, whose light-harvesting complexes (LHC) sub-fractions contained TGase. Spm was conjugated by TGase mainly to LHCII, more markedly in the light. Immunodetection of TGase revealed multiple proteins in young leaves, possibly representing different TGase isoforms when TGase activity was high, whereas in already senescent leaves, when its activity decreased, one high-molecular-mass band was found, possibly because of enzyme polymerisation. Spm thus protected senescing Lactuca leaves from the decay of their chloroplast photosystem complexes. The senescence-delaying effects of Spm could be mediated by TGase, as TGase was re-activated to the level in young leaves following Spm treatment.     

A prawn transglutaminase: Molecular characterization and biochemical properties

In this study, we report the bioinformatics characterization, gene expression, transglutaminase activity and coagulation assays of transglutaminase (TGase) of freshwater prawn Macrobrachium rosenbergii identified from the constructed cDNA library by GS FLX™ technology. Even though, TGase have sequence similarity, they differ extensively in their substrate specificity and are thought to play an important in variety of functions such as development, tissue differentiation and immune responses etc. Gene expression studies show that MrTGase is widely distributed in the tissues such as heart, muscle, intestine, brain, etc., but higher amounts are found in hemocyte. Results of TGase mRNA relative expression in hemocyte, before and after infected with white spot syndrome baculovirus (WSBV) and Vibrio harveyi show that the gene expression initially increases up to 24 h and then it falls down. Coagulation assay results showed that the endogenous TGase is involved in the rapid assembly of a specific, plasma clotting protein. Structural studies show that MrTGase contains a typical TGc domain between 323 and 424, and two putative integrin-binding motifs at Arg¹⁸⁰–Gly¹⁸¹–Asp¹⁸² and Arg²⁶⁹–Gly²⁷⁰–Asp²⁷¹. The predicted 3D model of MrTGase contains 47.04% coils (366 amino acid residues), 26.74% extended strand (208 residues), 21.72% α-helix (169 residues) and 4.5% beta turns (35 residues). BLASTp analysis of MrTGase exhibited high sequence similarities with other crustacean TGase, with the highest observed in white shrimp (77.1%). Moreover, the phylogenetic analysis also showed that MrTGase clustered with the other members of crustacean TGase. Overall, these results suggested that MrTGase is a major and functional TGase of M. rosenbergii for haemolymph coagulation and also in spread of infection.     

Deletion combined with saturation mutagenesis of N-terminal residues in transglutaminase from Streptomyces hygroscopicus results in enhanced activity and thermostability

Transglutaminase (TGase) is an important industrial enzyme that catalyzes the cross-linking of proteins. In this study, the N-terminal residues were deleted and substituted to improve the activity and thermostability of Streptomyces hygroscopicus TGase. Seven N-terminal residues of TGase were chosen to be deleted individually. The mutated TGase missing the first four residues showed an increase in specific activity of 32.92%. The fifth residue (E5) in the N-terminus was then selected for substitution with the 19 other amino acids. The mutant replacing the fifth residue with an aspartic acid exhibited a 1.85-fold higher specific activity and a 2.7-fold longer half-life at 50 °C when compared with the wild-type enzyme. The melting temperature of the mutated TGase increased from 68.9 to 79.1 °C by circular dichroism spectroscopy analysis. This study showed that substitution combined with deletion of the N-terminal amino acids could enhance the activity and thermostability of TGase.     

Diamine Oxidase Activity in Different Physiological Stages of Helianthus tuberosus Tuber

Diamine oxidase (DAO, EC 1.4.3.6) activity was examined in relation to polyamine content in Helianthus tuberosus L. during the first synchronous cell cycle induced in vitro by 2,4,-dichloro-phenoxyacetic acid in tuber slices and during the in vivo formation of the tuber. The optimal pH, buffer and dithiothreitol concentrations for the enzyme extraction and assay were determined. When added in the assay mixture, catalase enhanced DAO activity, while polyvinylpyrrolidone had no effect; both aminoguanidine and hydrazine inhibited enzyme activity. The time course of the reaction, based on the recovery of Δ¹-pyrroline from labeled putrescine in lipophilic solvents, showed that it was linear up to 30 minutes; the K_m of the enzyme for putrescine was of the order of 10⁻⁴ molar. During the first cell cycle, DAO activity exhibited a peak at 15 hours of activation while putrescine content gave a peak at 12 hours. During tuber formation (from August till October) DAO activity was relatively high during the first phase of growth (cell division), decreased until flowering (end of September-early October), and then newly increased during the cell enlargement phase preceding the entry into dormancy (November). Maximum putrescine content was observed at the end of October. The increase in DAO activity paralleled the accumulation of putrescine. This indicates a direct correlation between the biosynthesis and oxidation of putrescine which, as already demonstrated in animal systems, occur simultaneously in physiological stages of intense metabolism such as cell division or organ formation.     

Effect of Pepsin Inhibitor (S-PI) on the Growth of Rhodotorula glutinis No. K-24

A tissue-type transglutaminase (TGase) was purified from liver tissue of the red sea bream, Pagrus major, by ion-exchange chromatography and heparin-Sepharose affinity chromatography. Its activity was assessed using a fluorometric assay to measure the incorporation of monodansylcadaverine into N,N′-dimethyl casein. The molecular mass of purified TGase was estimated to be 78kDa by SDS–polyacrylamide gel electrophoresis. The enzyme required Ca²⁺ to express its activity, although 10 mM Sr²⁺ also activated the enzyme fully. TGase activity was maximal at pH 9.0–9.5, and the enzyme was strongly inhibited by sulfhydryl reagents. The purified enzyme catalyzed the cross-linking of myosin heavy chain obtained from Alaska pollack, resulting in gelation of an actomyosin solution. The partial amino acid sequence of this fish TGase showed divisionally significant similarity to TGase from guinea pig liver.     

Some properties of γ-tocopherol methyltransferase solubilized from spinach chloroplasts

γ-Tocopherol methyltransferase occurs in the chloroplast fraction of spinach leaves. Its specific activity with γ-tocopherol and S-adenosyl-l-methionine was 3.91 nmol hr⁻¹ mg⁻¹ protein. The enzyme was effectively solubilized by 6 mM sodium deoxycholate from the membrane fraction of chloroplasts. The activity was maximum at pH 7.5 and 35°. γ-Tocopherol was preferred to β-tocopherol (25:7). The K_m value for S-adenosyl-l-methionine as methyl donor was 9.1 μM.     

Effects of transglutaminase substrates and inhibitors on the motility of demembranated reactivated spermatozoa

The effects of transglutaminase (TGase) substrates putrescine, dansylcadaverine, spermine, etc., and the TGase inhibitor cystamine were tested on the motility of demembranated mammalian spermatozoa. These products blocked within a few seconds the motility of demembranated reactivated spermatozoa at concentrations ranging from 0.25 to 5 mM. These minimal inhibitory concentrations could be decreased 5–150-fold when TGase substrates and inhibitor were incubated with demembranated spermatozoa for 15 min prior to the addition of Mg·ATP. The inhibition was reversed by higher concentrations of Mg·ATP but none of these TGase substrates or inhibitor could inhibit bull sperm dynein ATPase. TGase activities, as measured by the incorporation of ³H-putrescine into TCA-precipitable proteins, were present in both sperm Triton-soluble and -insoluble fractions. On the other hand, amine acceptor protein substrates for the TGase-catalyzed reaction were present only in the insoluble fraction. The Triton-soluble TGase was similar to the known “tissue” TGases; the Triton-insoluble TGase activity was calcium independent. The same TGase substrates and inhibitor that blocked the motility of reactivated spermatozoa also blocked TGase activities. Linear relationships were observed between the concentrations of these substances required to block sperm motility and those to block TGase activities. These data suggest the involvement of a TGase activity in sperm motility.     

Suborganellar localisation and effect of light on <Emphasis Type="Italic">Helianthus tuberosus</Emphasis> chloroplast transglutaminases and their substrates

The light stimulation of transglutaminase (TGase EC 2.3.2.13) activity was verified by incubating isolated chloroplasts of Helianthus tuberosus L. continuously or for alternate periods of light or dark (light/dark and dark/light). The first 10 min of incubation always represented the critical period. Light-harvesting complexes of photosystem II (LHCII) were more intensely labelled by (14)C-polyamines under light and light/dark than under dark and dark/light conditions. Chloroplasts were fractionated into thylakoid- and stroma-enriched fractions in which multiple TGase forms and substrates were found. Antibodies against TGase recognised 58- and 24-kDa bands in thylakoids and a 150-kDa band in the stroma. The latter, and its 150-kDa fraction, catalysed the conjugation of 14C-polyamines to Rubisco. In both fractions (thylakoid-pre and stroma-pre) the analysis of polyamine glutamyl derivatives showed a significant light-affected conjugation of polyamines to endogenous proteins. Alternatively, entire chloroplasts were incubated and afterwards their sub-fractions were isolated (thylakoid-post and stroma-post). The PSII and LHCII complexes were more intensely immunodetected in thylakoid-post than in thylakoid-pre, especially under dark conditions. Conversely, the conjugation of polyamines to thylakoid proteins was clearly light-stimulated in thylakoid-post, and much less in thylakoid-pre. Stroma-pre proteins were poorly polyamine-conjugated and not light-affected; on the contrary, stroma-post proteins were much more polyamine-modified and strongly light-stimulated. Thus, the light-activated conjugation depends mainly on the presence of the thylakoid fraction during the assay. The protective effect on chloroplasts under photo-damage, stress or senescence conditions attributed in the literature to free polyamines is discussed with regard to the occurrence of polyamine conjugates catalysed by TGases.     

Conjugation of ubiquitin to proteins from green plant tissues

Conjugation of the polypeptide ubiquitin to endogenous proteins was studied in oat (Avena sativa L.) plants, and particularly in green tissues. Conjugating activity in leaf extracts was different from that in root extracts, and in both was less than in etiolated tissue. The conjugates were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and their formation was both time- and ATP-dependent and had a pH optimum of about 8.2. The assay had a high affinity for ATP with a probable K_m of less than 50 micromolar. The ubiquitin conjugating system was also shown to be present in isolated chloroplasts, and ubiquitin could be conjugated to endogenous proteins of lyzed chloroplasts in which the ATP concentrations were reduced by preincubation or desalting. SDS-PAGE analysis led to the suggestion that the large and small subunits of ribulose-1,5-bisphosphate carboxylase (RuBPCase) may be able to be ubiquitinated, and we have shown that ubiquitin can stimulate the in vitro breakdown of ¹²⁵I-labeled RuBPCase. These results invite the speculation that ubiquitin may be involved in the regulation of protein turnover in green plants.     

Transglutaminase in sporulating cells of Bacillus subtilis

We screened various Bacillus species producing transglutaminase (TGase), measured as labeled putrescine incorporated into N,N-dimethylcasein. As a result, we detected TGase activity in sporulating cells of B. subtilis, B. cereus, B. alvei and B. aneurinolyticus, and found TGase activity related to sporulation. TGase activity of Bacillus subtilis was detected in lysozyme-treated sporulating cells during late sporulation, but not in cells without lysozyme treatment or the supernatant of the culture broth. TGase was found to be localized on spores. TGase was preliminarily purified by gel filtration chromatography for characterization. Its activity was eluted in the fractions indicating a molecular weight of approximately 23 kDa. TGase could cross-link and polymerize a certain protein. The enzyme was strongly suggested to form epsilon-(gamma-glutamyl)lysine bonds, which were detected in the spore coat proteins of B. subtilis. The activity was Ca(2+)-independent like the TGases derived from Streptoverticillium or some plants. It is suggested that TGase is expressed during sporulation and plays a role in the assembly of the spore coat proteins of the genus Bacillus.     

First evidence for polyamine conjugation mediated by an enzymic activity in plants

An enzyme activity, found for the first time in plants, mainly located in the 22,000g supernatant of the crude extract of sprout apices of Helianthus tuberosus L. cv OB1 tubers, is able in vitro to covalently bind polyamines to endogenous substrates of different molecular weights. The major assay parameters, such as pH, dithiothreitol, and extract concentrations were optimized. The time course of the reaction, the dependence on putrescine concentration, its competition with histamine, the capacity to bind spermidine and spermine better than putrescine, the stability of the reaction product and analysis of the latter by sodium dodecyl sulfate polyacrylamide gel electrophoresis and thin-layer chromatography suggest that putrescine is linked to endogenous substrates by means of an enzyme reaction that shows some similarities with transglutaminase activities detected in animals. However, the activities of the crude extract and of a fraction eluted from a Sephadex G25 column were not affected by CaCl₂ concentrations lower or equal to 5 millimolar; 4 or 10 millimolar EGTA caused a very small reduction; higher concentrations of CaCl₂ and 15 millimolar or more of EDTA were inhibitory. N,N′-dimethylcasein was not recognized as a substrate. These data indicate that this activity also displays some characteristics which are different from those of animal transglutaminases.     

Characterization of a nuclear phosphatidylinositol 4-kinase in carrot suspension culture cells

We have shown previously that a nuclear phosphatidylinositol (PI) 4-kinase activity was present in intact nuclei isolated from carrot suspension culture cells (Daucus carota L.). Here, we further characterized the enzyme activity of the nuclear enzyme. We found that the pH optimum of the nuclear-associated PI kinase varied with assay conditions. The enzyme had a broad pH optimum between 6.5–7.5 in the presence of endogenous substrate. When the substrate was added in the form of phosphatidylinositol/phosphatidylserine (PI/PS) mixed micelles (1 mM PI and 400 μM PS), the enzyme had an optimum of pH 6.5. In comparison, the pH optimum was 7.0 when PI/Triton X-100 mixed micelles (1 mM PI in 0.025 %, v/v final concentration of Triton X-100) were used. The nuclear-associated PI kinase activity increased 5-fold in the presence of low concentrations of Triton X-100 (0.05 to 0.3 %, v/v); however, the activity decreased by 30 % at Triton X-100 concentrations greater than 0.3 % (v/v). Calcium at 10 μM inhibited 100 % of the nuclear-associated enzyme activity. The K_m for ATP was estimated to be between 36 and 40 μM. The nuclear-associated PI kinase activity was inhibited by both 50 μM ADP and 10 μM adenosine. Treatment of intact nuclei with DNase, RNase, phospholipase A₂ and Triton X-100 did not solubilize the enzyme activity. Based on sensitivity to calcium, ADP, detergent, pH optimum and the product analysis, the nuclear-associated PI 4-kinase was compared with previously reported PI kinases from plants, animals and yeast.     

Transglutaminase activity changes during the hypersensitive reaction, a typical defense response of tobacco NN plants to TMV

The occurrence of glutamyl polyamines (PAs) and changes in activity and levels of transglutaminase (TGase, EC 2.3.2.13), the enzyme responsible for their synthesis, are reported during the progression of the hypersensitive reaction (HR) of resistant NN tobacco plants ( Nicotiana tabacum L. cv. Samsun) to tobacco mosaic virus (TMV). Mature leaves of tobacco were collected over 0–72 h after inoculation with TMV or phosphate buffer (mock). In vivo synthesis of polyamine glutamyl derivatives (glutamyl PAs), catalyzed by TGase activity, was evaluated after supplying labeled putrescine (Pu, a physiological substrate of TGase) to leaves. Results show that, starting from 24 h, mono-( γ -glutamyl)-Pu and bis-( γ -glutamyl)-Sd were recovered in TMV-inoculated samples but not in mock-inoculated ones; 2 days later, in the former, the amount of glutamyl derivatives further increased. An in vitro radiometric assay showed that, in TMV-inoculated leaves, TGase activity increased from 24 h onwards relative to mock controls. An immunoblot analysis with AtPng1p polyclonal antibody detected a 72-kDa protein whose amount increased at 72 h in TMV-inoculated leaves and in the lesion-enriched areas. A biotin-labeled cadaverine incorporation assay showed that TGase activity occurred in S1 (containing soluble proteins), S2 (proteins released by both cell walls and membranes) and S3 (membrane intrinsic proteins) fractions. In S3 fraction, where changes were the most relevant, TGase activity was enhanced in both mock-inoculated and TMV-inoculated samples, but the stimulation persisted only in the latter case. These data are discussed in the light of a possible role of TGase activity and glutamyl PAs in the defense against a viral plant pathogen.     

Putrescine N-methyltransferase – The start for alkaloids

Putrescine N-methyltransferase (PMT) catalyses S-adenosylmethionine (SAM) dependent methylation of the diamine putrescine. The product N-methylputrescine is the first specific metabolite on the route to nicotine, tropane, and nortropane alkaloids. PMT cDNA sequences were cloned from tobacco species and other Solanaceae, also from nortropane-forming Convolvulaceae and enzyme proteins were synthesised in Escherichia coli. PMT activity was measured by HPLC separation of polyamine derivatives and by an enzyme-coupled colorimetric assay using S-adenosylhomocysteine. PMT cDNA sequences resemble those of plant spermidine synthases (putrescine aminopropyltransferases) and display little similarity to other plant methyltransferases. PMT is likely to have evolved from the ubiquitous enzyme spermidine synthase. PMT and spermidine synthase proteins share the same overall protein structure; they bind the same substrate putrescine and similar co-substrates, SAM and decarboxylated S-adenosylmethionine. The active sites of both proteins, however, were shaped differentially in the course of evolution. Phylogenetic analysis of both enzyme groups from plants revealed a deep bifurcation and confirmed an early descent of PMT from spermidine synthase in the course of angiosperm development.     

Enhancement of Streptomyces transglutaminase activity and pro-peptide cleavage efficiency by introducing linker peptide in the C-terminus of the pro-peptide

Streptomyces transglutaminase (TGase) has been widely used in food, pharmaceutical and textile industries. Streptomyces TGase is naturally synthesized as zymogen (pro-TGase), which is then processed to produce active enzyme by removing its N-terminal pro-peptide. Although the pro-peptide is essential for TGase folding and secretion, few studies have been reported on improving the properties of TGase by pro-peptide engineering. In this study, we developed a new approach to improve the properties of TGase based on pro-peptide engineering. When the α-helix^37G?42S in pro-peptide was substituted with three glycines and three alanines respectively, the mutants exhibited higher specific activity and the efficiency of pro-peptide cleavage was enhanced. To further improve the properties of TGase, relevant mutations were constructed by introducing linker peptides in the C-terminus of the pro-peptide. Mutants with GS (GGGGS) and PT (PTPPTTPT) linker peptide exhibited 1.28 fold and 1.5 fold higher specific activity than the wild-type enzyme, respectively. This new method could be used to improve the properties of TGase by pro-peptide modification, which is a promising technology for creating unique TGase with various beneficial properties.     

Acclimation of chloroplast transglutaminase to high NaClconcentration in a polyamine-deficient variant strain of Dunaliella salina and in its wild type

The wild type (Wt) and the polyamine-deficient strain (PA⁻vs) of the halotolerant Dunaliella salina were subjected to stress caused by 3.5 mol/L NaCl concentration. The chloroplasts were isolated and the molecular aspects of their reaction to salt stress were studied together with their recovery response to these hyper-saline conditions.In the Wt, the photosynthetic complexes were found to be severely affected by salt stress under light conditions. Transglutaminases, which are present in chloroplasts as two units of 25 and 50 kDa, were immunorecognized by antibodies raised against rat prostatic gland transglutaminase. The amount, in particular that of the 50 kDa unit, underwent an immediate change following hyper-saline stress. These concentration changes were found to coincide with variations in enzymic activity, which is also affected by the presence or absence of light.The PA⁻vs has a concentration of proteins and chlorophylls which is much lower than that of the Wt. In addition, the PA⁻vs appeared to be more severely affected by both salt and subculture stresses. Its recovery time was also longer. Its TGase activity increased after salt stress and was always higher in the light than in the dark, except soon after subculture, showing an additive stress effect of salt and light. In the PA⁻vs acclimated to high salinity, or immediately after stress application, the chloroplast content of chlorophyll a and b was considerably enhanced, like the TGase activity (by two-fold or more), and these changes exhibited almost coincident behaviours.Some transglutaminase substrates (proteins of 68, 55, 29 and 27 kDa) were found to be similar to those present in higher plants (thylakoid photosynthetic complexes and Rubisco). They were more markedly labelled by [1,4-¹⁴C] polyamines when the transglutaminase assay was performed in the light than in the dark, and much more in algae already acclimated to hyper-saline conditions than in those cultured in the optimal saline medium, or subjected to stress. The amount of 68 and 55 kDa polypeptides was particularly high in the 3.5 mol/L NaCl acclimated cells. The possible role of polyamine conjugation in the assembly of chloroplast proteins in cells affected by salt stress is discussed.     

γ-glutamylcysteine synthetase from Plasmodium berghei

γ-glutamylcysteine synthetase (l-glutamate-l-cysteine ligase, γ-GCS, EC 6.3.2.2.), the rate limiting enzyme in glutathione biosynthetic pathway has been analysed in the asexual erythrocytic stages of rodent malaria parasite, Plasmodium berghei and its host erythrocytes. Cell-free parasite isolated by saponin lysis contained about 2 and 8 times higher activity of γ-GCS compared to P. berghei-infected and normal mice erythrocytes respectively. Subcellular fractionation revealed that the enzyme was mainly confined to the cytosolic part of the parasite. γ-GCS from P. berghei was purified employing ammonium sulphate precipitation, Sephadex G-200 gel filtration and anionic exchange chromatography on DEAE-cellulose. There was 51.6 fold purification of enzyme and its specific activity was 39.5 U/mg. SDS-PAGE showed P. berghei γ-GCS as a heterodimer dissociating into two non-identical sub-units of 66 kDa and 57 kDa. The enzyme was observed as white band of activity on native polyacrylamide gel stained for specific γ-GCS activity. Km values for l-Cys, ATP and l-Glu were 0.53 mM, 0.92 mM and 0.75 mM, respectively. The inhibition of γ-GCS activity by glutathione was found to be competitive with respect to glutamate (Ki = 1.53 mM) and non competitive to ATP and cysteine. Antimalarial drugs did not show any significant effect on parasite γ-GCS. Parasite enzyme induced humoral response in mice demonstrated by ELISA, IFA and immunoblotting and exhibited partial protection against P. berghei infection suggesting a significant role of P. berghei γ-GCS in malaria control.     

Solid state bioreactor production of transglutaminase by Amazonian Bacillus circulans BL32 strain

In this work, we investigated the production of transglutaminase (TGase) by an Amazonian isolated strain of Bacillus circulans by solid-state cultivation (SSC). Several agro-industrial residues, such as untreated corn grits, milled brewers rice, industrial fibrous soy residue, soy hull, and malt bagasse, were used as substrates for microbial growth and enzyme production. Growth on industrial fibrous soy residue, which is rich in protein and hemicellulose, produced the highest TGase activity (0.74 U g⁻¹ of dried substrate after 48 h of incubation). A 2³ central composite design was applied to determine the optimal conditions of aeration, cultivation temperature and inoculum cell concentration to TGase production. The best culture conditions were determined as being 0.6 L air min⁻¹, 33 °C and 10 log ₁₀ CFU g⁻¹ of dried substrate, respectively. Under the proposed optimized conditions, the model predicted an enzyme production of 1.16 U g⁻¹ of dried substrate, closely matching the experimental activity of 1.25 U g⁻¹. Results presented in this work point to the use of this newly isolated B. circulans strain as a potential alternative of microbial source for TGase production by SSC, using inexpensive culture media.     

Identification of chlorophyll-a/b proteins as substrates of transglutaminase activity in isolated chloroplasts of Helianthus tuberosus L.

Endogenous substrates of transglutaminase (TGase; EC 2.3.2.13) have been identified in choloroplasts of Helianthus tuberosus leaves. The activity of TGase is Ca²⁺- and light-stimulated and catalyzes the incorporation of polyamines into thylakoid and stromal proteins. These proteins were separated by two-dimensional gel electrophoresis (first dimension: Deriphat-PAGE; second dimension: SDS-urea-PAGE) and Western-blotted. The thylakoid proteins were recognized by polyclonal antibodies as apoproteins of the chlorophyll-a/b antenna complex (LHCII, CP24, CP26 and CP29); a stromal protein was recognized by antibodies as the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. A possible localization of the acyl donor site for CP26 is proposed. A comparative analysis of polyamine incorporation into trichloroacetic-acid-precipitable material indicated that spermidine was a preferential acyl-acceptor substrate with respect to putrescine, even though the above-reported substrates are the same. The nature of the substrates, together with the light stimulation, support the working hypothesis of a possible role of TGase in regulating the light-harvesting function.Abbreviations CP chlorophyll protein - LHC light-harvesting complex - M_r relative molecular mass - PA polyamine - PU putrescine - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SD spermidine - TCA trichloroacetic acid - TGase transglutaminase We acknowledge the financial support provided by CNR contribution No. 91.00539.CTO 4 to D. Serafini-Fracassini and MAF grant No. 4.7240.90 to R. Bassi. We thank Prof. C. Bergamini (Istituto di Biochimica, Università di Ferrara, Ferrara, Italy) for the kind gift of antibodies against human erythrocyte TGase.     

Transglutaminase-catalyzed modification of cytoskeletal proteins by polyamines during the germination of Malus domestica pollen

 We investigated polyamine linkage to different structural proteins in pollen of Malus domestica Borkh. cv Red Chief at different phases of germination. This linkage has the characteristics of covalent linkages, indeed, it could be catalyzed by transglutaminase (TGase; EC 2.3.2.13). This assumption is supported by: (1) formation of a labelled TCA pellet and selective labelling of endogenous proteins by covalent binding of radioactive polyamines and (2) cross-reactivity of two different polyclonal antibodies against mammalian TGases; western blot analysis allowed us to detect a protein of about 80 kDa in both rehydrated ungerminated and germinated pollen. TGase activity was high at 90 min after germination and was influenced by Ca²⁺ supply only in the rehydrated ungerminated pollen. Extraction by Triton X-100 suggests that pollen TGase was at least partially membrane-bound. The enzyme catalyzed the incorporation of polyamines mainly into proteins having a molecular mass of 43 kDa and 52–58 kDa in both ungerminated and germinated pollen. These bands matched immunolabelled spots identified by mouse monoclonal anti-actin and anti-α-tubulin antibodies. Supplying exogenous actin and tubulin in a cell-free extract of rehydrated ungerminated and germinated pollen enhanced the activity. Autoradiography of the SDS-PAGE of these samples clearly showed that both actin and tubulin were substrates of TGase. Thus, the pollen TGase may be involved in the rapid cytoskeletal rearrangement which takes place during rehydration of ungerminated pollen and organization and growth of pollen tubes. Received: 9 August 1996 / Revision accepted: 26 October 1996