Changes In Polypeptide Composition Research Articles

Photoacclimation impacts the molecular features of photosystem supercomplexes in the centric diatom Thalassiosira pseudonana

In diatoms, light-harvesting processes take place in a specific group of proteins, called fucoxanthin chlorophyll a/c proteins (FCP). This group includes many members and represents the major characteristic of the diatom photosynthetic apparatus, with specific pigments bound (chlorophyll c, fucoxanthin, diadino- and diatoxanthin besides chlorophyll a). In thylakoids, FCP and photosystems (PS) form multimeric supercomplexes.In this study, we compared the biochemical properties of PS supercomplexes isolated from Thalassiosira pseudonana cells grown under low light or high light conditions, respectively. High light acclimation changed the molecular features of the PS and their ratio in thylakoids. In PSII, no obvious changes in polypeptide composition were observed, whereas for PSI changes in one specific group of FCP proteins were detected. As reported before, the amount of xanthophyll cycle pigments and their de-epoxidation ratio was increased in PSI under HL. In PSII, however, no additional xanthophyll cycle pigments occurred, but the de-epoxidation ratio was increased as well. This comparison suggests how mechanisms of photoprotection might take place within and in the proximity of the PS, which gives new insights into the capacity of diatoms to adapt to different conditions and in different environments.

Phenotypic subunit composition of the tobacco ( Nicotiana tabacum L.) vacuolar-type H +-translocating ATPase

The model plant tobacco ( Nicotiana tabacum L.) was chosen for a survey of the subunit composition of the V-ATPase at the protein level. V-ATPase was purified from tobacco leaf cell tonoplasts by solubilization with the nonionic detergent Triton X-100 and immunoprecipitation. In the purified fraction 12 proteins were present. By matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS) and amino acid sequencing 11 of these polypeptides could be identified as subunits A, B, C, D, F, G, c, d and three different isoforms of subunit E. The polypeptide which could not be identified by MALDI analysis might represent subunit H. The data presented here, for the first time, enable an unequivocal identification of V-ATPase subunits after gel electrophoresis and open the possibility to assign changes in polypeptide composition to variations in respective V-ATPase subunits occurring as a response to environmental conditions or during plant development.

Changes in total protein profiles of barley cultivars in response to toxic boron concentration

In this study, ten‐day‐old seedlings of barley {Hordeum vulgare L. cultivar Anadolu [boron (B)‐tolerant] and Hamidiye (B‐sensitive)} were used. Boron‐treated plants were grown on H3BO3 solution (final concentration of 10 mM) for five days. Control plants received no B treatment during this period. Total protein patterns were obtained by analysis of total protein extract from root and leaf tissues of control and B‐treated plants using two‐dimensional gel electrophoresis followed by silver staining. The protein profile of B‐treated seedlings of each cultivar was compared to the profile of control (no stress treatment) plants of the same cultivar. Silver‐stained gels showed that B stress caused increases or decreases in a number of proteins in root and leaf tissues. Moreover, as a result of B treatment, one newly synthesized protein with relative molecular weight (Mr) of 35.0 kDa was detected in root profile of the tolerant cultivar. This protein failed to show up in root profile of the B‐treated sensitive cultivar. Three proteins were quantitatively increased in B‐treated root profile of both cultivars. Following B treatment, three proteins were increased in root profile of the tolerant cultivar, but were not changed in the sensitive one. In leaf tissues, however, there were remarkable changes in total protein profiles after B treatment, relative to the control. Following B treatment, in leaf tissues, at least seven proteins were increased in amount in tolerant cultivar but were unchanged in the susceptible one. In tolerant and sensitive cultivars, amounts of two proteins were increased in B‐treated plants, relative to control seedlings. In addition, four proteins (Mr:29, 58, 58, and 22 kDa) were unchanged in control and B‐treated seedlings of the tolerant cultivar. In the susceptible cultivar however, among these four proteins, the first one (Mr:29) was very much reduced and the others (Mr: 58, 58, and 22 kDa) were completely lost in B‐treated seedlings. Moreover, following B treatment, a set of high‐molecular‐weight proteins was quantitatively decreased in the susceptible cultivar but was unchanged in the tolerant cultivar. These results indicate that in barley, certain proteins may be involved in tolerance to B toxicity. In this study, changes in polypeptide composition as a result of B toxic concentration in leaf tissues were more abundant than in roots. Therefore, it is suggested that these changes, especially at shoot level may form the basis of the tolerance mechanism to B toxicity.

Quantitative Changes in Maize Membrane Proteins Induced by Aluminium

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for monitoring Al-induced changes in polypeptide composition of membrane proteins isolated from 3-d-old maize seedlings subjected to aluminium stress. Analysis of peripheral membrane proteins isolated from maize root showed an Al-induced increase in accumulation of 14 polypeptides with apparent molecular mass from 10 to 135 kDa. Qualitative differences were found between peripheral membrane proteins isolated from root tip (increased accumulation of 4 polypeptides with Mr 42 000 - 135 000) and from root base (increased accumulation of 10 polypeptides with Mr 10 000 - 59 000). On the other hand, no Al-induced changes were observed in peripheral membrane proteins isolated from maize coleoptile and integral membrane proteins isolated either from root or coleoptile. These results indicate that peripheral membrane proteins undergo considerable changes during 24-h Al treatment while integral membrane proteins pattern is stable.

Structural and functional heterogeneity in the major light-harvesting complexes of higher plants

The major light-harvesting complex (LHC IIb) of higher plants plays a crucial role in capturing light energy for photosynthesis and in regulating the flow of energy within the photosynthetic apparatus. Multiple isoforms of the protein bind chlorophyll and xanthophyll chromophores, but it is commonly believed that the pigment-binding properties of different LHC IIb complexes are conserved within and between species. We have investigated the structure and function of different LHC IIb complexes isolated from Arabidopsis thaliana grown under different light conditions. LHC IIb isolated from low light-grown plants shows increased amounts of the Lhcb2 gene product, increased binding of chlorophyll a, and altered energy transfer characteristics. We suggest that Lhcb2 specifically binds at least one additional chlorophyll a compared to the Lhcb1 gene product, and that differences in the functioning of LHC IIb from high and low light-grown plants are a direct consequence of the change in polypeptide composition. We show that changes in LHC IIb composition are accompanied by changes in photosynthetic function in vivo and discuss the possible functional significance of LHC IIb heterogeneity.

Quantitative changes in maize cytoplasmic proteins induced by aluminium

Three-day-old maize seedlings were subjected to 100 µM AlCl3 for 24 h. Cytoplasmic proteins were isolated from root tips, root base and from coleoptiles. After fractionation of cytoplasmic proteins on anion chromatography column Bio-Scale Q2 sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used to monitor Al-induced changes in polypeptide composition of particular fractions. Four (root) and 7 (coleoptile) fractions were eluted from the column with linear 0 - 1.0 M NaCl gradient. In fraction 1 of cytoplasmic proteins from root tips Al induced accumulation of polypeptide with molecular mass of 16 kD and simultaneous reduction of two polypeptides (67.5 and 60 kD). In fraction 1 isolated from mature zone of maize roots Al-induced accumulation of 22 kD polypeptide and reduction of 67.5, 60, and 14 kD polypeptides. Most pronounced changes were revealed in coleoptile. In three protein fractions increased accumulation of polypeptides with molecular mass of 14, 17.5, 20, 24.5, 28, 30, and 37.5 kD were observed. In the remaining three root or four coleoptile fractions of cytoplasmic proteins, no differences were found between Al-treated and control maize seedlings.

Influence of GA3 and 4-PU-30 on Leaf Protein Composition, Photosynthetic Activity, and Growth of Maize Seedlings

The effects of gibberellic acid (GA3) and N1-(2-chloro-4-pyridyl)-N2 phenylurea (4-PU-30) on maize seedling growth, photosynthetic parameters, and leaf protein composition were investigated. The agents used alone or in combination increased leaf growth and photosynthetic rate of the seedlings. Chlorophyll and total nitrogen contents in leaves as well as the quantity of individual protein fractions increased simultaneously. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of soluble proteins (albumins and globulins) revealed quantitative differences between 4-PU-30-treated plants and the other experimental variants. They differed in polypeptide composition associated with changes in soluble proteins and amino acids. However, GA3 did not induce similar changes in polypeptide composition of soluble proteins.

Embryonic protein composition and synthesis during imbibition of white lupin seeds: effect of low temperatures

White lupin seed imbibition under low temperature conditions (8 °C) and their subsequent effects on embryonic protein composition and synthesis were investigated. The response to low temperatures was accompanied by changes in polypeptide composition and synthesis. The embryonic axes labelled in vivo with ( 35S)-methionine retained their capacity to synthesize proteins during imbibition at 8 °C. The synthesis of some polypeptides was increased during low temperature treatment as compared to that at 25 °C. These cold-induced polypeptides were essentially detected in a molecular mass range from 15 to 35 kDa and a pI range from 6.3 to 8.7.

Improved method for the extraction of proteins fromEucalyptusleaves. Application in leaf response to temperature

Most studies on protein metabolism require extraction and fractionation of the individual proteins or polypeptides. However, when the proteins from Eucalyptus leaves are extracted by a standard procedure, commonly utilized for the leaves of many plant species, and subsequently fractionated by sodium dodecylsulphate:polyacrylamide gel electrophoresis (SDS-PAGE), no clear polypeptide bands are visible in the gel. A method has been developed and optimized that is efficient in extracting the proteins from Eucalyptus leaves, producing an excellent resolution when the polypeptides are fractionated by SDS-PAGE. This procedure involves five consecutive steps: (i) lyophilization of the leaves followed by grinding to a fine powder in liquid nitrogen; (ii) washing with methanol:acetic acid:water (10:1:9); (iii) washing with hexane; (iv) washing with acetone; (v) solubilization of the proteins in the SDS-PAGE sample buffer containing SDS and 2-mercaptoethanol. This optimized procedure allows the detection, by SDS-PAGE or fluorography, of changes in polypeptide composition in attached or detached Eucalyptus leaves during cold acclimation or heat shock. It may also be utilized successfully in the extraction of leaf proteins from other tree species. In addition, it has been possible to induce cold acclimation in detached leaves of Eucalyptus, a methodology which will speed considerably the study of polypeptide composition in response to cold. © 1997 John Wiley & Sons, Ltd.

Zinc stress induces changes in apoplasmic protein content and polypeptide composition of barley primary leaves

Excessive zinc in the rooting medium, and consequently increasing zinc content in the shoot of barley (Hordeum vulgare) seedlings, strongly affected the protein content and the polypeptide composition of the leaf apoplasm. In contrast, only small changes in polypeptide composition (in the IEF range of pH 4-8 and between 14 and 80 kDa) were detected in mesophyll and epidermis protoplasts. The zinc stress-induced changes in apoplasmic protein content were analysed in detail. The apoplasmic protein content increased by more than 3-fold when the zinc concentration in the medium was increased from 0.02 to about 200 mu mol l(-1); at higher zinc concentrations no further changes occurred. The increase in apoplasmic protein content was due both to a general increase in abundance of the most dominant apoplasmic polypeptides and to a pronounced increase in the abundance of specific polypeptides as monitored in electrophoretic separations or by measuring the activity of apoplasmic enzymes. The largest induction was seen for four apoplasmic polypeptides of molecular masses 16, 23, 27, and 28 kDa with as yet unknown function.

Grapein vivoProtein Synthesis: Changes in Response to Anaerobiosis

Protein synthesis was examined <i>in vivo</i> in grape cell culture and ripe berries. Synthesized polypeptides from cell culture and from berries were labeled by ³⁵S-methionine. Protein fractions were analyzed by fluorography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Ripe berries were able to synthesize a full set of proteins. Further, transfer from air to anaerobic conditions resulted in qualitative and quantitative changes in polypeptide composition: labeling in certain proteins was constant, while others were labeled to lesser or greater extent during stress. Two-dimensional electrophoresis (IEF-SDS-PAGE) afforded greater resolution of the total polypeptides, describing common features between grape cell culture and berries, and confirming modification upon anaerobiosis in the <i>in vivo</i> protein pattern by increasing synthesis of some polypeptides. We obtained evidence that some of these polypeptides were common in the two models, and that common features between <i>in vivo</i> and <i>in vitro</i> protein synthesis occurred.

Ectomycorrhizin Synthesis and Polypeptide Changes during the Early Stage of Eucalypt Mycorrhiza Development

In functioning eucalypt ectomycorrhizas, biochemical alterations are accompanied by a differential accumulation of polypeptides including the synthesis of symbiosis-related proteins (JL Hilbert, Martin FM [1988] New Phytol 110: 339-346). In the present study, protein biosynthesis in the early stages of ectomycorrhiza formation on Eucalyptus globulus subsp. bicostata Kirkp. was examined using compatible and incompatible isolates of the basidiomycete Pisolithus tinctorius (Coker & Couch). Changes in polypeptide composition were observed within hours following contact of the compatible mycelium with the roots, well before the differentiation of typical symbiotic tissues. At this stage, at least seven symbiosis-related proteins (ectomycorrhizins) accumulated in root tissues. In vivo incorporation of [(35)S]methionine by ectomycorrhizas followed by electrophoresis of the labeled proteins revealed that most of these differences in polypeptide concentrations, including the ectomycorrhizin accumulation, are the result of differential protein biosynthesis rather than posttranslational modifications of the polypeptides. The initial development of eucalypt ectomycorrhizas, therefore, coincides with the synthesis of symbiosis-related proteins and the data presented here provide essential evidence to ascribe a functional developmental role to these proteins.

Molecular changes associated with tuberisation in Solanum tuberosum. Differential expression of α‐tubulin isotypes

Changes in gene expression that occur in the stolon tips of potato (Solanum tuberosum L.) cv. Record during tuberisation were investigated. Protein extracts from stolon tips at various stages in the tuberisation process were analysed by two‐dimensional gel electrophoresis. A number of quantitative and qualitative changes in polypeptide composition accompanied the very early stages of tuberisation. In vitro translation of RNA extracted from stolon tips also revealed quantitative and qualitative changes associated with tuberisation. Immunoblotting of protein extracts with monoclonal antibodies raised against α‐ and β‐tubulin showed quantitative changes in the relative level of β‐tubulin, but not α‐tubulin, as the stolon tips tuberised. Changes in the pattern of α‐tubulin isotype expression were shown to occur at early stages in the tuberisation process.

An improved SDS-PAGE method for the analysis of wheat endosperm storage proteins

An improved one-dimensional, one-step SDS polyacrylamide gel electrophoresis (SDS-PAGE) technique was developed for the analysis of wheat endosperm storage proteins (glutenins and gliadins). SDS-PAGE then was used to re-evaluate the glutenin and gliadin composition of intervarietal chromosomal substitution lines of wheat ( Triticum aestivum L.). Glutenins and gliadins were extracted from reciprocal substitution lines involving group 1 and group 6 chromosomes of the cultivars ‘Cheyenne’ and ‘Wichita’. Molecular-weight estimates were made for all proteins unique to each cultivar. Variation among lines was found to be greater for group 1 chromosome-encoded proteins, especially gliadins and low-molecular-weight subunits of glutenin, than for group 6 chromosome-encoded proteins. Both highmolecular-weight and putative low-molecular-weight glutenin subunits were found to be slightly soluble in aqueous ethanol solutions. Substituted chromosomes of the cultivars Cheyenne and Wichita produced simultaneous changes in polypeptide composition of both gliadin and glutenin fractions of the recipient cultivars. Thus, correlations between end-use quality changes (associated with substituted chromosomes) and endosperm storage proteins are reliable only when proteins of both gliadin and glutenin fractions are analyzed.

Comparison of soluble and membrane polypeptides ofSynechocystis 6308 by two-dimensional electrophoresis

Two-dimensional electrophoresis was carried out on fractions of the cyanobacteriumSynechocystis 6308 (ATCC 27150). Phycobilisomes isolated fromSynechocystis in 0.75M potassium phosphate buffer, pH 6.8, (KPi) plus Triton X-100 showed 5 prominent polypeptides when examined by two-dimensional electrophoresis. Ultracentrifugation of cells broken in KPi lacking Triton yielded three fractions, a membrane-containing pellet, a green supernatant, and a less dense yellow supernatant. The three fractions yielded a total of 272 polypeptides visualized by silver staining of two-dimensional gels. Fourteen polypeptides were found only in the yellow fraction, 14 polypeptides were found only in the green fraction, and 16 polypeptides were found only in the membrane fraction; 23 polypeptides were found in all three fractions. The crude Triton-containing KPi extract contained 62, and a 50 mM HEPES extract contained 55, of the 272 polypeptides visualized in these fractions. Two-dimensional electrophoresis combined with subcellular fractionation may be useful tools for examining changes in polypeptide composition caused by nitrogen starvation.

Changes in polypeptide composition of Synechocystis sp. strain 6308 phycobilisomes induced by nitrogen starvation

Phycobilisomes isolated from actively growing Synechocystis sp. strain 6308 (ATCC 27150) consist of 12 polypeptides ranging in molecular mass from 11.5 to 95 kilodaltons. The phycobilisome anchor and linker polypeptides are glycosylated. Nitrogen starvation causes the progressive loss of phycocyanin and allophycocyanin subunits with molecular masses between 16 and 20 kilodaltons and of two linker polypeptides with molecular masses of 27 and 33 kilodaltons. Nitrogen starvation also leads to enrichment of four additional polypeptides with molecular masses of 46, 53, 57, and 61 kilodaltons and a transient enrichment of 35- and 41-kilodalton polypeptides in isolated phycobilisomes. The 57-kilodalton additional polypeptide was identified by immunoblotting as the large subunit of ribulosebisphosphate carboxylase/oxygenase. Proteins with the same molecular weights as the additional polypeptides were also coisolated with the 12 phycobilisome polypeptides in the supernatant of nitrogen-replete Synechocystis thylakoid membranes extracted in high-ionic-strength buffer and washed with deionized water. These observations suggest that the additional polypeptides in phycobilisomes from nitrogen-starved cells may be soluble or loosely bound membrane proteins which associate with phycobilisomes. The composition and degree of association of phycobilisomes with soluble and adjacent membrane polypeptides appear to be highly dynamic and specifically regulated by nitrogen availability. Possible mechanisms for variation in the strength of association between phycobilisomes and other polypeptides are suggested.

Induced Changes in Chloroplast Protein Accumulation during Heat Bleaching in Euglena gracilis

When growing cultures of light-grown Euglena gracilis Z are exposed to slightly elevated temperatures (33 degrees C) there is a time-dependent decrease in chlorophyll (bleaching) and a gradual transformation of chloroplasts into rudimentary plastids. A study was undertaken whose primary objective was to document major changes in polypeptide composition in the stroma and in thylakoids of cells that have been exposed to the bleaching temperature for up to 57 hours. A novel polypeptide of about 60,000 to 63,000 M(r) whose function is presently unknown, accumulates in the stroma and in thylakoids in response to growth at the bleaching temperature. The levels of the large and small subunit of ribuolosebisphosphate carboxylase, on the other hand, decrease to very low levels at about 33 hours and remain very low for the duration of the temperature treatment. Of two polypeptides associated with the light-harvesting chlorophyll-protein complex of photosystem II (28,000 and 24,500 M(r)) only the level of the smaller polypeptide decreases at the elevated temperature. The levels of 28,000 M(r) species remain virtually unchanged throughout the temperature treatment period. Changes in chloroplast polypeptide composition were also studied in cells that were allowed to recover at room temperature from an initial treatment at 33 degrees C. Bleaching Euglena could provide a useful tool for studying the interaction between the nucleus and chloroplast genetic system that govern the development and maintenance of this vital organelle to plants.

Preparation and Characterization of Membranes from a Coryneform Isolated from Ratoon Stunted Sugarcane

AbstractMembranes from the coryneform were prepared by an optimized chemical procedure using EDTA and lysozyme and isolated upon equilibrium sucrose density gradient ultracentrifugation. Four bands, I to IV, of peak densities 1.15, 1.19, 1.21 and 1.245 g. cm−3 respectively, were reproducibly obtained. Immunodiffusion of the isolated membrane bands against bacterial antiserum showed that the immunological integrity of the isolated membranes was retained. Membrane bands I and II had similar precipitin arcs having one major component. A shift towards greater immunological complexity in band IV was observed. Similarly, a change in polypeptide composition established by polyacrylamide gel electrophoresis was found. The major group of polypeptides inband I ranging in size from 39 to 67 kdal were successively reduced in bands II, III and IV whereas the polypeptide of 15.7 kdal and of those in the region of 140 kdal showed respective increases. Transmission electron microscopy revealed similar membrane, structures in all membrane bands with only minor differences between them.

Changes in polypeptide composition and grain quality due to sulfur deficiency in wheat

Qualitative and quantitative differences in protein composition were examined in 12 samples of flour milled from wheat (cultivar Olympic) grown under a wide range of sulfur and nitrogen inputs. Two-dimensional fractionation, comprising isoelectric focusing in one dimension and electrophoresis at pH 3 in the other, indicated that the changes in gliadin composition associated with sulfur deficiency involved the proportions of individual gliadins but not their charge, size or isoelectric point characteristics. Quantitative Polyacrylamide gel electrophoresis in the presence of SDS showed that sulfur deficiency resulted in increases in the proportions of polypeptides in the mol.wt. ranges 51,000 to 80,000 and above, but decreases in the proportions of lower mol.wt. polypeptides (8000 to 28,000, mainly albumins). The proportions of polypeptides in five size ranges were highly correlated with the sulfur to nitrogen ratios in the flour samples and also with their dough quality characteristics, particularly as measured with the Extensograph. Doughs produced from the variety, Olympic, usually are extensible but the increased proportion of high mol.wt. polypeptides, found in the sulfur-deficient samples, was associated with an increase in the toughness and a decrease in the extensibility of the dough.

Modification of human epidermal prekeratin in the region of granular and transient cell layers.

ABSTRACTTo investigate the process of keratin formation, changes in polypeptide composition of epidermal keratin and of prekeratin were examined using serial slices of human skin. In most of the skin surface, prekeratin was composed of 70K, 58K, 56K, 49.5K and occasionally 55K polypeptides, and an additional 63K polypeptide was present in the skin of the palm and sole. The decrease in molecular weight of these prekeratins to that of keratins was observed in the region of the granular and transient cell layers, especially in that of palm and sole. This was partially confirmed by preliminary digestion experiments in vitro. Our present studies suggest that limited proteolysis of higher molecular weight prekeratin(s) is a step in keratin formation of the stratum corneum.