Two-Dimensional Electrophoretic Analysis of Salicylic Acid-Induced Changes in Polypeptide Pattern of Barley Leaves

The salicylic acid-induced changes in the polypeptide patterns of barley (Hordeum vulgare L.) leaves have been analysed using two-dimensional gel electrophoresis. An optimized 2-D PAGE protocol was used and gave reproducible 2-D gels from leaf crude protein extracts with a high number of detected polypeptides. When applied for 24 h SA affected the expression of a number of soluble proteins. Most of them appeared to be down-regulated. Although no abundant expression of specific proteins was observed, we detected three polypeptides that were present only in SA-treated leaves.     

Regulation of polypeptide synthesis during Caulobacter development: two-dimensional gel analysis.

The gram-negative bacterium Caulobacter crescentus progresses through three distinct morphological transitions, including both motile and nonmotile cell types, during its cell cycle. Assessment of the extent of regulation of polypeptide synthesis during these transitions was carried out with two-dimensional gel electrophoresis of whole-cell extracts. Synchronous cells were pulse-labeled with 14C-amino acids for 10-min intervals throughout the entire 2-h cell cycle. The radioactively labeled polypeptides were analyzed by two-dimensional polyacrylamide gel electrophoresis. Autoradiograms resulting from fluorography of the second dimension provided the detection of approximately 1,000 unique spots. The 600 predominant polypeptide spots, representing approximately 40% of the coding capacity of Caulobacter deoxyribonucleic acid, were analyzed for major changes in their synthetic rates. Quantitation by densitometric scanning of individual polypeptide spots represented on the sequential fluorograms demonstrated significant changes in the temporal synthesis of 6% of the polypeptides. Extracts from asynchronous cells were fractionated to obtain total-membrane and deoxyribonucleic acid-binding polypeptide fractions. Subsequent electrophoresis of these cellular fractions revealed approximately 100 membrane polypeptides and 25 deoxyribonucleic acid-binding polypeptides. Eight of the regulated polypeptides were identified as membrane or deoxyribonucleic acid-binding proteins. The regulated polypeptides can be grouped into three main categories based on their interval of synthesis. The three categories are in direct correlation with the three distinct cell cycle stages. This analysis has also revealed a unique transition period in the cell cycle in which a significant proportion of gene expression is regulated.     

Two-dimensional electrophoretic analyses of proteins synthesized during differentiation of 3T3-L1 preadipocytes.

The changes in protein composition and cell surface proteins that occur during the adipocyte conversion of 3T3-L1 preadipocytes were monitored by two-dimensional polyacrylamide gel electrophoresis folowing incubation of cells with [35S]methionine for periods of 3 and 24 h. Alterations in the biosynthesis of more than 30 cytoplasmic proteins, 9 non-histone, chromosome-associated proteins, and 24 membrane proteins, were detected. Although the methodological limitations of the electrophoretic systems employed result in an underestimate of the total number of differences, the alterations observed exceed the enzyme changes known to occur during differentiation of these cells. One major alteration occurring during differentiation is a decrease in the content of a protein whose position following two-dimensional electrophoresis tentatively identified it as actin. A fall in actin content accompanying adipocyte conversion was confirmed by direct analysis of the DNase 1 inhibitory activity in homogenates prepared from cells during the course of differentiation. Studies of cell surface proteins by lactoperoxidase-catalyzed iodination reveal a number of changes during differentiation including an increase in a polypeptide(s) in the molecular weight range of 16,500 to 18,500, a decrease in at least four proteins of molecular weights greater than 100,000, and in a protein of molecular weight 95,000.     

Changes in protein and mRNA populations during the senescence of carnation petals

Developmental changes in polypeptide and mRNA popultions in carnation ( Dianthus caryophyllus L. cv. White Sim) petals were investigated during the senescence of harvested flowers. Total proteins were extracted from flower petals at various stages of senescence and subjected to separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Analysis of the Coomassie blue stained gels revealed polypeptides with apparent molecular weights of 76, 62, 35.5 and 24 kDa which increased, while those with molecular weights of 70.5, 67.5, 46.5 and 31 kDa decreased during petal senescence. Changes in mRNA populations were investigated by translating poly (A)⁺RNA, isolated from carnation petals, in vitro using the rabbit reticulocyte lysate system. Polypeptides synthesized in vitro were separated by one- and two-dimensional gel electrophoresis and visualized by fluorography. Three classes of mRNA's were associated with the senescence of carnation petals. The majority of the mRNA's were constitutive at all stages of senescence. Another class of mRNA's increased with the climacteric rise in ethylene production, which accompanied the onset of senescence. Their translation products were 81, 58, 42, 38 and 35 kDa. In addition, several mRNA's appeared to decrease in abundance during the course of petal senescence. These results indicate that senescence of carnation flower petals is associated with changes in gene expression.     

Polypeptide changes associated with loss of proliferative potential during the terminal event in differentiation

The differentiation of murine mesenchymal stem cells occurs in nonterminal and terminal phases. In previous reports we established the characteristics of nonterminally differentiated cells and showed that transition from the nonterminal to the terminal state of differentiation can be induced by human plasma. We also showed that this transition is blocked by protein synthesis inhibitors and other pharmacological agents. In this paper, we have employed two-dimensional gel electrophoresis to evaluate changes in specific polypeptides that are induced when cells lose proliferative capacity associated with the terminal event in differentiation. Using silver staining procedures for analysis of electrophoretograms, we detected only seven major polypeptide differences between nonterminally differentiated and terminally differentiated cells. Six polypeptides were expressed only in preparations of terminally differentiated cells; these included two polypeptides identified in cytosolic fractions and four polypeptides identified in nuclear fractions. One polypeptide was also found to be selectively expressed only in nuclear fractions of nonterminally differentiated cells. Based on these observations we conclude that the loss of proliferative potential that occurs during the terminal event in mesenchymal stem cell differentiation is associated with changes in the composition of a limited number of specific polypeptides. We suggest that one or more of these polypeptides may be important in the regulation of cellular proliferation.     

Posttranslational modifications as a cause of gonadal polypeptide diversity in the rat

The testicular and ovarian protein patterns of prepubertal rats were studied by two-dimensional gel electrophoresis after both in vivo and in vitro translation. Analysis of the approximately 750 polypeptide spots detectable in gonadal extracts (in vivo translated polypeptides) revealed 14 testis-specific, and 2 ovary-specific spots. Conversely, only 4 testis- and 2 ovary-specific polypeptide spots (of a total of about 600) were detected after in vitro translation of the respective polyadenylated RNAs using the reticulocyte lysate system. Simultaneous in vitro translation plus microsome-mediated cotranslational modification increased the number (of testicular spots to 8, and of ovarian spots to 3 (of a total of 600). The polypeptides synthesized in vitro were well comparable with respect to molecular weight and isoelectric point to those synthesized in vivo. Therefore, testicular polypeptide diversity is mainly the result of co/posttranslational modifications. Many of these proteins appeared to be glycosylated as indicated by their specific binding to Concanavalin A. The system used demonstrates that co/posttranslational modifications play a role in the establishment of polypeptide and thus organ diversity.     

Plastid Development in Pisum sativum Leaves during Greening : I. A Comparison of Plastid Polypeptide Composition and in Organello Translation Characteristics

Changes in plastid polypeptide composition during greening of etiolated peas were investigated by two-dimensional gel electrophoresis. One hundred of the more than 250 polypeptides which could be detected upon silver staining were followed during plastid development. Thirty-nine polypeptides decreased in abundance on a per organelle basis. Twentythree of the 46 polypeptides which increased in abundance upon greening could be identified as proteins of the thylakoid membrane. The changes in proteins observed during greening of etiolated leaves corresponded largely to those observed during normal leaf expansion. The origin of some of the polypeptides was traced back by comparing the two-dimensional gels of plastid proteins with in organello translation products and with polypeptides which had been synthesized in vitro from poly(A⁺) mRNA preparations and posttranslationally imported by chloroplasts. Some polypeptides were specifically identified in two-dimensional gels by Western blot analysis.     

Basic plasticity of protein expression in tobacco leaf plasma membrane

Highly purified plasma membrane fractions were prepared using top-, middle- or bottom-leaf sets of tobacco. Plasma membrane polypeptides were analysed by two-dimensional gel electrophoresis and the abundances of 90 polypeptides non-washed out by Triton X-100 were quantified by image analysis. Under constant environmental conditions, the relative abundances of approximately 75% of polypeptides were shown to change during plant development, irrespective of the position of the leaves on the plant. The greatest plasticity of the plasma membrane polypeptide pattern was observed, first during the early stages of plant development, including floral induction, and, to a lesser extent, during the late stages, including seed formation. Some of the leaf plasma membrane polypeptides were abundant only at specific developmental stages and in specific leaf sets. A similar situation existed during the nycthemeron, where the abundances of approximately 75% of the polypeptides were observed to change during a 24 h period. Some polypeptides appeared to be essentially dark- or light-specific. Overall data analysis supports the conclusion that, under constant environmental conditions, approximately 90% of plant plasma membrane polypeptides are simultaneously subjected to both long-term (day-scaled) and short-term (hour-scaled) dynamics. This unexpected steady-state dynamics (i) corresponds to a new kind of plasticity which has to be distinguished from the phenotypic plasticity shown by plants in response to various changes in environmental factors, (ii) provides new insights into the functioning of membranes and is proposed to constitute a signature of the plant physiological state.     

Analysis of pea chloroplast inner and outer envelope membrane proteins by two-dimensional gel electrophoresis and their comparison with stromal proteins

Analysis of inner and outer pea (Pisum sativum var. Laxtons Progress No. 9) chloroplast envelope membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, although the two membranes have distinct polypeptide compositions, there are several comigrating polypeptides in the two membrane fractions. To determine whether these comigrating polypeptides were identical by criteria other than molecular weight, the membrane proteins were analyzed by two-dimensional gel electrophoresis. The results demonstrated that an 86-kilodalton band found in both membranes represents at least two different polypeptides, one an outer membrane protein and the other an inner membrane protein. Several other polypeptide bands found in both membranes appear to be of stromal origin. Two of these polypeptides were shown to be the large and small subunits of ribulose 1,5-bisphosphate carboxylase. The large subunit was identified by two-dimensional electrophoresis of envelope membranes to which stromal proteins were added. Additionally, the large and small subunits of ribulose 1,5-bisphosphate carboxylase were immunologically identified using an electrophoretic transfer procedure coupled with an enzyme-linked immunosorbent assay. Various treatments, including sonication, resulted in no significant loss of the stromal polypeptides from the outer envelope membranes. Based on these results, it is suggested that the stromal proteins are not simply bound to the outer surface of the vesicles.     

Microsomal polypeptides in sunflower (Helianthus annum). Comparison between normal type before and after cold-induction, and a high oleic acid mutant

Differences in high-resolution two-dimensional gel electrophoresis patterns of micro-somal proteins from developing normal sunflower ( Helianthus annuus L.) seeds before and after cold-induction, and also from normal and a high oleic sunflower mutant have been studied in order to detect the polypeptides associated with the microsomal Δ^l2-desaturase activity and its regulation by temperature. Proteins were obtained from developing seeds of two isogenic sunflower lines HA-89 (normal) and HA-OL9 (high oleic) which greatly differed in linoleic acid content and "in vitro" oleate desaturase activity. In the high oleic mutant, four polypeptides of about 32 kDa and two of 33 kDa were found to change in position, to the same extent, toward a lower isoelectric point in the high oleic mutant. Also, two polypeptides, of 32 and 49 kDa each, appeared in the mutant. Quantitative differences between cold-induced seeds (10°C, 24 h) and their non-induced controls were found. One polypeptide of 43 kDa decreased in the cold-treated seeds and two others, of 30 and 32 kDa each, increased markedly after cold induction. Some of these polypeptides could be related to oleate desatnrase activity or its regulation by temperature.     

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. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis:

Summary Approximately 250 phytohemagglutinin (PHA)-stimulated peripheral blood lymphocyte polypeptides from three unrelated healthy males were compared by high-resolution two-dimensional gel electrophoresis and double-label autoradiography. Comparisons by all possible pairwise combinations of [¹⁴C]leucine-labeled proteins from an individual and [³H]leucine-labeled proteins from another revealed that only three polypeptides differed qualitatively among the three individuals. The degree of variation in lymphocyte polypeptides between different individuals was similar to that in fibroblast polypeptides reported previously. Among the three variant polypeptides, two polypeptides with mol.wt. 64,000 and mol. wt. 37,000 coexisted with a polypeptide with the same molecular weight, and they showed the behavior expected of two allelic gene products separated in the isoelectric focusing dimension by charge differences. Analysis of [¹⁴C]leucine labeled peripheral blood lymphocyte proteints, from the parents of each individual, by two-dimensional gel electrophoresis indicated that the variant polypeptides with mol. wt. 64,000 and mol. wt. 37,000 in the propositus were inherited from one of his parents. The data indicate that genetic analysis of PHA-stimulated peripheral blood lymphocyte proteins is feasible by high-resolution two-dimensional gel electrophoresis in combination with double-label autoradiography and pedigree analysis.     

Nature and timing of some sporulation-specific protein changes in Saccharomyces cerevisiae.

During meiosis and spore formulation in Saccharomyces cerevisiae, changes that occur in a/alpha diploids, but not in isogenic nonsporulating a/a diploids, have been detected in cellular polypeptides. These were found by the technique of prelabeling growing cells with 35SO4(2-) and suspending them in sulfur-free sporulation medium. Under the conditions used, about 400 polypeptides were detected by two-dimensional gel electrophoresis, and 45 were altered during sporulation; of these, 21 changes were specific to a/alpha strains. These alterations were mainly due to the appearance of new polypeptides or to marked increases in the concentrations of a few polypeptides produced during vegetative growth. They could have been due either to modifications of existing polypeptides present in growing cells or to de novo synthesis of new gene products. They occurred at characteristic times during sporulation; whereas the majority of changes took place early (within the first 6 h in sporulation conditions), there were several changes characterizing the later stages of sporulation. Ten of the 35SO4(2-)-labeled polypeptides were also labeled with 32P in the presence of [32P]orthophosphate; of these, three were previously found to be sporulation specific. One of these was phosphorylated at all stages of sporulation and was labeled when [32P]orthophosphate was added either during growth of the culture of 1 h after transfer to sporulation medium. Another was labeled in the same way by adding 32P at either time, so that by 7 h in sporulation medium it was phosphorylated, but was dephosphorylated by 24 h. The third sporulation-specific peptide was labeled in extracts prepared at 7 h in sporulation medium (but not at 24 h) when [32P]-orthophosphate was added during presporulation growth, but not when [32P]-orthophosphate was added 1 h after transfer of the culture to sporulation medium. This polypeptide appeared early during sporulation; it is probably phosphorylated as it appears and is dephosphorylated at some time between 7 h and 24 h of sporulation.     

Correlated induction of nitrate uptake and membrane polypeptides in corn roots

Induction of corn (Zea mays L.) seedling root membrane polypeptides was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis in relation to induction of nitrate uptake. When nitrate uptake was studied using freshly harvested roots from 4-day old corn seedlings, a steady state rate of uptake was achieved after a lag of 2 to 3 hours. The plasma membrane fraction from freshly harvested roots (uninduced) and roots pretreated in 5 millimolar nitrate for 2.5 or 5 hours (induced) showed no differences in the major polypeptides with Coomassie blue staining. Autoradiography of the ³⁵S-methionine labeled proteins, however, showed four polypeptides with approximate molecular masses of 165, 95, 70, and 40 kilodaltons as being induced by both 2.5 and 5-hour pretreatment in 5 millimolar nitrate. All four polypeptides appeared to be integral membrane proteins as shown by Triton X-114 (octylphenoxypolyethoxyethanol) washing of the membrane vesicles. Autoradiography of the two-dimensional gels revealed that several additional low molecular weight proteins were induced. A 5-hour pretreatment in 5 millimolar chloride also induced several of the low molecular weight polypeptides, although a polypeptide of about 30 kilodaltons and a group of polypeptides around 40 kilodaltons appeared to be specifically induced by nitrate. The results are discussed in relation to the possibility that some of the polypeptides induced by nitrate treatment may be directly involved in nitrate transport through the plasma membrane.     

Compositional changes in soluble proteins of cerebral mantle,cerebellum, and brain stem of rat brain during development

Soluble proteins from the cerebral mantle, cerebellum, and brain stem of rat brains were analyzed at various developmental stages by a two-dimensional gel electrophoresis technique. The electrophoretic technique resolved the soluble proteins into 100–150 polypeptide spots on two-dimensional gels and gave reproducible and highly resolved profiles of them. Although most of major polypeptides were commonly found in all the three brain regions, some polypeptides were shown to be unique to a specific brain region. Each brain region was different in the electrophoretic profile of soluble proteins at every developmental stage examined, although there was considerable similarity in the profiles of each of the three brain regions in fetal animals (16–17 days), indicating that soluble proteins undergo different compositional changes in each of the three brain regions during postnatal development. In addition, the number of polypeptide spots on the electrophoretic profile increased remarkably during postnatal development in all of the three brain regions, suggesting that soluble proteins become more heterogeneous during postnatal development in each of the three brain regions.     

Protein Synthesis Initiated by Cell Fusion in Dictyostelium discoideum

D. discoideum has two alternative developmental pathways. If cells of two complement mating-type strains, NC4 and HM1, fuse sexually, a giant cell is produced which subsequently develops into a macrocyst, the sexual structure of this organism. However, if fusion fails to occur and cells are starved, a fruiting-body is produced instead of a macrocyst. In this paper, a two-dimensional polypeptide gel electrophoresis study showed that giant cells produce specific polypeptides which may possibly be involved in macrocyst development. Out of total 497 polypeptides which appeared in a giant cell during an incubation period of 13 hr, 92 were the specific for giant cells. Four of these polypeptides were appeared within only 1 hr after the cell fusion. The other 405 were non-specific polypeptides which appeared in both giant cells and NC4 or/and HM1 cells. However, the patterns and the rates of production of each polypeptide during the incubation period were different between these cells.     

Identification of herpesvirus sylvilagus-induced polypeptides in productively infected cells.

Polypeptides synthesized in cell cultures infected with high multiplicities of herpesvirus sylvilagus were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled cell extracts. Initiation of polypeptide synthesis was detected by 6 h after infection. The maximum intensity of many [35S]methionine-labeled viral bands was observed at 45 h after infection. Production of detectable infectious virus began between 18 and 24 h and reached a plateau at 48 h after infection. Immunoprecipitation of cell extracts identified a minimum of 45 virus-induced polypeptides ranging in molecular weight from 230,000 to 27,000. The major polypeptide appeared to have a molecular weight of 150,000. The pattern of these extracts suggested that the synthesis of host polypeptides is stimulated during the first 12 h and thereafter reduced, but not completely inhibited, during the remaining course of infection.     

Changes in protein phosphorylation during the maturation of mammalian oocytes in vitro

Cumulus-enclosed sheep oocytes were cultured in gonadotrophin-containing medium for up to 9 hr and were then incubated for 3 hr in the presence of [32P]phosphate. The incorporation of 32P into TCA-insoluble material was measured, and oocyte proteins were separated by one- and two-dimensional gel electrophoresis. Incorporation of [32P]phosphate into protein increased after 3 hr culture and again after 9 hr, the time of germinal vesicle breakdown (GVBD). Qualitative and quantitative changes in the phosphorylation of proteins occurred over the 12-hr period studied. One of the most prominent changes was the appearance of a band of Mr 33,000, which was absent at 0-3 hr but appeared with increasing intensity with longer periods of culture. Two-dimensional electrophoresis revealed that the bulk of material in this band was a neutral polypeptide. No significant incorporation of [32P]phosphate was found in ribosomal extracts of oocytes.     

Floral-specific polypeptides of the Japanese morning glory

Polypeptides from stems, leaves, sepals, corollas, stamens and pistils of the Japanese morning glory (Ipomoea nil Roth (Pharbitis nil Chois.)) were separated by one- and two-dimensional gel electrophoresis and visualized by silver staining. The majority of polypeptides were expressed in two or more organs, while those specific to only one organ were comparatively rate. Among the polypeptides of the former class were two which appeared to be floral-specific. A 46-kDa (kilodalton) polypeptide was expressed in corollas, stamens and pistils, whereas a 32-kDa polypeptide was observed only in extracts prepared from reproductive organs. Polypeptide spots from the various organs were compared with those from leaves, and it was found that sepals and stems shared 40–50% of their polypeptides with leaves, whereas corollas, stamens and pistils shared 20% or less. The latter organs shared 120 polypeptides or roughly 15% of those identified in the floral extracts. Floralorgan-specific polypeptides comprised nearly 10% of the total floral polypeptides identified.Abbreviations kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate     

Role of germinal vesicle on protein synthesis in rat oocyte during in vitro maturation

To investigate the role of the germinal vesicle (GV) on in vitro maturation (IVM) of rat oocytes, we examined protein synthesis during IVM by comparing polypeptide patterns in control and enucleated oocytes using one and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separation of polypeptides extracted from the cytoplasm of GV by one-dimensional SDS-PAGE revealed that a 55 kDa polypeptide was present only in the GVs of rat oocytes. At 0, 12, 24, 36, and 44 hr after PMSG injection, prior to the initiation of maturation, enucleated oocytes synthesized the same major polypeptides as cumulus intact (CI) oocytes. During meiotic maturation, no major changes were detected in protein synthesis from prophase (GV stage) to prometaphase I (0–6 hr IVM). However, after entry into prometaphase I (7 hr IVM), striking changes were seen; a 24 kDa polypeptide disappeared and expression of a 34 kDa polypeptide became stronger. This pattern lasted until metaphase II. We detected no major differences in the pattern of protein synthesis between CI and enucleated oocytes using two-dimensional PAGE. These results indicate that protein synthesis in the maturing rat oocyte is controlled by cytoplasmic regulators rather than intrinsic nuclear components. © 1996 Wiley-Liss, Inc.