A rice protein library: a data-file of rice proteins separated by two-dimensional electrophoresis

Proteins extracted from embryos, endosperms and leaves of rice were separated by two-dimensional electrophoresis and relative molecular weights and isoelectric points were determined. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane and 85 electroblotted proteins were analyzed by a gas-phase protein sequencer. The N-terminal amino-acid sequences of 27 out of 85 proteins were determined in this manner. The N-terminal regions of the remaining proteins could not be sequenced and they were inferred to have a blocking group at the N-terminus. Among proteins, 11 could be sequenced after deblocking by in situ treatment with pyroglutamyl peptidase. The internal amino-acid sequences of 23 proteins were determined by sequence analysis of peptides obtained by Cleveland peptide mapping. The amino-acid sequences determined here were compared with those of known plant and animal proteins. The concanavalin A-peroxidase method was used to determine whether the 85 proteins were glycosylated and the diagonal electrophoresis method was used to determine whether they contained disulphide bonding. Finally, we constructed a data-file of rice proteins including information on relative molecular weight, isoelectric point, amino-acid sequence, sequence homology, glycosylation, and the presence of disulphide bonding.     

A proteomics approach towards understanding blast fungus infection of rice grown under different levels of nitrogen fertilization

Proteins extracted from leaf blades of rice plants infected with blast fungus, Magnaporthe grisea, were separated by two-dimensional polyacrylamide gel electrophoresis. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane, and 63 proteins were analyzed by a gas-phase protein sequencer. The N-terminal amino acid sequences of 33 out of 63 proteins were determined in this manner. N-terminal regions of the remaining proteins could not be sequenced. The internal amino acid sequences of 12 proteins were determined by sequence analysis of peptides obtained by the Cleveland peptide mapping method. The amino acid sequences were compared with those of known plant and animal protein sequences to understand the nature of these proteins. As expected, leaf blades revealed predominantly the presence of photosynthetic proteins. Using this experimental approach named as proteome analysis, the functional proteins during blast fungus infection of rice with different levels of nitrogen nutrient were analyzed. Twelve proteins which appeared to change with different levels of nitrogen nutrient were identified. It was revealed that the level of ribulose-1,5-bisphosphate carboxylase/oxygenase was increased by top-dressing with nitrogen nutrient. Additionally, the pathogenesis related protein were observed following blast fungus infection using immunoblot analysis. It was conjectured that these proteins might be involved in incompatible interaction in rice plants following blast fungus infection. The information obtained on the amino acid sequences and antibodies interaction is expected to be helpful in predicting the function of these proteins.     

Rice proteomics: a step toward functional analysis of the rice genome

The technique of proteome analysis with two-dimensional PAGE has the power to monitor global changes that occur in the protein expression of tissues and organisms and/or expression that occurs under stresses. In this study, the catalogues of the rice proteome were constructed, and a functional characterization of some of these proteins was examined. Proteins extracted from tissues of rice and proteins extracted from rice under various kinds of stress were separated by two-dimensional PAGE. An image analyzer was used to reveal a total of 10,589 protein spots on 10 kinds of two-dimensional PAGE gels stained by Coomassie Brilliant Blue. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane, and the N-terminal amino acid sequences of 272 of 905 proteins were determined. The internal amino acid sequences of 633 proteins were determined using a protein sequencer or mass spectrometry after enzyme digestion of the proteins. Finally, a data file of rice proteins that included information on amino acid sequences and sequence homologies was constructed. The major proteins involved in the growth and development of rice can be identified using the proteome approach. Some of these proteins, including a calcium-binding protein that turned out to be calreticulin and a gibberellin-binding protein, which is ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice, have functions in the signal transduction pathway. The information thus obtained from the rice proteome will be helpful in predicting the function of the unknown proteins and will aid in their molecular cloning.     

Microsequence analysis of winged bean seed proteins electroblotted from two-dimensional gel

Electroblotting method employing a semidry blotting apparatus for the subsequent protein microsequence analysis (Hirano, 1987) was improved. This method is convenient and allows rapid and efficient transfer of the proteins from a polyacrylamide gel (1 mm thick) onto the Polybrene-coated glass-fiber sheet or polyvinylidene difluoride membrane filter in only 20 min. The electroblotted proteins could be sequenced directly with the gas-phase protein sequencer at a 20-pmole level. This method was applied to the sequence analysis of winged bean seed proteins. A portion of the crude extracts from only one-twentieth of a seed of the winged bean was separated by two-dimensional polyacrylamide gel electrophoresis and electroblotted, and the N-terminal amino acid sequences of the blotted proteins were analyzed. The sequences of about 60% of the blotted major proteins, including nine Kunitz trypsin inhibitor-like proteins with heterogeneity in the N-terminal sequences, a protein that has a homologous sequence to the leghaemoglobin, nitrogen-fixing root nodule-specific protein, and a soybean basic 7S globulin-like protein could be easily identified.     

Deblocking and subsequent microsequence analysis of N alpha-blocked proteins electroblotted onto PVDF membrane.

A method was developed for direct microsequencing of N alpha-acetylated proteins electroblotted onto polyvinylidene difluoride membranes from polyacrylamide gels. N alpha-Acetylated proteins (greater than 32 pmol), including horse heart cytochrome c, five mutants of yeast cytochrome c, and bovine erythrocyte superoxide dismutase, were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The portions of the membrane carrying the bands were cut out and treated with 0.5% polyvinylpyrrolidone in acetic acid solution at 37 degrees C for 30 min. The protein was digested on the membrane with 5-10 micrograms of trypsin at 37 degrees C for 24 h. During tryptic digestion, the resultant peptides were released from the membrane and the N-terminal peptide was efficiently deblocked with 50 mU of acylamino acid-releasing enzyme at 37 degrees C for 12 h. Picomole levels of the deblocked proteins could be sequenced directly by use of a gas-phase protein sequencer.     

用有限酶切法分析蛋白质的氨基酸序列

报道了一个通过有限酶切蛋白质产生多肽片段的方法.蛋白质经单向SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离和用考马斯亮蓝短暂染色后,切下所需的蛋白质带,将其放入另一个SDS-PAGE凝胶的样品槽内,在电泳过程中该蛋白质被蛋白酶如蛋白酶V8降解,所产生的多肽片段随之被分离.电泳结束后,将多肽片段电印迹至聚偏二氟乙烯(polyvinylidene difluoride,PVDF)膜上.这些多肽片段从PVDF膜上切下后可以直接被用于分析氨基酸序列.该方法能广泛适用于分析一般蛋白质和N端被修饰蛋白质的氨基酸序列.     

Electroblotting onto glass-fiber filter from an analytical isoelectrofocusing gel: a preparative method for isolating proteins for N-terminal microsequencing

A new method has been developed for the isolation of proteins for microsequencing. Proteins were separated by isoelectric focusing on polyacrylamide slab gels. Ampholytes in the gel were washed out with 3.5% (v/v) perchloric acid, and the proteins were electroblotted onto unmodified glass-fiber sheets. The immobilized proteins on the glass-fiber sheet were detected with Coomassie blue dye staining. The protein bands were then excised from the sheet and inserted into a gas phase sequenator for direct sequencing. They could also be extracted with sodium dodecyl sulfate buffer for molecular weight determination. Bovine serum albumin, beta-lactoglobulin A, and soybean trypsin inhibitor have been used as standard proteins for the test of this technique. Using this technique, we have determined the partial N-terminal sequence (26 residues) of an acidic (pI 5.6) glutathione S-transferase isolated from the chicken liver.     

Proteome Analysis of Light-induced Proteins in Synechocystis sp. PCC 6803: Identification of Proteins Separated by 2D-PAGE Using N-terminal Sequencing and MALDI-TOF MS

The cyanobacterium Synechocystis sp. PCC 6803 is an ideal model organism for the proteome study of light-induced gene expression because the whole genomic sequence has been determined. The soluble proteins extracted from light- and dark-cultured cells were separated by two-dimensional polyacrylamide gel electrophoresis. Light-induced protein spots electroblotted on a polyvinyldiene difluoride membrane were analyzed by N-terminal Edman sequence determination and followed by CyanoBase. The tryptic digests of some proteins were also confirmed by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) and MS-Fit search. Interestingly, eight proteins were related to photosynthesis and respiration (RbcS/L, CbbA, Gap2, AtpB, CpcB, PsbO, and PsbU). Four proteins (SodB, DnaK, GroEL2, and Tig) were involved in cellular processes and the functions of another two proteins (rehydrin and membrane protein) were unknown. The proteome analysis by N-terminal Edman sequencing and MALDI-TOF enabled us to characterize one-shot protein profiles expressed under different physiological conditions.     

Structural homology between semidwarfism-related proteins and glutelin seed protein in rice (Oryza sativa L.)

Summary Two semidwarfism-related proteins, SRP-1 and SRP-2, were detected as major spots in a long-culm rice cultivar, Norin 29 and its semidwarf near-isogenic line, SC-TN1, respectively, by two-dimensional gel electrophoresis (2D-PAGE). The testcross showed that SRP-1 and SRP-2 are controlled by codominant alleles, Srp-1 and Srp-2, respectively, at a single locus Srp. This locus was considered to be closely linked with the semidwarfing gene locus sd-1. SRP-1 and SRP-2 were separated by 2D-PAGE, electroblotted onto a polyvinylidene difluoride membrane, and sequenced by a gas-phase protein sequencer. The N-terminal amino acid sequences, however, could not be determined due to the blockage of the N-terminals of these proteins. After removal of the N-terminal residue with pyroglutamyl peptidase given to the membrane, the amino acid sequence in the N-terminal region was determined. The N-terminal and internal amino acid sequences of SRP-1 and SRP-2 were highly homologous with those of the glutelin -subunits of seed endosperm storage protein, which were deduced by the cDNA sequences. In the seed endosperms of Norin 29 and SC-TN1, a total of eight glutelin -subunits was identified by 2D-PAGE. The amino acid sequences in the N-terminal and internal regions of these proteins were determined. This experiment confirmed that SRP-1 and SRP-2 are almost identical in structure with the glutelin _5a- and _5b-subunits, respectively, which were identified in several organs such as endosperms, embryos, and leaves, unlike the other glutelin -subunits.     

Proteome Analysis of Light-induced Proteins in Synechocystis sp. PCC 6803: Identification of Proteins Separated by 2D-PAGE Using N-terminal Sequencing and MALDI-TOF MS

The cyanobacterium Synechocystis sp. PCC 6803 is an ideal model organism for the proteome study of light-induced gene expression because the whole genomic sequence has been determined. The soluble proteins extracted from light- and dark-cultured cells were separated by two-dimensional polyacrylamide gel electrophoresis. Light-induced protein spots electroblotted on a polyvinyldiene difluoride membrane were analyzed by N-terminal Edman sequence determination and followed by CyanoBase. The tryptic digests of some proteins were also confirmed by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) and MS-Fit search. Interestingly, eight proteins were related to photosynthesis and respiration (RbcS/L, CbbA, Gap2, AtpB, CpcB, PsbO, and PsbU). Four proteins (SodB, DnaK, GroEL2, and Tig) were involved in cellular processes and the functions of another two proteins (rehydrin and membrane protein) were unknown. The proteome analysis by N-terminal Edman sequencing and MALDI-TOF enabled us to characterize one-shot protein profiles expressed under different physiological conditions.     

Characterization of a lectin-binding storage protein from pea (Pisum sativum)

From the storage proteins of the pea (Pisum sativum), the fraction which interacts with the pea lectin by the sugar-binding site was studied. By electrophoretical subunit patterns and other criteria, this fraction resembles the group of the 7S storage proteins (vicilins). The fraction was resolved into subunits by micropreparative SDS PAGE. The N-terminal sequences of the individual subunits were determined. Most of these are identical with published vivilin subunit sequences; therefore this lectin-binding fraction belongs to the vicilins. Selected subunits and tryptic fragments were analysed for amino-acid compositions. Though unequivocal assignments to vicilin segments were possible, significant differences could be recognized, in particular in the tryptic fragments.     

A putative acyl-CoA-binding protein is a major phloem sap protein in rice (Oryza sativa L.)

The N-terminal amino-acid sequence of a major rice phloem-sap protein, named RPP10, was determined. RPP10 is encoded by a single gene in the rice genome. Its complete amino-acid sequence, predicted from the corresponding rice full-length cDNA, showed high similarity to plant acyl-CoA-binding proteins (ACBPs). Western blot analysis using anti-ACBP antiserum revealed that putative ACBP is abundant in the phloem sap of rice plants, and is also present in sieve-tube exudates of winter squash (Cucurbita maxima), oilseed rape (Brassica napus), and coconut palm (Cocos nucifera). These findings give rise to the idea that ACBP may involve lipid metabolism and regulation in the phloem.     

槲寄生蛋白抗癌活性研究及新成分鉴定

目的评价槲寄生蛋白的抗癌活性,鉴定其中的新成分。方法以H22肝癌移植瘤为模型,评价槲寄生蛋白对肿瘤生长的抑制作用。采用CMSepharoseF．F．弱阳离子交换色谱,分离出一种低含量的槲寄生蛋白。基质辅助激光解吸飞行时间质谱测定其分子量。蛋白电泳后转印PVDF膜,采用Edman降解,测定该成分A,B两链的N端序列。结果槲寄生蛋白对H22的抑制率达80．2％,其中的CMO为未见报道的新成分。结论槲寄生总蛋白主要含有3种成分,且具有显著的抗癌活性。     

Cloning and characterization of a gene encoding wheat starch synthase I

 A cDNA clone, and a corresponding genomic DNA clone, containing full-length sequences encoding wheat starch synthase I, were isolated from a cDNA library of hexaploid wheat (Triticum aestivum) and a genomic DNA library of Triticum tauschii, respectively. The entire sequence of the starch synthase-I cDNA (wSSI-cDNA) is 2591 bp, and it encodes a polypeptide of 647 amino-acid residues that shows 81% and 61% identity to the amino-acid sequences of SSI-type starch synthases from rice and potato, respectively. In addition, the putative N-terminal amino-acid sequence of the encoded protein is identical to that determined for the N-terminal region of the 75-kDa starch synthase present in the starch granule of hexaploid wheat. Two prominent starch synthase activities were demonstrated to be present in the soluble fraction of wheat endosperm by activity staining of the non-denaturing PAGE gels. The most anodal band (wheat SSI) shows the highest staining intensity and results from the activity of a 75-kDa protein. The wheat SSI mRNA is expressed in the endosperm during the early to mid stages of wheat grain development but was not detected by Northern blotting in other tissues from the wheat plant. The gene encoding the wheat SSI (SsI-D1) consists of 15 exons and 14 introns, similar to the structure of the rice starch synthase-I gene. While the exons of wheat and rice are virtually identical in length, the wheat SsI-D1 gene has longer sequences in introns 1, 2, 4 and 10, and shorter sequences in introns 6, 11 and 14, than the corresponding rice gene. Received: 5 June 1998 / Accepted: 29 September 1998     

A proteomic analysis of leaf sheaths from rice

The proteins extracted from the leaf sheaths of rice seedlings were separated by 2-D PAGE, and analyzed by Edman sequencing and mass spectrometry, followed by database searching. Image analysis revealed 352 protein spots on 2-D PAGE after staining with Coomassie Brilliant Blue. The amino acid sequences of 44 of 84 proteins were determined; for 31 of these proteins, a clear function could be assigned, whereas for 12 proteins, no function could be assigned. Forty proteins did not yield amino acid sequence information, because they were N-terminally blocked, or the obtained sequences were too short and/or did not give unambiguous results. Fifty-nine proteins were analyzed by mass spectrometry; all of these proteins were identified by matching to the protein database. The amino acid sequences of 19 of 27 proteins analyzed by mass spectrometry were similar to the results of Edman sequencing. These results suggest that 2-D PAGE combined with Edman sequencing and mass spectrometry analysis can be effectively used to identify plant proteins.     

Screening of Rice Genes from the cDNA Catalog Using the Data Obtained by Protein Sequencing

The partial amino acid sequences of 121 rice proteins separated by two-dimensional gel electrophoresis (2D-PAGE), were determined for a protein sequence data file. In the Rice Genome Research Program (RGP), more than 20,000 cDNA clones randomly selected from rice cDNA libraries have been sequenced to construct a cDNA catalog. Complimentary DNAs encoding about 30% of proteins in the protein sequence data file could be identified in the catalog by computer search. It was deduced that 20,000–40,000 genes are present in the rice genome. Only half of about 20,000 cDNAs sequenced in the RGP, corresponding to 1/4–1/2 of genes present in the entire rice genome, should have unique sequences after considering gene redundancy. This is consistent with the fact that the cDNAs encoding about 30% of the sequenced proteins could be identified in the catalog. If the size of the cDNA catalog is enlarged further, cDNAs encoding all proteins separated by 2D-PAGE could be easily identified from the catalog by using the protein sequence data.     

Amino-acid and cDNA nucleotide sequences of human Clara cell 10 kDa protein

A human lung cDNA expression library was screened by using a rabbit antiserum specific for a human Clara cell 10 kDa protein. The cDNA from two positive clones was sequenced by the dideoxy chain termination method. The nucleotide and primary amino-acid sequence deduced therefrom are presented. The N-terminal amino-acid sequence of the Clara cell 10 kDa protein, purified from bronchoalveolar lavage, was also determined. The deduced and experimentally determined sequences were identical where data for both were available. From the amino-acid composition, deduced and experimentally determined amino-acid sequences, it was determined that the 10 kDa protein in bronchoalveolar lavage consists of two identical 70-amino-acid long polypeptide chains joined by two cystine residues. The size of mRNA for the protein was found to be about 0.6 kb and the monomeric nascent protein, obtained by in vitro translation of lung mRNA was about 7.3 kDa in size. The 10 kDa protein recovered from bronchoalveolar lavage has 61% sequence identity with rabbit uteroglobin, the two proteins have common predicted secondary structures with marked surface differences when comparing predicted and actual structure determined by X-ray diffraction. The differences imply similarity of structure but, not identity of function.     

The homology between the serum proteins PO2 in pig, Xk in horse and alpha 1B-glycoprotein in human

1. Pig serum Po2 protein and horse Xk protein were purified by FPLC, non-denaturing 2D agarose-PAGE and 2D IPG-PAGE. 2. The separated fractions were electroblotted to poly(4-vinyl-N-methylpyridinium iodide) coated GF/C glass fiber sheets. 3. The partial amino acid sequences and amino acid compositions of different genetic variants of the proteins were determined. 4. The results proved that previously reported polymorphic serum post-albumins in each of these species were homologous to human plasma alpha 1B-glycoprotein.     

Identification of several new classes of low-molecular-weight wheat gliadin-related proteins and genes

During the initial phases of a wheat endosperm Expressed-Sequence-Tag (EST) project, several clones were determined to be related to wheat gliadin sequences, but not similar enough to be classified into any of the traditional gliadin families [α-, γ-, and ω-gliadins, low-molecular-weight (LMW) glutenins]. Complete sequences of these cDNA clones revealed four new classes of gliadin-related endosperm proteins, but lacking a prominent repeat domain which until now has been characteristic of the gliadins. Two of these classes are related to different minimally described groups of Triticeae endosperm proteins. One class of proteins, which has N-terminal amino-acid sequences matching members of a reported 25-kDa globulin family from wheat, is shown by amino-acid sequencing to match to a family of 25-kDa endosperm proteins, is encoded by a multigene family, and is most similar to the LMW-glutenins. A second new class shows N-terminal homologies to LMW secalins from rye, and has an amino-acid composition similar to wheat and barley LMW proteins with extraction properties similar to prolamins. The third class is most similar to α-gliadins, and the fourth class has no close association to previously described wheat endosperm proteins. Received: 20 October 2000 / Accepted: 20 November 2000     

Amino-terminal amino acid sequences of electron transfer proteins from Gram-negative bacteria as indicators of their cellular localization: the sulfate-reducing bacteria

Abstract Data are presented that all known periplasmic redox proteins from the sulfate reducing bacteria included in the genus, Desulfovibrio have aminoterminal (N-terminal) amino-acid sequences commonly found in other Gram-negative bacteria and are indicative of recognition sites for signal peptides. In contrast, none of the cytoplasmic redox proteins exhibited these unique N-terminal amino-acid sequences. It is proposed that the N-terminal amino-acid residues of a given protein can be used as an indicator of its cellular localization within the bacterial cell.