Purification and characterization of a membrane-bound ATP diphosphohydrolase from Cicer arietinum (chick-pea)roots.
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ABSTRACT:
SUBMITTER: Vara F
PROVIDER: S-EPMC1163175 | biostudies-other | 1981 Sep
REPOSITORIES: biostudies-other
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Project description:1. A trypsin and chymotrypsin inhibitor was isolated by extraction of chick-pea meal at pH8.3, followed by (NH4)2SO4 precipitation and successive column chromatography on CM-cellulose and calcium phosphate (hydroxyapatite). 2. The inhibitor was pure by polyacrylamide-gel and cellulose acetate electrophoresis and by isoelectric focusing in polyacrylamide gels. 3. The inhibitor had a molecular weight of approx. 10000 as determined by ultracentrifugation and by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. A molecular weight of 8300 was resolved from its amino acid composition. 4. The inhibitor formed complexes with trypsin and chymotrypsin at molar ratios of 1:1. 5. Limited proteolysis of the inhibitor with trypsin at pH3.75 resulted in hydrolysis of a single-Lys-X-bond and in consequent loss of 85% of the trypsin inhibitory activity and 60% of the chymotrypsin inhibitory activity. Limited proteolysis of the inhibitor with chymotrypsin at pH3.75 resulted in hydrolysis of a single-Tyr-X-bond and in consequent loss of 70% of the trypsin inhibitory activity and in complete loss of the chymotrypsin inhibitory activity. 6. Cleavage of the inhibitor with CNBr followed by pepsin and consequent separation of the products on a Bio Gel P-10 column, yielded two active fragments, A and B. Fragment A inhibited trypsin but not chymotrypsin, and fragment B inhibited chymotrypsin but not trypsin. The specific trypsin inhibitory activity, on a molar ratio, of fragment A was twice that of the native inhibitor, suggesting the unmasking of another trypsin inhibitory site as a result of the cleavage. On the other hand, the specific chymotrypsin inhibitory activity of fragment B was about one-half of that of the native inhibitor, indicating the occurrence of a possible conformational change.
| S-EPMC1163917 | biostudies-other
Project description:The lectin isolated from mature seeds of Cicer arietinum (CAL) agglutinates pronase-treated rabbit and human erythrocytes and its haemagglutination activity is inhibited by fetuin and desialated fetuin but not by simple monosaccharides or oligosaccharides. The purified lectin is a dimer of molecular weight 43,000 Da composed of two identical subunits (MW 21,500), as confirmed by SDS-PAGE. The lectin has been crystallized using the hanging-drop vapour-diffusion method at 295 K over a well solution containing 0.2 M sodium acetate, 0.1 M sodium phosphate buffer pH 6.5 and 14%(w/v) polyethylene glycol 8000. The triangular prism-shaped crystals belong to space group R3 and have unit-cell parameters a = b = 81.2, c = 69.4 A. The diffraction data are 93.8% complete to 2.3 A Bragg spacing with an Rmerge of 0.103.
| S-EPMC1952404 | biostudies-literature
Project description:UNLABELLED: BACKGROUND:Compartmentalization is a unique feature of eukaryotes that helps in maintaining cellular homeostasis not only in intra- and inter-organellar context, but also between the cells and the external environment. Plant cells are highly compartmentalized with a complex metabolic network governing various cellular events. The membranes are the most important constituents in such compartmentalization, and membrane-associated proteins play diverse roles in many cellular processes besides being part of integral component of many signaling cascades. RESULTS:To obtain valuable insight into the dynamic repertoire of membrane proteins, we have developed a proteome reference map of a grain legume, chickpea, using two-dimensional gel electrophoresis. MALDI-TOF/TOF and LC-ESI-MS/MS analysis led to the identification of 91 proteins involved in a variety of cellular functions viz., bioenergy, stress-responsive and signal transduction, metabolism, protein synthesis and degradation, among others. Significantly, 70% of the identified proteins are putative integral membrane proteins, possessing transmembrane domains. CONCLUSIONS:The proteomic analysis revealed many resident integral membrane proteins as well as membrane-associated proteins including those not reported earlier. To our knowledge, this is the first report of membrane proteome from aerial tissues of a crop plant. The findings may provide a better understanding of the biochemical machinery of the plant membranes at the molecular level that might help in functional genomics studies of different developmental pathways and stress-responses.
| S-EPMC3558352 | biostudies-literature
Project description:A haemoprotein was purified to apparent homogeneity from Bengal-gram seeds. The purified protein exhibited an absorption maximum at 412 nm (Soret band) that upon reduction with dithionite gave rise to a shift in the Soret band to 426 nm with concomitant appearance of an alpha-band at 559 nm and a beta-band at 530 nm. In the reduced state the Bengal-gram haemoprotein showed reactivity towards CO, nitrite and hydroxylamine. SDS/polyacrylamide-slab-gel electrophoresis showed that the haemoprotein has Mr 78,000. Gel-filtration and ultracentrifugal analyses suggest that the Bengal-gram haemoprotein is oligomeric in nature. Since it differs from photosynthetic membrane cytochrome b-559 in solubility in buffer, in reactivity towards CO and in molecular size, it appears to be a novel haemoprotein b-559.
| S-EPMC1147918 | biostudies-other
Project description:The total RNA were extracted from pooled tissues of leaves and flowers from several plants of chickpea (Cicer arietinum) using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Then small RNAs ranging in 18–30 nucleotides were size fractionated electrophoretically, isolated from the gel, ligated with the 5′ and 3′ RNA adapters. The ligated product was reverse transcribed and subsequently amplified using 10–12 PCR cycles. The purified PCR product was sequenced using Illumina Genome Analyzer II. The qualified reads were used to predict microRNAs and phased small interfering RNAs from chickpea. Identification of microRNAs and phased small inferfering RNAs in chickpea (Cicer arietinum) by analyzing small RNA sequencing profiles of leaves and flowers using Illumina GAII.
2015-05-08 | E-GEOD-62216 | biostudies-arrayexpress
Project description:The total RNA were extracted from pooled tissues of leaves and flowers from several plants of chickpea (Cicer arietinum) using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Then small RNAs ranging in 18–30 nucleotides were size fractionated electrophoretically, isolated from the gel, ligated with the 5′ and 3′ RNA adapters. The ligated product was reverse transcribed and subsequently amplified using 10–12 PCR cycles. The purified PCR product was sequenced using Illumina Genome Analyzer II. The qualified reads were used to predict microRNAs and phased small interfering RNAs from chickpea.
2015-05-08 | GSE62216 | GEO
Project description:Chickpea has a profound nutritional and economic value in vegetarian society. Continuous decline in chickpea productivity is attributed to insufficient genetic variability and different environmental stresses. Chickpea like several other legumes is highly susceptible to terminal drought stress. Multiple genes control drought tolerance and ASR gene plays a key role in regulating different plant stresses. The present study describes the molecular characterization and functional role of Abscissic acid and stress ripening (ASR) gene from chickpea (Cicer arietinum) and the gene sequence identified was submitted to NCBI Genbank (MK937569). Molecular analysis using MUSCLE software proved that the ASR nucleotide sequences in different legumes show variations at various positions though ASR genes are conserved in chickpea with only few variations. Sequence similarity of ASR gene to chickpea putative ABA/WDS induced protein mRNA clearly indicated its potential involvement in drought tolerance. Physiological screening and qRT-PCR results demonstrated increased ASR gene expression under drought stress possibly enabled genotypes to perform better under stress. Conserved domain search, protein structure analysis, prediction and validation, network analysis using Phyre2, Swiss-PDB viewer, ProSA and STRING analysis established the role of hypothetical ASR protein NP_001351739.1 in mediating drought responses. NP_001351739.1 might have enhanced the ASR gene activity as a transcription factor regulating drought stress tolerance in chickpea. This study could be useful in identification of new ASR genes that play a major role in drought tolerance and also develop functional markers for chickpea improvement.
| S-EPMC7360048 | biostudies-literature
Project description:The membrane-bound NAC transcription (NTL) factors have been demonstrated to participate in the regulation of plant development and the responses to multiple environmental stresses. This study is aimed to functionally characterize soybean NTL transcription factors in response to Al-toxicity, which is largely uncharacterized. The qRT-PCR assays in the present study found that thirteen out of fifteen GmNTL genes in the soybean genome were up-regulated by Al toxicity. However, among the Al-up-regulated GmNTLs selected from six duplicate gene pairs, only overexpressing GmNTL1, GmNTL4, and GmNTL10 could confer Arabidopsis Al resistance. Further comprehensive functional characterization of GmNTL4 showed that the expression of this gene in response to Al stress depended on root tissues, as well as the Al concentration and period of Al treatment. Overexpression of GmNTL4 conferred Al tolerance of transgenic Arabidopsis in long-term (48 and 72 h) Al treatments. Moreover, RNA-seq assay identified 517 DEGs regulated by GmNTL4 in Arabidopsis responsive to Al stress, which included MATEs, ALMTs, PMEs, and XTHs. These results suggest that the function of GmNTLs in Al responses is divergent, and GmNTL4 might confer Al resistance partially by regulating the expression of genes involved in organic acid efflux and cell wall modification.
| S-EPMC8657865 | biostudies-literature
Project description:Small RNA Solexa sequencing of chickpea (Cicer arietinum)
| PRJNA263491 | ENA
Project description:Comparative transcriptome analysis of nodules of two Mesorhizobium-chickpea associations with differential symbiotic nitrogen fixation capacity under phosphate deficiency
| PRJDB5244 | ENA