scholarly journals Mitochondrial сomplexome of etiolated pea shoots

2022 ◽  
Vol 11 (4) ◽  
pp. 570-580
Author(s):  
I. V. Ukolova ◽  
I. G. Kondratov ◽  
M. A. Kondakova ◽  
I. V. Lyubushkina ◽  
O. I. Grabelnykh ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Pyruvate Dehydrogenase Complex ◽  
Energy System ◽  
Physiological Status ◽  
Major Protein ◽  
Functional Categories ◽  
Sds Page ◽  
Acid Processing ◽  
Oxphos System ◽  
Protein Group

Studies into mitochondrial сomplexomes in various organisms provide an insight into the native organization of proteins and metabolic pathways in the organelles of the subject under study. “Complexome” is a relatively recent concept describing the proteome of protein complexes, supercomplexes, and oligomeric proteins. Complexome analysis is performed using current electrophoretic and mass spectrometric techniques, in particular, by two-dimensional electrophoresis (2D BN/SDS-PAGE) in combination with mass spectrometry (MS). Unlike 2D IEF/SDS-PAGE, this method enables analysis of not only hydrophilic proteins of the mitochondrial matrix, but also membrane proteins and their associations, thus expanding the possibilities of studying the organelle proteome. In the present work, the complexome of etiolated pea shoots was studied for the first time using 2D BN/SDS-PAGE followed by MALDI-TOF MS. To this end, 145 protein spots excised from the gel were analyzed; 110 polypeptides were identified and assigned to different functional groups. A densitometric analysis revealed that the major protein group comprised the enzymes of the mitochondrial energy system (1), accounting for an average of 43% of the total polypeptide content. The remaining 57% was primarily distributed among the following functional categories: pyruvate dehydrogenase complex and citric acid cycle (2); amino acid metabolism (3); nucleic acid processing (4); protein folding (5); antioxidant protection (6); carrier proteins (7); other proteins (8); proteins having unknown functions (9). The obtained data indicate the complex organization of the pea proteome. In addition to the enzymes of the OXPHOS system, the proteins of other functional categories are found to form supramolecular structures. It is suggested that the presence of proteins from other cellular compartments may indicate the interaction of mitochondria with the enzymes or structures of corresponding organelles. In general, the obtained data on the pea complexome represent a kind of a mitochondrial “passport” that reflects the native state of the proteome of organelles corresponding to their physiological status.

scholarly journals Unveiling Putative Functions of Mucus Proteins and Their Tryptic Peptides in Seven Gastropod Species Using Comparative Proteomics and Machine Learning-Based Bioinformatics Predictions

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3475
Author(s):  
Viroj Tachapuripunya ◽  
Sittiruk Roytrakul ◽  
Pramote Chumnanpuen ◽  
Teerasak E-kobon
Keyword(s):  
Bioactive Peptides ◽  
Nearest Neighbor ◽  
Peptide Identification ◽  
Comparative Proteomics ◽  
Mass Spectrometric ◽  
Functional Categories ◽  
K Nearest Neighbor ◽  
Gastropod Species ◽  
Bioactive Properties ◽  
Sds Page

Gastropods are among the most diverse animals. Gastropod mucus contains several glycoproteins and peptides that vary by species and habitat. Some bioactive peptides from gastropod mucus were identified only in a few species. Therefore, using biochemical, mass spectrometric, and bioinformatics approaches, this study aimed to comprehensively identify putative bioactive peptides from the mucus proteomes of seven commonly found or commercially valuable gastropods. The mucus was collected in triplicate samples, and the proteins were separated by 1D-SDS-PAGE before tryptic digestion and peptide identification by nano LC-MS/MS. The mucus peptides were subsequently compared with R scripts. A total of 2818 different peptides constituting 1634 proteins from the mucus samples were identified, and 1218 of these peptides (43%) were core peptides found in the mucus of all examined species. Clustering and correspondence analyses of 1600 variable peptides showed unique mucous peptide patterns for each species. The high-throughput k-nearest neighbor and random forest-based prediction programs were developed with more than 95% averaged accuracy and could identify 11 functional categories of putative bioactive peptides and 268 peptides (9.5%) with at least five to seven bioactive properties. Antihypertensive, drug-delivering, and antiparasitic peptides were predominant. These peptides provide an understanding of gastropod mucus, and the putative bioactive peptides are expected to be experimentally validated for further medical, pharmaceutical, and cosmetic applications.

Application of 2D BN/SDS-PAGE coupled with mass spectrometry for identification of VDAC-associated protein complexes related to mitochondrial binding sites for type I brain hexokinase

Mitochondrion ◽  
2013 ◽  
Vol 13 (6) ◽  
pp. 823-830 ◽  
Author(s):  
Carla Rossini Crepaldi ◽  
Phelipe Augusto Mariano Vitale ◽  
Andrea Cristina Tesch ◽  
Hélen Julie Laure ◽  
José César Rosa ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Binding Sites ◽  
Protein Complexes ◽  
Type I ◽  
Sds Page

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements

1992 ◽  
Vol 12 (5) ◽  
pp. 2302-2314
Author(s):  
J D Trawick ◽  
N Kraut ◽  
F R Simon ◽  
R O Poyton
Keyword(s):  
Transcription Factor ◽  
Carbon Source ◽  
Protein Binding ◽  
Glucose Repression ◽  
Protein Complexes ◽  
Carbon Sources ◽  
Major Protein ◽  
Mobility Shift ◽  
Gel Mobility Shift ◽  
In Cells

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

scholarly journals Binding of recombinant interleukin-1 beta to the third complement component and alpha 2-macroglobulin after activation of serum by immune complexes

Blood ◽  
1990 ◽  
Vol 75 (12) ◽  
pp. 2388-2395 ◽  
Author(s):  
W Borth ◽  
A Urbanski ◽  
R Prohaska ◽  
M Susanj ◽  
TA Luger
Keyword(s):  
Complex Formation ◽  
Immune Complexes ◽  
Protein Complexes ◽  
Interleukin 1 ◽  
Complement Component ◽  
Interleukin 1 Beta ◽  
Thiol Groups ◽  
Thiol Ester ◽  
The Third ◽  
Sds Page

Abstract Activation of human normal serum with tetanus/antitetanus immune complexes (TAT-IC) resulted in increased binding of 125I-labeled interleukin-1 beta (IL-1 beta) to serum factors, as opposed to untreated serum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography showed labeling of two large molecular mass factors of an apparent molecular weight (Mr) of 200,000 and 400,000, respectively. These complexes could be dissociated by reduction. No complexes were formed when reducing compounds were added to serum-TAT-IC-125I-IL-1 beta mixtures. Complex formation was largely prevented by alkylating compounds. Molecular sieve chromatography of TAT-IC-activated serum confirmed that 125I-IL-1 beta became bound to high Mr serum proteins. Fractions containing high molecular 125I-IL-1 serum protein complexes partially retained IL-1- like activity since they induced proliferation of an IL-1-dependent murine T helper (D10G4) cell lineage. The 125I-IL-1 beta binding factors could be immunoprecipitated from TAT-IC-activated serum 125I-IL- 1 beta solutions by antisera to alpha 2-macroglobulin (alpha 2M) or to the third complement component (C3). SDS-PAGE of the immunoprecipitates showed radioactive bands corresponding to the expected Mr resulting from complex formation between 125I-IL-1 beta and these two proteins. Treatment of purified plasma alpha 2M and C3 with trypsin or activation with methylamine, which causes cleavage of the internal thiol ester and the appearance of free thiol groups in these proteins, mediated binding of 125I-IL-1 beta to alpha 2M and C3b. The results suggest that cleavage of the internal thiol ester in C3 and alpha 2M makes these plasma proteins susceptible to binding of 125I-IL-1 beta and that free thiol groups do play a role in the formation of 125I-IL-1 beta plasma protein complexes. Activated C3 and alpha 2M may function as IL-1 beta carrier proteins in biologic fluids, in addition to their other physiologic roles.

Cytoplasmic surface and intramembrane components of rat heart gap junctional proteins

1984 ◽  
Vol 246 (6) ◽  
pp. H865-H875 ◽  
Author(s):  
C. K. Manjunath ◽  
G. E. Goings ◽  
E. Page
Keyword(s):  
Electron Microscopy ◽  
Gap Junctions ◽  
Electron Micrographs ◽  
Rat Heart ◽  
Major Protein ◽  
Thin Sections ◽  
Cytoplasmic Surface ◽  
Sds Page ◽  
Junction Protein ◽  
Major Protein Band

Gap junctions were purified from rat hearts in the presence of absence of proteolysis inhibitors and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopy of thin sections. In absence of proteolysis inhibitors or in presence of ethylenediaminetetraacetic acid or leupeptin, gap junctions contained a single major protein band at relative molecular weight (Mr) 29,500 and minor bands at Mr 44,000–47,000, 17,750, and 16,500 and showed smooth cytoplasmic surfaces in electron micrographs. SDS-PAGE of junctions prepared with phenylmethylsulfonylfluoride (PMSF) showed markedly decreased intensity of the Mr 29,500 band and increased intensity of bands at Mr 44,000, 45,500, and 47,000; electron microscopy of these gap junctions showed presence of a fuzzy layer on their cytoplasmic surfaces. Urea (8 M) could not remove this fuzzy layer. In electron micrographs of rat ventricular myocytes, cytoplasmic surfaces of gap junctions were fuzzy. We conclude that rat heart gap junction protein consists of an intramembrane component (Mr 29,500) that extends into the “gap” and a cytoplasmic surface component (Mr 14,500–17,500) that corresponds to the fuzzy layer and is hydrolyzable by a serine protease.

Cloning, expression and immunogenic characterization of theapxIIAgene ofActinobacillus pleuropneumoniae

2007 ◽  
Vol 4 (1) ◽  
pp. 63-67
Author(s):  
Yan Ke-Xia ◽  
Liu Jian-Jie ◽  
Wu Bin ◽  
Tang Xi-Biao ◽  
Cai Li-Jun ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Lethal Dose ◽  
Serum Antibody ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gene Encoding ◽  
Lethal Challenge ◽  
Sds Page ◽  
Prokaryotic Expression Vector ◽  
Protection Rate ◽  
Protein Group

AbstractThe structural gene encoding ApxII toxin (apxIIA) was amplified from the genomic DNA ofActinobacillus pleuropneumoniae(APP) strain HB08 (serotype 2) and cloned into the prokaryotic expression vector pET-28a. SDS-PAGE and Western blot analysis showed that theapxIIAgene was expressed inEscherichia coliBL21 (DE3) and the expressed products could react with ApxII antibodies. The recombinant ApxIIA was purified from the inclusion bodies. Kunming mice were intraperitoneally vaccinated twice, with an interval of 2 weeks, using unfolded/refolded recombinant proteins, the native ApxII toxin extracted from the cultural supernatant of a strain of APP serotype 7 (APP-7) or phosphate-buffered saline (PBS). Serum antibody was examined by ApxIIA-specific enzyme-linked immunosorbent assay (ELISA) 2 weeks after every vaccination. Two weeks after the second vaccination, mice were challenged intraperitoneally with a lethal dose of APP-7 (1.08 × 108cfu per mouse). The protection rate reached 91.7% in the native ApxII group, 83.3% in the refolded recombinant protein group and 58.3% in the unfolded recombinant protein group, while all mice in the PBS group died within 36 h after challenge. Our data revealed that the refolded recombinant ApxIIA had excellent immunogenicity and could elicit protection against a lethal challenge of APP.

scholarly journals Purification of human gamma interferon receptors by sequential affinity chromatography on immobilized monoclonal antireceptor antibodies and human gamma interferon.

1987 ◽  
Vol 165 (4) ◽  
pp. 988-999 ◽  
Author(s):  
M Aguet ◽  
G Merlin
Keyword(s):  
Competitive Inhibition ◽  
Raji Cell ◽  
Gamma Interferon ◽  
Major Protein ◽  
Ifn Gamma ◽  
Gamma Receptor ◽  
Sds Page ◽  
Number Of Binding Sites ◽  
Direct Binding ◽  
Triton X

mAbs against human IFN-gamma (huIFN-gamma) receptors were obtained by immunizing a BALB/c mouse with eluates from immobilized recombinant huIFN-gamma (rhuIFN-gamma) on which lysates of enriched Raji cell membranes had been adsorbed. mAbs were selected for competitive inhibition of receptor binding of 125I-labeled rhuIFN-gamma. The following additional properties suggest that these antibodies are specific for huIFN-gamma receptors: they bind to the surface of human cells expressing IFN-gamma receptors but not to heterologous cells; this binding is inhibited competitively by addition of rhuIFN-gamma; the number of binding sites revealed by direct binding of 125I-labeled rhuIFN-gamma correlates with the amount of antigen recognized by the mAbs on different cell lines. A Triton X-100 extract of a membrane-enriched fraction of human Raji cells was affinity purified with these mAbs and the eluates from such columns were further purified on immobilized rhuIFN-gamma. As revealed by SDS-PAGE, the final eluate contained two major protein bands with approximate Mr of 90,000 (p90) and 50,000 (p50), respectively. Both proteins were able to specifically bind 125I-labeled rhuIFN-gamma upon electroblotting to nitrocellulose. This binding could be inhibited by the huIFN-gamma receptor mAbs, suggesting that the same epitopes are recognized on p90, p50, and on the cell surface. Therefore, these proteins most likely represent at least a part of huIFN-gamma receptors.

scholarly journals Activated Charcoal—A Potential Material in Glucoamylase Recovery

2011 ◽  
Vol 2011 ◽  
pp. 1-4 ◽  
Author(s):  
S. O. Kareem ◽  
I. Akpan ◽  
T. O. S. Popoola ◽  
L. O. Sanni
Keyword(s):  
Molecular Weight ◽  
Activated Charcoal ◽  
Contact Time ◽  
Enzyme Purification ◽  
Downstream Processing ◽  
Single Step ◽  
Major Protein ◽  
Rhizopus Oligosporus ◽  
Sds Page ◽  
Effects Of Ph

The potential of activated charcoal in the purification of fungal glucoamylase was investigated. Various concentrations of activated charcoal (1–4% w/v) were used to concentrate crude glucoamylase from Rhizopus oligosporus at different temperature values (30–50°C). Effects of pH (3.0–6.0) and contact time (0–60 min) on enzyme purification were also monitored. Activated charcoal (3% w/v) gave a 16-fold purification in a single-step purification at 50°C for 20 min and pH 5.5. The result of SDS-PAGE analysis of purified glucoamylase showed two major protein bands with corresponding molecular weight of 36 kDa and 50 kDa. The method is inexpensive, rapid, and simple which could facilitate downstream processing of industrial enzyme.

scholarly journals Separation and Identification of HSP-Associated Protein Complexes from Pancreatic Cancer Cell Lines Using 2D CN/SDS-PAGE Coupled with Mass Spectrometry

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Zhiyun Zhao ◽  
Hui Liu ◽  
Xinli Wang ◽  
Xiaodong Wang ◽  
Zhili Li
Keyword(s):  
Pancreatic Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Protein Function ◽  
Pancreatic Cancer Cell ◽  
Protein Complexes ◽  
Early Stage ◽  
Cancer Cell Lines ◽  
Building Models ◽  
Sds Page

Protein complexes are a cornerstone of many biological processes and together they form various types of molecular machinery. A broad understanding of these protein complexes is crucial for revealing and building models of protein function and regulation. Pancreatic cancer is a highly lethal disease which is difficult to diagnose at early stage and even more difficult to cure. In this study, we applied a gradient clear native gel system combined with subsequent second-dimensional SDS-PAGE to separate protein complexes from cell lysates of SW1990 and PANC-1 pancreatic cancer cell lines with different degrees of differentiation. Ten heat-shock-protein- (HSP-) associated protein complexes were separated and identified, and the differentially expressed proteins related to cancers were also found, such as HSP60, protein disulfide-isomerase A4 (ERp72), and transitional endoplasmic reticulum ATPase (TER ATPase).

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