Isolation of ubiquitin–E2 (ubiquitin-conjugating enzyme) complexes from erythroleukaemia cells using immunoaffinity techniques

2001 ◽  
Vol 356 (1) ◽  
pp. 199-206
Author(s):  
Koji TAKADA ◽  
Tae HIRAKAWA ◽  
Hideyoshi YOKOSAWA ◽  
Yutaka OKAWA ◽  
Hideki TAGUCHI ◽  
...  
Keyword(s):  
Heat Shock ◽  
Gel Filtration ◽  
Simple Method ◽  
Ubiquitinated Proteins ◽  
Ubiquitin System ◽  
Ubiquitin Conjugating Enzyme ◽  
Molecular Masses ◽  
Ubiquitin Molecule ◽  
E2 Enzymes ◽  
Enzyme Complexes

A variety of ubiquitin-associated (or conjugated) proteins, including substrates and enzymes for the ubiquitin system, are present in eukaryotic cells. In the present study we developed a simple method for their isolation, consisting of immunoaffinity chromatography using the monoclonal antibody FK2, which recognizes the conjugated ubiquitin molecule. Using this method followed by gel filtration, we isolated multi-ubiquitinated proteins with high molecular masses (> 30kDa) and also ubiquitinthioester-linked and mono-ubiquitinated forms of ubiquitin-conjugating (E2) enzymes, UbcH7 and UBE2N, together with mono-, di- and tri-ubiquitin molecules, from the cytoplasmic extract of heat-shock-treated K562 erythroleukaemia cells. We also demonstrated that the FK2 antibody was capable of precipitating a ubiquitin–UbcH7 thioester, but not free UbcH7, which enabled the measurement of the respective cellular levels separately. The immunoprecipitable ubiquitin–UbcH7 thioester was found only when the cells were treated with heat-shock. These results suggest the usefulness of the immunoaffinity techniques for identifying and analysing the cellular enzyme/protein–ubiquitin complexes.

scholarly journals Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

1997 ◽  
Vol 325 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Isabelle GARCIA ◽  
Matthew RODGERS ◽  
Catherine LENNE ◽  
Anne ROLLAND ◽  
Alain SAILLAND ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Gel Filtration ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Carrot Cells ◽  
Expression Studies ◽  
Sds Page ◽  
Molecular Masses ◽  
The Difference ◽  
Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

scholarly journals Plasma heat shock protein response to euglycemia in type 2 diabetes

2021 ◽  
Vol 9 (1) ◽  
pp. e002057
Author(s):  
Alexander S Atkin ◽  
Abu Saleh Md Moin ◽  
Ahmed Al-Qaissi ◽  
Thozhukat Sathyapalan ◽  
Stephen L Atkin ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Heat Shock ◽  
Protein Interactions ◽  
Glucose Variability ◽  
Interacting Protein ◽  
Ubiquitin Conjugating Enzyme ◽  
Shock Proteins ◽  
Protein Response ◽  
Design And Methods

IntroductionGlucose variability is associated with mortality and macrovascular diabetes complications. The mechanisms through which glucose variability mediates tissue damage are not well understood, although cellular oxidative stress is likely involved. As heat shock proteins (HSPs) play a role in the pathogenesis of type 2 diabetes (T2D) complications and are rapidly responsive, we hypothesized that HSP-related proteins (HSPRPs) would differ in diabetes and may respond to glucose normalization.Research design and methodsA prospective, parallel study in T2D (n=23) and controls (n=23) was undertaken. T2D subjects underwent insulin-induced blood glucose normalization from baseline 7.6±0.4 mmol/L (136.8±7.2 mg/dL) to 4.5±0.07 mmol/L (81±1.2 mg/dL) for 1 hour. Control subjects were maintained at 4.9±0.1 mmol/L (88.2±1.8 mg/dL). Slow Off-rate Modified Aptamer-scan plasma protein measurement determined a panel of HSPRPs.ResultsAt baseline, E3-ubiquitin-protein ligase (carboxyl-terminus of Hsc70 interacting protein (CHIP) or HSPABP2) was lower (p=0.03) and ubiquitin-conjugating enzyme E2G2 higher (p=0.003) in T2D versus controls. Following glucose normalization, DnaJ homolog subfamily B member 1 (DNAJB1 or HSP40) was reduced (p=0.02) in T2D, with HSP beta-1 (HSPB1) and HSP-70-1A (HSP70-1A) (p=0.07 and p=0.09, respectively) also approaching significance relative to T2D baseline levels.ConclusionsKey HSPRPs involved in critical protein interactions, CHIP and UBE2G2, were altered in diabetes at baseline. DNAJB1 fell in response to euglycemia, suggesting that HSPs are reacting to basal stress that could be mitigated by tight glucose control with reduction of glucose variability.

scholarly journals Targeting neddylation E2s: a novel therapeutic strategy in cancer

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yi-Chao Zheng ◽  
Yan-Jia Guo ◽  
Bo Wang ◽  
Chong Wang ◽  
M. A. A. Mamun ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
Therapeutic Strategy ◽  
Neural Precursor Cell ◽  
Catalytic Core Domain ◽  
Catalytic Core ◽  
Core Domain ◽  
E3 Ligases ◽  
Ubiquitin Conjugating Enzyme ◽  
E2 Enzymes ◽  
Conjugating Enzyme

AbstractUbiquitin-conjugating enzyme E2 M (UBE2M) and ubiquitin-conjugating enzyme E2 F (UBE2F) are the two NEDD8-conjugating enzymes of the neddylation pathway that take part in posttranslational modification and change the activity of target proteins. The activity of E2 enzymes requires both a 26-residue N-terminal docking peptide and a conserved E2 catalytic core domain, which is the basis for the transfer of neural precursor cell-expressed developmentally downregulated 8 (NEDD8). By recruiting E3 ligases and targeting cullin and non-cullin substrates, UBE2M and UBE2F play diverse biological roles. Currently, there are several inhibitors that target the UBE2M-defective in cullin neddylation protein 1 (DCN1) interaction to treat cancer. As described above, this review provides insights into the mechanism of UBE2M and UBE2F and emphasizes these two E2 enzymes as appealing therapeutic targets for the treatment of cancers.

scholarly journals Structural features of the exocellular polysaccharides of Mycobacterium tuberculosis

1994 ◽  
Vol 297 (2) ◽  
pp. 351-357 ◽  
Author(s):  
A Lemassu ◽  
M Daffé
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Mimicry ◽  
Degradation Products ◽  
Cell Envelope ◽  
Gel Filtration ◽  
Structural Features ◽  
Two Dimensional ◽  
Phagocytic Cells ◽  
Molecular Masses ◽  
Branched Structure

The cell envelope which surrounds pathogenic mycobacteria is postulated to be a defence barrier against phagocytic cells and its outermost constituents have a tendency to accumulate in the culture medium. The present work demonstrates that the exocellular material of Mycobacterium tuberculosis contains large amounts of polysaccharides with only traces, if any at all, of lipids. Three types of polysaccharides were purified by anion-exchange and gel-filtration chromatography; all were found to be neutral compounds devoid of acyl substituents. They consisted of D-glucan, D-arabino-D-mannan and D-mannan, which were eluted from gel-filtration columns in positions corresponding to molecular masses of 123, 13 and 4 kDa respectively. Their predominant structural features were determined by the characterization of the per-O-methyl derivatives of enzymic, acetolysis and Smith-degradation products and by 1H- and 13C-n.m.r. spectroscopy of the purified polysaccharides, using mono- and two-dimensional homonuclear chemical-shift correlated spectroscopy and two-dimensional heteronuclear (1H/13C) spectroscopy. The glucan which represented up to 90% of the polysaccharides was composed of repeating units of five or six-->4-alpha-D-Glcp-1--> residues and a -->4-alpha-D-Glcp substituted at position 6 with an alpha-D-Glcp, indicating a glycogen-like highly branched structure not related to the so-called polysaccharide-II previously identified in tuberculin. The arabinomannan consisted of a mannan segment composed of a -->6-alpha-D-Man-1--> core substituted at some positions 2 with an alpha-D-Manp. The arabinan termini of the arabinomannan were found to be extensively capped with mannosyl residues. The possibility that these polysaccharides contribute to the persistence of the tubercle bacillus in the macrophage by molecular mimicry is discussed.

Evaluation of dialysis treatment in uremic patients by gel filtration of serum

1990 ◽  
Vol 36 (11) ◽  
pp. 1906-1910 ◽  
Author(s):  
J Osada ◽  
T Gea ◽  
C Sanz ◽  
I Millan ◽  
J Botella
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
The Other ◽  
Control Group ◽  
Dialysis Treatment ◽  
Additional Information ◽  
Molecular Masses ◽  
Two Peaks

Abstract A group of substances of molecular masses between 300 and 1500 Da have been found to be toxic metabolites in patients with uremia. We determined the concentration in serum of these molecules in the following groups of patients: two hemodialyzed groups (one with cuprophane and the other with polyacrylonitrile dialyzers), one group treated with continuous ambulatory peritoneal dialysis, one group of nondialyzed azotemic patients, and one control group of healthy persons. Ultrafiltrates of the subjects' sera were fractionated on Sephadex G-15 followed by ion-exchange chromatography. Eluates were monitored by absorbance at 254 and 206 nm. Partially characterized peaks P1 and P2, obtained by gel filtration, correlated with the concentration of creatinine in serum; their concentrations were significantly (P less than 0.01) larger in hemodialyzed groups than in peritoneal dialyzed or in nondialyzed azotemic patients. After ion-exchange chromatography, two peaks (P'5 and P'6) correlated with serum creatinine and also were larger in hemodialyzed patients than in the other groups. Apparently, adequate discrimination is obtained by gel-filtration analysis and further analysis by ion-exchange chromatography does not provide additional information in most of the affected patients.

scholarly journals Heat shock induces protein tyrosine phosphorylation in cultured cells.

1989 ◽  
Vol 108 (6) ◽  
pp. 2029-2035 ◽  
Author(s):  
P A Maher ◽  
E B Pasquale
Keyword(s):  
Heat Shock ◽  
Tyrosine Phosphorylation ◽  
Cultured Cells ◽  
Protein Tyrosine Phosphorylation ◽  
Animal Cells ◽  
Protein Tyrosine ◽  
Rous Sarcoma ◽  
Wide Range ◽  
Sarcoma Virus ◽  
Molecular Masses

We examined the effect of heat shock on protein tyrosine phosphorylation in cultured animal cells using antiphosphotyrosine antibodies in immunoblotting and immunofluorescence microscopy experiments. Heat shock significantly elevated the level of phosphotyrosine in proteins in most of the cultured cells examined, including fibroblasts, epithelial cells, nerve cells, and muscle cells, but not in Rous sarcoma virus-transformed fibroblasts. The increase in protein tyrosine phosphorylation induced by heat shock occurred in proteins with a wide range of molecular masses and was dependent on the temperature and duration of the heat shock.

scholarly journals OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination

2018 ◽  
Vol 293 (47) ◽  
pp. 18285-18295 ◽  
Author(s):  
Nagesh Pasupala ◽  
Marie E. Morrow ◽  
Lauren T. Que ◽  
Barbara A. Malynn ◽  
Averil Ma ◽  
...  
Keyword(s):  
Polyubiquitin Chains ◽  
Protein Ubiquitination ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzymes ◽  
E2 Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
In Cells ◽  
Conjugating Enzyme

OTUB1 is a deubiquitinating enzyme that cleaves Lys-48–linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2∼ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1–E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1−/− knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.

scholarly journals Purification of a Crenarchaeal ATP Synthase in the Light of the Unique Bioenergetics ofIgnicoccusSpecies

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Lydia J. Kreuter ◽  
Andrea Weinfurtner ◽  
Alexander Ziegler ◽  
Julia Weigl ◽  
Jan Hoffmann ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Cell Envelope ◽  
Gel Filtration ◽  
Cellular Membrane ◽  
Content Type ◽  
The Pacific ◽  
Native Electrophoresis ◽  
Molecular Masses ◽  
The Pacific Ocean

ABSTRACTIn this study, the ATP synthase ofIgnicoccus hospitaliswas purified, characterized, and structurally compared to the respective enzymes of the otherIgnicoccusspecies, to shed light on energy conservation in this unique group of archaea. The crenarchaeal genusIgnicoccuscomprises three described species, i.e.,I. hospitalisandIgnicoccus islandicusfrom hot marine sediments near Iceland andIgnicoccus pacificusfrom a hydrothermal vent system in the Pacific Ocean. This genus is unique among all archaea due to the unusual cell envelope, consisting of two membranes that enclose a large intermembrane compartment (IMC).I. hospitalisis the best studied member of this genus, mainly because it is the only known host for the potentially parasitic archaeonNanoarchaeum equitans.I. hospitalisgrows chemolithoautotrophically, and its sole energy-yielding reaction is the reduction of elemental sulfur with molecular hydrogen, forming large amounts of hydrogen sulfide. This reaction generates an electrochemical gradient, which is used by the ATP synthase, located in the outer cellular membrane, to generate ATP inside the IMC. The genome ofI. hospitalisencodes nine subunits of an A-type ATP synthase, which we could identify in the purified complex. Although the maximalin vitroactivity of theI. hospitalisenzyme was measured around pH 6, the optimal stability of the A1AOcomplex seemed to be at pH 9. Interestingly, the soluble A1subcomplexes of the differentIgnicoccusspecies exhibited significant differences in their apparent molecular masses in native electrophoresis, although their behaviors in gel filtration and chromatography-mass spectrometry were very similar.IMPORTANCETheCrenarchaeotarepresent one of the major phyla within theArchaeadomain. This study describes the successful purification of a crenarchaeal ATP synthase. To date, all information about A-type ATP synthases is from euryarchaeal enzymes. The fact that it has not been possible to purify this enzyme complex from a member of theCrenarchaeotauntil now points to significant differences in stability, possibly caused by structural alterations. Furthermore, the study subjectI. hospitalishas a particular importance among crenarchaeotes, since it is the only known host ofN. equitans. The energy metabolism in this system is still poorly understood, and our results can help elucidate the unique relationship between these two microbes.

Purification and characterization of a lectin from Xanthomonas campestris NCIM 5028

1996 ◽  
Vol 42 (6) ◽  
pp. 609-612 ◽  
Author(s):  
Bhagyashree Joshi ◽  
Jayant M. Khire ◽  
Hephzibah SivaRaman ◽  
M. Islam Khan
Keyword(s):  
Molecular Mass ◽  
Host Plant ◽  
Xanthomonas Campestris ◽  
Simple Procedure ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Ammonium Sulphate Precipitation ◽  
Molecular Masses ◽  
Lectin Purification

A lectin was isolated from culture filtrates of Xanthomonas campestris NCIM 5028, by a simple procedure of hydrophobic chromatography on phenyl-Sepharose after ammonium sulphate precipitation. The lectin was a heterodimer, with subunit molecular masses of 30 000 and 28 000. Gel filtration on S-300 column, calibrated with markers, showed its molecular mass to be approximately 70 000. Its isoelectric point was 7.2. The agglutination of the rabbit erythrocytes by the lectin was inhibited by fetuin glycopeptides and host plant (Brassica oleracea) extracts.Key words: Xanthomonas campestris, lectin, purification.

The copurification of β-glucosidase, β-xylosidase, and 1,3-β-glucanase in two separate enzyme complexes isolated from Trichoderma harzianum E58

1987 ◽  
Vol 65 (9) ◽  
pp. 822-832 ◽  
Author(s):  
Larry U. L. Tan ◽  
Paul Mayers ◽  
Michelle Illing ◽  
John N. Saddler
Keyword(s):  
Isoelectric Point ◽  
Mercuric Chloride ◽  
Trichoderma Harzianum ◽  
Inhibition Constant ◽  
Activity Ratios ◽  
Molecular Masses ◽  
Inhibition Constants ◽  
Inhibition Studies ◽  
Enzyme Complexes

Two enzyme complexes, each with β-glucosidase (β-D-glucoside glucohydrolase, EC 3.2.1.21), β-xylosidase (β-D-xylan xylohydrolase, EC 3.2.1.37), and 1,3-β-glucanase (laminarinase, EC 3.2.1.39) activity, were purified to near homogeneity from the cellulolytic fungus Trichoderma harzianum E58. The two complexes had the same isoelectric point of pH 8.3 and identical subunit molecular masses of 75 400 daltons. The two complexes were also similar in that all activities were sensitive to inhibition by mercuric chloride (2 mM) and D-glucono-1,5-lactone (0.2% w/v). The activity ratios of the major and minor complexes were 1:1.7:4.3 and 1:1.6:3.1 for the β-xylosidase, β-glucosidase, and 1,3-β-glucanase, respectively. Both complexes had approximately the same Km values for p-nitrophenyl β-D-glucopyranoside and salicin. The pH optima of corresponding activities of the two complexes were also similar. The major and minor complexes differed in that the Km of the former for laminarin was almost threefold lower than that of the latter. Whereas all three activities of the minor complexes were inhibited by D-glucono-1,5-lactone with the same inhibition constant, the β-glucosidase and 1,3-β-glucanase of the major complex had inhibition constants which differed by more than 80 000 times. In addition, the inhibition on the 1,3-β-glucanase in the major and minor complexes using D-glucono-1,5-lactone were noncompetitive and competitive, respectively. From the inhibition studies, the β-glucosidase, β-xylosidase, and 1,3-β-glucanase activities in the minor complex were deduced to be more interdependent than the same activities in the major complex.

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