scholarly journals Extra Purified Exosomes from Human Placenta Contain an Unpredictable Small Number of Different Major Proteins

2019 ◽  
Vol 20 (10) ◽  
pp. 2434 ◽  
Author(s):  
Evgeniya E. Burkova ◽  
Alina E. Grigor’eva ◽  
Dmitrii V. Bulgakov ◽  
Pavel S. Dmitrenok ◽  
Valentin V. Vlassov ◽  
...  
Keyword(s):  
Interleukin 1 ◽  
Gel Filtration ◽  
Normal Pregnancy ◽  
Maldi Mass Spectrometry ◽  
2D Electrophoresis ◽  
Cytoplasmic Actin ◽  
Sds Page ◽  
Two Peaks ◽  
Sepharose 4B ◽  
Different Sources

Exosomes are nanovesicles (30–100 nm) containing various RNAs and different proteins. Exosomes are important in intracellular communication, immune function, etc. Exosomes from different sources including placenta were mainly obtained by different types of centrifugation and ultracentrifugations and were reported to contain from a few dozen to thousands of different proteins. First crude exosome preparations from four placentas (normal pregnancy) were obtained here using several standard centrifugations but then were additionally purified by gel filtration on Sepharose 4B. Individual preparations demonstrated different gel filtration profiles showing good or bad separation of exosome peaks from two peaks of impurity proteins and their complexes. According to electron microscopy, exosomes before gel filtration contain vesicles of different size, ring-shaped structures forming by ferritin and clusters of aggregated proteins and their complexes. After filtration through 220 nm filters and gel filtration exosomes display typically for exosome morphology and size (30–100 nm) and do not contain visible protein admixtures. Identification of exosome proteins was carried out by MS and MS/MS MALDI mass spectrometry of proteins’ tryptic hydrolyzates after their SDS-PAGE and 2D electrophoresis. We have obtained unexpected results. Good, purified exosomes contained only 11–13 different proteins: CD9, CD81, CD-63, hemoglobin subunits, interleukin-1 receptor, annexin A1, annexin A2, annexin A5, cytoplasmic actin, alkaline phosphatase, serotransferin, and probably human serum albumin and immunoglobulins. We assume that a possible number of exosome proteins found previously using crude preparations may be very much overestimated. Our data may be important for study of biological functions of pure exosomes.

IDENTIFICATION AND PURIFICATION OF A NEW PROTEIN FROM WATER-SOLUBLE EXTRACTS OF HUMAN ADRENAL GLAND

1979 ◽  
Vol 82 (3) ◽  
pp. 383-NP ◽  
Author(s):  
M. A. AL-AWQATI ◽  
Y. B. GORDON ◽  
T. CHARD
Keyword(s):  
Molecular Weight ◽  
Adrenal Gland ◽  
Gel Filtration ◽  
Water Soluble ◽  
Double Immunodiffusion ◽  
Molecular Weights ◽  
Physicochemical Methods ◽  
Two Peaks ◽  
Sepharose 4B ◽  
New Protein

An homogenate of human foetal adrenal gland was subjected to negative immunoabsorption by column chromatography using anti-whole human serum coupled to Sepharose 4B. Two peaks were eluted and used to immunize rabbits. The antisera produced were absorbed and tested for specificity by double immunodiffusion. Two antigens, which appeared to be specific to the adrenal gland, were identified having molecular weights of 25 000 and 65 000 as determined by gel filtration. The lower molecular weight antigen was isolated by physicochemical methods and found to be a protein. The amino acid composition is reported.

Studies On Glycocalicin And Glycoprotein Gp Ib

1981 ◽  
Author(s):  
N O Solum ◽  
T Sletbakk ◽  
I Hagen ◽  
G Gogstad
Keyword(s):  
Fast Moving ◽  
Gel Filtration ◽  
Integral Membrane Protein ◽  
Human Platelets ◽  
Characteristic Change ◽  
Extraction Buffer ◽  
Sds Page ◽  
Slow Moving ◽  
Triton X ◽  
Two Peaks

Crossed immunoelectrophoresis (CIE) of extracts of human platelets in 1% Triton X-100 using antiserum to purified glycocalicin shows an immunopre- cipitate consisting of two peaks. Previous experiments have shown the small fast-moving peak to represent free glycocalicin whereas the slow-moving one corresponds to a larger amphiphilic protein, probably the integral membrane protein GP Ib. Glycocalicin is probably derived from the latter secondary to an activation of a calsium-dependent protease during platelet lysis. Further studies on these problems are presented. A gradual reduction of the concentration of Triton X-100 in the extraction buffer (tris-glycine, pH 8.7, 135 mOsM) gradually reduced the area of the slow-moving peak and increased that of the fast-moving one untill all was present as free glycocalicin. Reduction of the concentration of Triton in an extract already prepared with 1% Triton X-100 by adsorption to Bio-Beads SM-2 had no such effect. The presence of the protease inhibitor leupeptin during extraction at a low concentration of Triton (0.2%) reduced the peak corresponding to free glycocalicin. GP Ib was purified from Triton extracts by precipitation with con A, affinity chromatography of the supernatant on WGA-Sepharose and elution with NAGA, and gel filtration of the eluate on Ultrogel AcA 22. Triton X-100, EDTA and sodium azide were present at all steps. The characteristic change in mobility of GP Ib on SDS PAGE comparing unreduced to reduced sampLes deduced from studies on whole platelet proteins, was confirmed with the purified material, as was the correspondence between the GP Ib band on SDS and the slow-moving component of the CIE.

scholarly journals Studies on gastric mucoproteins. The isolation and characterization of the mucoprotein of the water-soluble mucus from pig cardiac gastric mucosa

1971 ◽  
Vol 123 (5) ◽  
pp. 845-853 ◽  
Author(s):  
D. Snary ◽  
A. Allen
Keyword(s):  
Sialic Acid ◽  
Gastric Mucosa ◽  
Gel Filtration ◽  
Water Soluble ◽  
Group Activities ◽  
Isolation And Characterization ◽  
Two Peaks ◽  
Sepharose 4B

1. Gel filtration of the water-soluble radioactive mucus produced three radioactive fractions, fraction A excluded on Sepharose 4B, fraction B included on Sepharose 4B but excluded on Sephadex G-200, and fraction C included on Sephadex G-200. 2. The specific radioactivities of fractions A and B were the same, with fraction C a little lower, whether the material was labelled with 14C-labelled carbohydrate or with 3H-labelled protein prepared by incubation of mucosal scrapings in vitro with [U-14C]glucose or [G-3H]threonine respectively. 3. Fractions A and B had an analysis of protein 22%, hexose 28%, hexosamine 28%, fucose 10% and sialic acid 1%; fraction C had an analysis closely similar to this, except that it contained about 10% of a protein contaminant. 4. All three fractions had closely similar A and H blood-group activities. 5. Ultracentrifuge studies showed fractions A, B and C were polydisperse with s025,w values of 18.7S, 4.9S and 3.9S respectively. 6. The unfractionated water-soluble mucus contained only two peaks, fraction A 18.7S and a peak of 4.4S, which was a combination of fractions B and C. 7. The radioactive mucoprotein accounted for 85% by weight of the soluble mucus and the results show that it consisted of two distinct fractions A and B–C, which were chemically, biosynthetically and immunologically very similar.

PURIFICATION AND CHARACTERIZATION OF THE PROCOAGULANT OF THE VENOM OF TROPIDECHIS CARINATUS

1987 ◽  
Author(s):  
P P Masci ◽  
A N Whitaker ◽  
J J Morrison ◽  
E A Bennett
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Factor Xa ◽  
Factor V ◽  
Crude Venom ◽  
Adult Rats ◽  
Sds Page ◽  
Sepharose 4B ◽  
Blue Sepharose

Tropidechis carinatus is a venomous elapid snake distributed throughout Eastern Queensland. It has been considered as a tropical relative of Notechis scutatus and, similarly, the crude venom contains an indirect prothrombin activator, which will clot plasma provided that Factor V is present. Myotoxins and neurotoxins are also present. Envenomated patients regularly develop disseminated intravascular coagulation. The crude whole venom of T.carinatus was shown to have five major components by gel filtration, SDS PAGE and HPLC, and even more components by isoelectric focusing. The procoagulant eluted with a molecular weight of 55,000, being found in peak II on gel filtration on Sephadex-G150. The procoagulant was purified using a combination of Sephadex-G150 chromatography and ion-exchange on DEAE Sephadex-A50 and shown to migrate as a single band of molecular weight 55.000 by SDS PAGE. On reduction by β -mercaptoethanol this component was resolvec into u heavy chain of molecular weight 30.000 and a light chain of 25,000. The procoagulant was shown to bind to con A-Sepharose 4B and Blue Sepharose 4B. Coagulation studies using this purified procoagulant confirmed a factor Xa-like activity activating prothrombin in the presence of factor V. The purified fraction is unstable in buffer solutions at 4°C, probably because of trypsin - like autodigestion. Ouchterlony studies of the procoagulants of T.carinatus and N.scutatus show both lines of homogeneity and spurring, indicating similarities but also significant differences between the two proteins. The purified procoagulant was lethal to adult rats, an IV injection of 10 μg killing in 1 - 2 minutes. Death was prevented by prior heparinization, suggesting that the procoagulant is toxic in the absence of neurotoxin and other components.

scholarly journals Neurofilament protein is phosphorylated in the squid giant axon.

1978 ◽  
Vol 78 (2) ◽  
pp. R23 ◽  
Author(s):  
H C Pant ◽  
G Shecket ◽  
H Gainer ◽  
R J Lasek
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Giant Axon ◽  
Squid Giant Axon ◽  
Neurofilament Protein ◽  
Acid Hydrolysate ◽  
Sds Page ◽  
Discontinuous Sucrose Gradient ◽  
Sepharose 4B

We have observed the phosphorylation of neurofilament protein from squid axoplasm. Phosphorylation is demonstrated by 32P labeling of protein during incubation of axoplasm with [gamma-32P]ATP. When the labeled proteins are separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two bands, at 2.0 x 10(5) daltons and greater than 4 x 10(5) daltons, contain the bulk of the 32P. The 2.0 x 10(5)-dalton phosphorylated polypeptide comigrates on SDS-PAGE with one of the subunits of squid neurofilament protein. Both major phosphorylated polypeptides co-fractionate with neurofilaments in discontinuous sucrose gradient centrifugation and on gel filtration chromatography on Sepharose 4B. The protein-phosphate bond behaves like a phospho-ester, and labeled phospho-serine is identified in an acid hydrolysate of the protein. The generality of this phenomenon in various species and its possible physiological significance are discussed.

scholarly journals Extraction and partial Purification of ipopolysaccaride (LPS) from E. coli O157:H7 ioslate

2009 ◽  
Vol 3 (1) ◽  
pp. 10-14
Author(s):  
Ashwak B. J. Al-Hashimi ◽  
Essam F. A. Al-Jumaily ◽  
Amina N. Al-Thiwini ◽  
Hitham A. Al-Omari
Keyword(s):  
Molecular Weight ◽  
Nucleic Acids ◽  
Petroleum Ether ◽  
Wave Length ◽  
Gel Filtration ◽  
Partial Purification ◽  
E Coli ◽  
Ether Mixture ◽  
Two Peaks ◽  
Sepharose 4B

Lipopolysaccharide (LPS) was partialy purified from E. coli O157:H7 local isolate by Sepharose – 4B gel filtration chromatography, after extraction by phenol-chloroform –petroleum ether mixture. The results show the presence of two peaks of proteins; while one peak of carbohydrates when tested at wave length 490nm. Nucleic acids were not found in the sample after partial purification. Electrophoresis pattern shows one large band with a molecular weight of 69000 daltons.

scholarly journals Purification and Characterization of Glucose-6-Phosphate Dehydrogenase from Camel Liver

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Mahmoud A. Ibrahim ◽  
Abdel-Hady M. Ghazy ◽  
Ahmed M. H. Salem ◽  
Mohamed A. Ghazy ◽  
Mohamed M. Abdel-Monsef
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Divalent Cations ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Native Form ◽  
Native Page ◽  
Sds Page ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Sepharose 4B

Glucose-6-phosphate dehydrogenase from camel liver was purified to homogeneity by ammonium sulfate precipitation and a combination of DEAE-cellulose, Sephacryl S-300 gel filtration, and 2′, 5′ ADP Sepharose 4B affinity chromatography columns. The specific activity of camel liver G6PD is increased to 1.80438 units/mg proteins with 63-fold purification. It turned out to be homogenous on both native PAGE and 12% SDS PAGE, with a molecular weight of 64 kDa. The molecular weight of the native form of camel liver G6PD was determined to be 194 kDa by gel filtration indicating a trimeric protein. The Km value was found to be 0.081 mM of NADP+. Camel liver G6PD displayed its optimum activity at pH 7.8 with an isoelectric point (pI) of pH 6.6–6.8. The divalent cations MgCl2, MnCl2, and CoCl2 act as activators; on the other hand, CaCl2 and NiCl2 act as moderate inhibitors, while FeCl2, CuCl2, and ZnCl2 are potent inhibitors of camel liver G6PD activity. NADPH inhibited camel liver G6PD competitively with Ki value of 0.035 mM. One binding site was deduced for NADPH on the enzyme molecule. This study presents a simple and reproducible purification procedure of G6PD from the camel liver.

Preparation and Characterization of NH2-Terminal Fibrinogen Bβ Fragments from N-DSK of Human Fibrinogen

1984 ◽  
Vol 51 (01) ◽  
pp. 016-021 ◽  
Author(s):  
S Birken ◽  
G Agosto ◽  
B Lahiri ◽  
R Canfield
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Human Fibrinogen ◽  
Human Volunteers ◽  
Cnbr Cleavage ◽  
Two Peaks

SummaryIn order to investigate the early release of NH2-terminal plasmic fragments from the Bβ chain of fibrinogen, substantial quantities of Bβ 1-42 and Bβ 1-21 are required as immunogens, as radioimmunoassay standards and for infusion into human volunteers to determine the half-lives of these peptides. Towards this end methods that employ selective proteolytic cleavage of these fragments from fibrinogen have been developed. Both the N-DSK fragment, produced by CNBr cleavage of fibrinogen, and Bβ 1-118 were employed as substrates for plasmin with the finding of higher yields from N-DSK. Bβ 1-42 and Bβ 1-21 were purified by gel filtration and ion-exchange chromatography on SP-Sephadex using volatile buffers. When the purified preparation of Bβ 1-42 was chromatographed on reverse-phase high performance liquid chromatography, two peaks of identical amino acid composition were separated, presumably due either to pyroglutamate or to amide differences.

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 Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

2005 ◽  
Vol 387 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Seonghun KIM ◽  
Sun Bok LEE
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Genome Database ◽  
Sds Page ◽  
Key Enzyme ◽  
Bivalent Metal Ions ◽  
Ammonium Sulphate Fractionation ◽  
Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

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