scholarly journals Exosomes from human placenta purified by affinity chromatography on sepharose bearing immobilized antibodies against CD81 tetraspanin contain many peptides and small proteins

IUBMB Life ◽  
2018 ◽  
Vol 70 (11) ◽  
pp. 1144-1155 ◽  
Author(s):  
Evgeniya E. Burkova ◽  
Pavel S. Dmitrenok ◽  
Dmitrii V. Bulgakov ◽  
Valentin V. Vlassov ◽  
Elena I. Ryabchikova ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Human Placenta ◽  
Immobilized Antibodies ◽  
Small Proteins ◽  
Cd81 Tetraspanin

scholarly journals Small protein 26 interacts and enhances glutamine synthetase activity in Methanosarcina mazei

2020 ◽  
Author(s):  
Miriam Gutt ◽  
Britta Jordan ◽  
Katrin Weidenbach ◽  
Mirja Gudzuhn ◽  
Claudia Kiessling ◽  
...  
Keyword(s):  
Glutamine Synthetase ◽  
Affinity Chromatography ◽  
Nitrogen Limitation ◽  
Functional Characterization ◽  
Size Exclusion ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity ◽  
Small Protein ◽  
Methanosarcina Mazei ◽  
Small Proteins

ABSTRACTSmall ORFs (sORF) encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding and non-coding predicted sORFs and in their biochemical detection and characterization. We report on the first biochemical and functional characterization of a small protein (sP26) in the archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved within numerous Methanosarcina strains on the amino acid as well as on nucleotide level strongly arguing for a cellular function of the small protein. spRNA26 is significantly enhanced under nitrogen limitation, but also under oxygen and salt stress conditions. His-tagged sP26 was heterologously expressed and purified by fractionated ammonium sulfate precipitation, affinity chromatography and size exclusion centrifugation. Using independent biochemical approaches (pull-down by affinity chromatography followed by MS analysis, revers pull-down, microscale thermophoresis and size exclusion chromatography) we observed that sP26 interacts and forms complexes with M. mazei glutamine synthetase (GlnA1) with high affinity (app. KD = 45 +/− 14 µM). Upon interaction with sP26, GlnA1 activity was significantly stimulated independently and in addition to the known activation by the metabolite 2-oxoglutarate. Besides strong interaction of sP26 with the PII-like protein GlnK1 was demonstrated (KD= 1.4 µM +/− 0.9 µM). On the basis of these findings, we hypothesize that in addition to 2-oxoglutarate, sP26 activates GlnA1 activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA1.

scholarly journals Affinity chromatography: Large-scale purification of the soluble oestradiol-17-β dehydrogenase of human placenta

FEBS Letters ◽  
1972 ◽  
Vol 23 (2) ◽  
pp. 175-179 ◽  
Author(s):  
J.C. Nicolas ◽  
M. Pons ◽  
B. Descomps ◽  
A.Crastes de Paulet
Keyword(s):  
Affinity Chromatography ◽  
Human Placenta ◽  
Large Scale

AMP and IMP dephosphorylation by soluble high- and low-Km 5'-nucleotidases

1989 ◽  
Vol 256 (3) ◽  
pp. E386-E391 ◽  
Author(s):  
J. Spychala ◽  
V. Madrid-Marina ◽  
P. J. Nowak ◽  
I. H. Fox
Keyword(s):  
Complex System ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Human Placenta ◽  
Mammalian Cells ◽  
Relative Content ◽  
Ph Optimum ◽  
Optimum Ph ◽  
Sepharose 4B ◽  
Soluble Fractions

Three distinct 5'-phosphomonoesterase activities were isolated from soluble fractions of human placenta, cultured human T and B lymphoblasts, and rat liver using 5'-AMP-sepharose 4B affinity chromatography. We define these activities as "low-Km" 5'-nucleotidase, "high-Km" 5'-nucleotidase, and nonspecific phosphatase. High-Km 5'-nucleotidase was eluted with 0.5 M NaCl, low-Km 5'-nucleotidase was eluted with 10 mM ADP, and nonspecific phosphatase was not retained on the column. We have found significant variability in the relative content of high- to low-Km activities in the tissues studied with the ratios ranging from 5.5 to 264. The properties were studied after further purification. The molecular mass of the low-Km enzymes ranged from 72.5 to 209 kDa, optimum pH ranged from 7.4 to 9.0, Km for AMP ranged from 7 to 15 microM, and Km for IMP ranged from 10 to 26 microM. The molecular mass of the high-Km enzymes ranged from 182 to 210 kDa, pH optimum was at 6.5, Km for AMP ranged from 3.0 to 9.4 mM, and the Km for IMP ranged from 0.3 to 0.5 mM. The data indicate that the soluble low- and high-Km 5'-nucleotidase coexist in the mammalian cells and tissues studied. These observations suggest a complex system for the regulation of nucleoside 5'-monophosphate dephosphorylation.

scholarly journals S-Adenosylhomocysteine hydrolase from human placenta. Affinity purification and characterization

1985 ◽  
Vol 230 (1) ◽  
pp. 43-52 ◽  
Author(s):  
M S Hershfield ◽  
V N Aiyar ◽  
R Premakumar ◽  
W C Small
Keyword(s):  
Affinity Chromatography ◽  
Human Placenta ◽  
Binding Sites ◽  
Specific Activity ◽  
Affinity Purification ◽  
Reduced Form ◽  
Purification And Characterization ◽  
Adenosylhomocysteine Hydrolase ◽  
Disulphide Bonds ◽  
Nadh Ratio

S-Adenosylhomocysteine hydrolase (EC 3.3.1.1) was purified to homogeneity from human placenta by using S-adenosylhomocysteine-agarose affinity chromatography. The enzyme is a tetramer with a native Mr of 189 000 and subunit Mr of 47 000-48 000; there were nine cysteine residues per subunit and no disulphide bonds. The pI was 5.7. H.p.l.c. analysis revealed that the enzyme contained four molecules of tightly bound cofactor (NAD) per tetramer, of which 10-50% was in the reduced form. The enzyme had four binding sites per tetramer for adenosine, of which 10-35% were found to be occupied. Two types of adenosine-binding sites could be distinguished on the basis of differences in rates of dissociation of the enzyme-adenosine complex, and by examining binding of adenosine at 0 degree C and 37 degrees C. The enzyme catalysed the interconversion of adenosine and 4′,5′-dehydroadenosine; the equilibrium constant for this reaction was 2.1 and favoured 4′,5′-dehydroadenosine formation. Variability in the specific activity of preparations of S-adenosylhomocysteine hydrolase was related to the NAD+/NADH ratio of the preparation. The capacity to bind radioactively labelled adenosine depended on the adenosine content of the purified enzyme. The rate of adenosine binding and the sensitivity of S-adenosylhomocysteine hydrolase to inactivation by adenosine were both diminished in the absence of dithiothreitol.

scholarly journals Purification of elongation factor 2 from human placenta and evidence of its fragmentation patterns in various eukaryotic sources

1987 ◽  
Vol 244 (2) ◽  
pp. 337-344 ◽  
Author(s):  
A Giovane ◽  
L Servillo ◽  
L Quagliuolo ◽  
C Balestrieri
Keyword(s):  
Affinity Chromatography ◽  
Human Placenta ◽  
Degradation Product ◽  
Diphtheria Toxin ◽  
Intact Cell ◽  
Elongation Factor ◽  
Purification Step ◽  
Elongation Factor 2 ◽  
Fragmentation Patterns ◽  
Chromatography Step

While preparing human placenta elongation factor 2 (EF-2), whose purification and some molecular properties are reported, we noticed the presence of numerous protein fractions which did not have EF-2 activity, but were ADP-ribosylated by diphtheria toxin in the presence of NAD+. All these proteins, like EF-2, were selectively retained by a heparin-Sepharose column, which we used as an affinity-chromatography step. This was also observed when EF-2 was prepared, by this purification step, from other sources, i.e. ox liver and two species of yeasts. In order to assess whether these proteins were a degradation product of EF-2, independent proteins or a mixture of both, they were analysed by subjecting them, after [14C]ADP-ribosylation, to exhaustive trypsinolysis. Only one radioactive peptide was found, thus suggesting that those proteins originate from EF-2 by some proteolytic process. Our findings indicate that this proteolysis does not occur after cell disruption, but is more or less active in the intact cell, depending on the system considered.

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