scholarly journals Biochemical characterization and pharmacological properties of a phospholipase A2 myotoxin inhibitor from the plasma of the snake Bothrops asper

1997 ◽  
Vol 326 (3) ◽  
pp. 853-859 ◽  
Author(s):  
Sergio LIZANO ◽  
Bruno LOMONTE ◽  
Jay W. FOX ◽  
José Maréa GUTIÉRREZ
Keyword(s):  
Phospholipase A2 ◽  
Molecular Mass ◽  
Biochemical Characterization ◽  
Biological Activities ◽  
Sequence Similarity ◽  
Gel Filtration ◽  
Cytolytic Activity ◽  
Bothrops Asper

A protein that neutralizes the biological activities of basic phospholipase A2 (PLA2) myotoxin isoforms from the venom of the snake Bothrops asper was isolated from its blood by affinity chromatography with Sepharose-immobilized myotoxins. Biochemical characterization of this B. asper myotoxin inhibitor protein (BaMIP) indicated a subunit molecular mass of 23–25 kDa, an isoelectric point of 4, and glycosylation. Gel-filtration studies revealed a molecular mass of 120 kDa, suggesting that BaMIP possesses an oligomeric structure composed of five 23–25 kDa subunits. Functional studies indicated that BaMIP inhibits the PLA2 activity of B. asper basic myotoxins I and III, as well as the myotoxicity and edema-forming activity in vivo and cytolytic activity in vitro towards cultured endothelial cells, of all four myotoxin isoforms (I–IV) tested. Sequence analysis of the first 63 amino acid residues from the N-terminus of BaMIP indicated more than 65% sequence similarity to the PLA2 inhibitors isolated from the blood of the crotalid snakes Trimeresurus flavoviridis and Agkistrodon blomhoffii siniticus. These inhibitors also share sequences similar to the carbohydrate-recognition domains of human and rabbit cellular PLA2 receptors, suggesting a common domain evolution among snake plasma PLA2 inhibitors and mammalian PLA2 receptors. Despite this similarity, this is the first description of a natural anti-myotoxic factor from snake blood.

scholarly journals Hematopoetic precursors respond to a unique B lymphocyte-derived factor in vivo

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1784-1789 ◽  
Author(s):  
E Niskanen ◽  
J Gorman ◽  
PC Isakson
Keyword(s):  
B Cells ◽  
Colony Formation ◽  
High Performance ◽  
B Lymphocyte ◽  
Biochemical Characterization ◽  
Gel Filtration ◽  
Plasma Clot ◽  
Lentil Lectin

Abstract In this study we detected a factor that stimulates the proliferation of bone marrow-derived hematopoietic precursors in diffusion chambers implanted in mice. This factor, called diffusible colony-stimulating factor (D-CSF), was found in medium conditioned in the presence of spleen and peripheral blood cells from mice with B cell leukemia (BCL1). After the administration of D-CSF, the number of colonies formed in the plasma clot inside the chamber (CFU-DG) was increased, as were the number of hematopoietic precursors (CFU-MIX, CFU-S, CFU-C, and BFU-E) as judged by a subculture of diffusion chamber contents. Depletion of macrophages and T cells from the spleen cell suspension did not decrease the production of D-CSF, thereby indicating that it was derived from B cells. Neoplastic BCL1 cells appear to be the source because D-CSF could not be detected in medium conditioned with normal B cells. BCL1-conditioned medium (CM) did not enhance CFU-MIX, BFU-E, and CFU-C colony formation in vitro, which suggested that D-CSF is different from multi-CSF, EPA, or CSF. The addition of BCL1 CM to multi- CSF-, erythroid potentiating activity (EPA), and CSF (EL-4CM)- containing cultures had no effect on CFU-MIX, BFU-E, and CFU-C colony formation, thus indicating the absence of a synergistic or inhibitory activity. On the other hand, EL-4 CM, which stimulates CFU-MIX, BFU-E, and CFU-C in vitro, had no effect on CFU-DG in vivo. Biochemical characterization of BCL1 CM revealed that D-CSF is relatively heat stable and loses its bioactivity with protease treatments. It binds to lentil-lectin, according to gel-filtration chromatography has a relative molecular weight of approximately 43,000, and on reverse-phase high-performance liquid chromatography elutes with acetonitrile. These data also indicate that transformed B cells may serve as a source for hematopoietic regulators that act on hematopoietic precursors in vivo.

scholarly journals Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria

2000 ◽  
Vol 345 (2) ◽  
pp. 271-278 ◽  
Author(s):  
Bruno ANTONSSON ◽  
Sylvie MONTESSUIT ◽  
Sandra LAUPER ◽  
Robert ESKES ◽  
Jean-Claude MARTINOU
Keyword(s):  
Cytochrome C ◽  
Molecular Mass ◽  
Lipid Membranes ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Cytochrome C Release ◽  
Octyl Glucoside ◽  
Apoptotic Activity

Bax is a Bcl-2-family protein with pro-apoptotic activity that can form channels in lipid membranes. The protein has been shown to trigger cytochrome c release from mitochondria both in vitro and in vivo. Recombinant human Bax isolated in the presence of detergent was found to be present as an oligomer with an apparent molecular mass of approx. 160000 Da on gel filtration. When Bax was isolated in the absence of detergent the purified protein was monomeric with an apparent molecular mass of 22000 Da. Bax oligomers formed channels in liposomes and triggered cytochrome c release from isolated mitochondria, whereas monomeric Bax was inactive in both respects. Incubation of the monomeric Bax with 2% octyl glucoside induced formation of oligomers that displayed channel-forming activity in liposomes and triggered cytochrome c release from mitochondria. Triton X-100, Nonidet P-40 and n-dedecyl maltoside also activated monomeric Bax, whereas CHAPS had no activating effect. In cytosolic extracts from mouse liver, Bax migrated at a molecular mass of 24000 Da on gel filtration, whereas after incubation of the cytosol with 2% octyl glucoside Bax migrated at approximately 140000 Da. These results show that oligomeric Bax possesses channel-forming activity whereas monomeric Bax has no such activity.

scholarly journals Molecular Characterization of Saccharomyces cerevisiae TFIID

2002 ◽  
Vol 22 (16) ◽  
pp. 6000-6013 ◽  
Author(s):  
Steven L. Sanders ◽  
Krassimira A. Garbett ◽  
P. Anthony Weil
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Molecular Mass ◽  
Molecular Characterization ◽  
Biochemical Characterization ◽  
Gel Filtration ◽  
Calculated Molecular Mass ◽  
General Transcription Factor

ABSTRACT We previously defined Saccharomyces cerevisiae TFIID as a 15-subunit complex comprised of the TATA binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). In this report we give a detailed biochemical characterization of this general transcription factor. We have shown that yeast TFIID efficiently mediates both basal and activator-dependent transcription in vitro and displays TATA box binding activity that is functionally distinct from that of TBP. Analyses of the stoichiometry of TFIID subunits indicated that several TAFs are present at more than 1 copy per TFIID complex. This conclusion was further supported by coimmunoprecipitation experiments with a systematic family of (pseudo)diploid yeast strains that expressed epitope-tagged and untagged alleles of the genes encoding TFIID subunits. Based on these data, we calculated a native molecular mass for monomeric TFIID. Purified TFIID behaved in a fashion consistent with this calculated molecular mass in both gel filtration and rate-zonal sedimentation experiments. Quite surprisingly, although the TAF subunits of TFIID cofractionated as a single complex, TBP did not comigrate with the TAFs during either gel filtration chromatography or rate-zonal sedimentation, suggesting that TBP has the ability to dynamically associate with the TFIID TAFs. The results of direct biochemical exchange experiments confirmed this hypothesis. Together, our results represent a concise molecular characterization of the general transcription factor TFIID from S. cerevisiae.

scholarly journals Biochemical characterization of androgen receptor-interacting protein 4

2006 ◽  
Vol 393 (3) ◽  
pp. 789-795 ◽  
Author(s):  
Andrii Domanskyi ◽  
Katja T. Virtanen ◽  
Jorma J. Palvimo ◽  
Olli A. Jänne
Keyword(s):  
Androgen Receptor ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Protein Complexes ◽  
Sequence Similarity ◽  
Protein Family ◽  
Interacting Protein ◽  
Attachment Sites

ARIP4 [AR (androgen receptor)-interacting protein 4] is a member of the SNF2-like family of proteins. Its sequence similarity to known proteins is restricted to the centrally located SNF2 ATPase domain. ARIP4 is an active ATPase, and dsDNA (double-stranded DNA) and ssDNA (single-stranded DNA) enhance its catalytic activity. We show in the present study that ARIP4 interacts with AR and binds to DNA and mononucleosomes. The N-terminal region of ARIP4 mediates interaction with AR. Kinetic parameters of the ARIP4 ATPase are similar to those of BRG-1 and SNF2h, two members of the SNF2-like protein family, but the specific activity of ARIP4 protein purified to >90% homogeneity is approximately ten times lower, being 120 molecules of ATP hydrolysed by an ARIP4 molecule per min in contrast with approx. 1000 ATP molecules hydrolysed per min by ATP-dependent chromatin remodellers. Unlike other members of the SNF2 family, ARIP4 does not appear to form large protein complexes in vivo or remodel mononucleosomes in vitro. ARIP4 is covalently modified by sumoylation, and mutation of six potential SUMO (small ubiquitin-related modifier) attachment sites abolished the ability of ARIP4 to bind DNA, hydrolyse ATP and activate AR function. We conclude that, similar to its closest homologues in the SNF2-like protein family, ATRX (α-thalassemia, mental retardation, X-linked) and Rad54, ARIP4 does not seem to be a classical chromatin remodelling protein.

Hematopoetic precursors respond to a unique B lymphocyte-derived factor in vivo

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1784-1789
Author(s):  
E Niskanen ◽  
J Gorman ◽  
PC Isakson
Keyword(s):  
B Cells ◽  
Colony Formation ◽  
High Performance ◽  
B Lymphocyte ◽  
Biochemical Characterization ◽  
Gel Filtration ◽  
Plasma Clot ◽  
Lentil Lectin

In this study we detected a factor that stimulates the proliferation of bone marrow-derived hematopoietic precursors in diffusion chambers implanted in mice. This factor, called diffusible colony-stimulating factor (D-CSF), was found in medium conditioned in the presence of spleen and peripheral blood cells from mice with B cell leukemia (BCL1). After the administration of D-CSF, the number of colonies formed in the plasma clot inside the chamber (CFU-DG) was increased, as were the number of hematopoietic precursors (CFU-MIX, CFU-S, CFU-C, and BFU-E) as judged by a subculture of diffusion chamber contents. Depletion of macrophages and T cells from the spleen cell suspension did not decrease the production of D-CSF, thereby indicating that it was derived from B cells. Neoplastic BCL1 cells appear to be the source because D-CSF could not be detected in medium conditioned with normal B cells. BCL1-conditioned medium (CM) did not enhance CFU-MIX, BFU-E, and CFU-C colony formation in vitro, which suggested that D-CSF is different from multi-CSF, EPA, or CSF. The addition of BCL1 CM to multi- CSF-, erythroid potentiating activity (EPA), and CSF (EL-4CM)- containing cultures had no effect on CFU-MIX, BFU-E, and CFU-C colony formation, thus indicating the absence of a synergistic or inhibitory activity. On the other hand, EL-4 CM, which stimulates CFU-MIX, BFU-E, and CFU-C in vitro, had no effect on CFU-DG in vivo. Biochemical characterization of BCL1 CM revealed that D-CSF is relatively heat stable and loses its bioactivity with protease treatments. It binds to lentil-lectin, according to gel-filtration chromatography has a relative molecular weight of approximately 43,000, and on reverse-phase high-performance liquid chromatography elutes with acetonitrile. These data also indicate that transformed B cells may serve as a source for hematopoietic regulators that act on hematopoietic precursors in vivo.

scholarly journals Distinct effects of radicicol on myotoxic activity of crotoxin and Bothrops asper phospholipase A2 myotoxins in vivo and in vitro

2011 ◽  
Vol 25 (S1) ◽  
Author(s):  
Débora Cardoso Rodrigues ◽  
Lygia Gonçalves ◽  
Talita Conte ◽  
Igor Baptista ◽  
Anselmo Moriscot ◽  
...  
Keyword(s):  
Phospholipase A2 ◽  
Bothrops Asper

scholarly journals Transferrin-binding protein B isolated from Neisseria meningitidis discriminates between apo and diferric human transferrin

1998 ◽  
Vol 334 (1) ◽  
pp. 269-273 ◽  
Author(s):  
Ian C. BOULTON ◽  
Andrew R. GORRINGE ◽  
Nigel ALLISON ◽  
Andrew ROBINSON ◽  
Beatrice GORINSKY ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Molecular Mass ◽  
Binding Protein ◽  
Protein A ◽  
Gel Filtration ◽  
Free Form ◽  
Stable Complex ◽  
Receptor Complex

Neisseria meningitidis utilization of human serum transferrin (hTF)-bound iron is an important pathogenicity determinant. The efficiency of this system would clearly be increased through preferential binding of diferric hTF over the iron-free form. To characterize this process, functionally active meningococcal transferrin-binding protein A (TbpA) and TbpB have been purified from N. meningitidis using a novel purification procedure. The association of isolated Tbps and Tbps in the presence of hTF was investigated by gel filtration. Co-purified TbpA+B formed a complex of molecular mass 300 kDa which bound 1–2 molecules of hTF. Purified TbpA formed a complex of 200 kDa, indicating association as a dimer, whereas TbpB aggregated to form multimers of variable sizes. On recombining TbpA and TbpB, a stable complex of equivalent size to co-purified TbpA+B was formed. This complex may be composed of a single TbpA dimer and 1 molecule of TbpB. The technique of surface plasmon resonance (SPR) was used to demonstrate clearly that TbpB of either high (85 kDa) or low (68 kDa) molecular-mass preferentially bound diferric hTF in comparison with iron-free hTF. This selectivity was not observed with TbpA, but was found at low levels with co-purified TbpA+B. Individual TbpA and TbpB, recombined in a 1:1 molecular ratio, showed iron-mediated discriminatory binding at an intermediate level. SPR was also used to show that TbpA and TbpB bound to distinct regions of hTF, and that prior saturation with TbpB reduced subsequent TbpA binding. The results demonstrated that hTF bound more TbpA than TbpB, with an approximate ratio of 2:1. We have demonstrated that in vitro, TbpA+B exists as a receptor complex composed of a TbpA dimer and one molecule of TbpB, and that TbpB selectively binds diferric hTF. We propose that, in vivo, TbpA and TbpB also exist as a receptor complex, with TbpB selectively binding diferric hTF, bringing it close to TbpA, the transmembrane component, where the ferric iron can be transported to the periplasm.

scholarly journals Purification, characterization and function of bacterioferritin from the cyanobacterium Synechocystis P.C.C. 6803

1992 ◽  
Vol 281 (3) ◽  
pp. 785-793 ◽  
Author(s):  
J P Laulhère ◽  
A M Labouré ◽  
O Van Wuytswinkel ◽  
J Gagnon ◽  
J F Briat
Keyword(s):  
Molecular Mass ◽  
Sequence Similarity ◽  
Iron Status ◽  
Iron Concentration ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Cellular Iron ◽  
And Function

Storage and buffering of iron is achieved by a class of proteins, the ferritins, widely distributed throughout the living kingdoms. All ferritins have in common their three-dimensional structure and their ability to store large amounts of iron in their central cavity. However, eukaryotic ferritins from plants and animals and bacterioferritins have no sequence similarity, and besides non-haem iron bacterioferritins contain haem residues whereas eukaryotic ferritins do not. In this paper we report the first purification and characterization of a bacterioferritin from a cyanobacterium. It has a molecular mass of 400 kDa and is built up from 19 kDa subunits. Its N-terminal sequence shows 73% identity with that of the Escherichia coli bacterioferritin subunit. It contains 2300 atoms of iron and 1500 molecules of phosphate per ferritin molecule and 0.25 haem residue per subunit; the alpha-peak of the cytochrome has its maximum at 559 nm. In contrast with what is known for eukaryotic ferritins, we found that bacterioferritin from Synechocystis is not inducible by iron under the conditions that we have tested and that it has a constant concentration whatever the iron status of the cells, even at very low iron concentration. Bacterioferritin from Synechocystis P.C.C. 6803 is fully assembled in vivo and it is shown by labelling with 59Fe that it is able to load iron in vitro as well as in vivo. Bacterioferritin from Synechocystis is shown to have an iron-buffering function while the bulk of cellular iron is found associated with a pool of low-molecular-mass electronegative molecules. The role of Synechocystis bacterioferritin in iron metabolism is discussed.

The Biophysical and Functional Properties of Antithrombin III (AT III) in Vitro and in Vivo

1975 ◽  
Author(s):  
S. Chandra ◽  
N. U. Bang
Keyword(s):  
Antithrombin Iii ◽  
Biological Activities ◽  
Gel Filtration ◽  
Biological Properties ◽  
Molecular Forms ◽  
Step Procedure ◽  
At Iii ◽  
Exclusion Chromatography

Human and rabbit AT III were purified by a two-step procedure of heparin-agarose affinity chromatography and Sephadex G150 gel filtration. The resulting preparation purified approximately 900 fold over plasma was homogeneous by SDS gel electrophoresis (SDSE), immunoelectrophoresis and gel exclusion chromatography (GEC). Both human and rabbit AT III possessed mol wt’s of approximately 68,000 daltons. As previously observed (Rosenberg and Damus: J. Biol. Chern., 248, 0490, 1973), we noted that human as well as rabbit AT III form firm stoichiometric complexes with thrombin demonstrable by SDSE and GEC. 125I labeled rabbit AT III possessing biophysical and biological properties identical to the unlabeled product was injected into rabbits. Radiolabeled AT III displayed complex multiphasic disappearance curves with a half-life of the major component of 36 hours. The apparent mol wt of AT III in vivo was reduced to approximately 30,000 daltons within minutes after its administration into rabbits as evidenced by GEC and SDSE. The 30,000 dalton AT III was as capable as the 68.000 dalton AT III to form firm stoichiometric complexes with thrombin. Thus, AT III may exist in different molecular forms possessing similar biological activities.

scholarly journals Effect of calcineurin inhibitors on myotoxic activity of crotoxin and Bothrops asper phospholipase A2 myotoxins in vivo and in vitro

2006 ◽  
Vol 143 (3) ◽  
pp. 284-294 ◽  
Author(s):  
E.H. Miyabara ◽  
I.L. Baptista ◽  
B. Lomonte ◽  
H.S. Selistre-de-Araújo ◽  
J.M. Gutiérrez ◽  
...  
Keyword(s):  
Phospholipase A2 ◽  
Calcineurin Inhibitors ◽  
Bothrops Asper
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