scholarly journals Chemical modification of the lectin of the marine coral Gerardia savaglia by marine quinone avarone

2007 ◽  
Vol 72 (12) ◽  
pp. 1271-1274 ◽  
Author(s):  
Ivana Pajic ◽  
Zoran Vujcic ◽  
Miroslava Vujcic ◽  
Irena Novakovic ◽  
Dusan Sladic ◽  
...  
Keyword(s):  
Chemical Modification ◽  
Isoelectric Focusing ◽  
Marine Sponge ◽  
Protein A ◽  
Hemagglutination Activity ◽  
Sds Page ◽  
Dysidea Avara

The quinone avarone, isolated from the marine sponge Dysidea avara, possesses the ability to chemically modify proteins. In this work, modification of lectin isolated from the coral Gerardia savaglia by avarone was examined. The techniques used for studying the modification were: SDS PAGE, isoelectric focusing and hemagglutination testing. The results of the SDS PAGE indicate dimerization of the protein. A shift of the pI toward lower value occurs upon modification. The change of the hemagglutination activity of the protein confirms that chemical modification of G. savaglia lectin by avarone changes its ability to interact with the membrane of erythrocytes.

scholarly journals Studies on regulation of IGF (insulin-like growth factor)-binding protein (IGFBP) 4 proteolysis by pregnancy-associated plasma protein-A (PAPP-A) in cells treated with phorbol ester

2004 ◽  
Vol 379 (1) ◽  
pp. 57-64 ◽  
Author(s):  
Arun S. SIVANANDAM ◽  
Subburaman MOHAN ◽  
Hirohito KITA ◽  
Sanjay KAPUR ◽  
Shin-Tai CHEN ◽  
...  
Keyword(s):  
Growth Factor ◽  
Proteolytic Activity ◽  
Plasma Protein ◽  
Binding Protein ◽  
Protein A ◽  
Insulin Like Growth Factor ◽  
Factor Binding ◽  
Fold Reduction ◽  
Sds Page ◽  
Eosinophil Major Basic Protein

PAPP-A (pregnancy-associated plasma protein-A) is produced by hSFs (human skin fibroblasts) and hOBs (human osteoblasts) and enhances the mitogenic activity of IGFs (insulin-like growth factors) by degradation of IGFBP-4 (insulin-like growth factor-binding protein 4). PKC (protein kinase C) activation in these cells led to reduction in IGFBP-4 proteolysis. This study was undertaken to determine the mechanism by which activation of PKC suppresses IGFBP-4 proteolysis. Treatment of hSFs/hOBs with TPA (PMA; 100 nM) reduced IGFBP-4 proteolysis without significantly decreasing the PAPP-A level in the CM (conditioned medium). Immunodepletion of the proform of eosinophil major basic protein (proMBP), a known PAPP-A inhibitor, from CM of TPA-treated cells (TPA CM) failed to increase IGFBP-4 proteolytic activity. Transduction of hSFs with proMBP retrovirus increased the concentration of proMBP up to 30 ng/ml and led to a moderate reduction in IGFBP-4 proteolysis. In contrast, TPA treatment blocked IGFBP-4 proteolysis but failed to induce a detectable amount of proMBP in the CM. While proMBP overexpression led to the formation of a covalent proMBP–PAPP-A complex and reduced the migration of PAPP-A on SDS/PAGE, TPA treatment dose- and time-dependently increased the conversion of a ≈470 kDa PAPP-A form (PAPP-A470) to a ≈400 kDa PAPP-A form (PAPP-A400). Since unreduced PAPP-A400 co-migrated with the 400 kDa recombinant PAPP-A homodimer and since PAPP-A monomers from reduced PAPP-A470 and PAPP-A400 co-migrated on SDS/PAGE, conversion of PAPP-A470 to PAPP-A400 is unlikely to be caused by proteolytic cleavage of PAPP-A. Consistent with the data showing that the increase in the ratio of PAPP-A400/PAPP-A470 is correlated with the extent of reduction in IGFBP-4 proteolysis, partially purified PAPP-A400 exhibited a 4-fold reduction in IGFBP-4 proteolytic activity compared with PAPP-A470. These data suggest that a novel mechanism, namely conversion of PAPP-A470 to the less-active PAPP-A400, could account for the TPA-induced suppression of PAPP-A activity.

Isoelectric focusing analysis of soluble immune complexes bound to Protein A-sepharose

1981 ◽  
Vol 111 (2) ◽  
pp. 336-342 ◽  
Author(s):  
Bert W. Maidment ◽  
Lawrence D. Papsidero ◽  
Marie Gamarra ◽  
Takuma Nemoto ◽  
T.Ming Chu
Keyword(s):  
Isoelectric Focusing ◽  
Immune Complexes ◽  
Protein A ◽  
Soluble Immune Complexes

FIBRINOGEN MILANO III, A NEW ABNORMAL HEREDITARY FIBRINOGEN VARIANT WITH A DEFECT IN THE Aα-CHAIN

1987 ◽  
Author(s):  
C Bögli ◽  
A Hofer ◽  
M Furlan ◽  
E A Beck ◽  
F Baudo ◽  
...  
Keyword(s):  
Isoelectric Focusing ◽  
Structural Defect ◽  
Reversed Phase ◽  
Amino Terminus ◽  
Molecular Defect ◽  
Functional Assay ◽  
Fibrinogen Concentration ◽  
Reversed Phase Hplc ◽  
Sds Page ◽  
Fibrin Monomers

A new congential dysfibrinogenemia, denoted as Milano III, was found in a 13-year-old girl with recurrent thrombophlebitis. Plasma of the patient exhibited prolonged thrombin and reptilase times. Plasma fibrinogen concentration, determined by a functional assay, was less than 0.2 g/1 while the immunological method revealed normal fibrinogen levels. Fibrinopeptide release, induced by thrombin, was normal whereas polymerization of fibrin monomers was delayed. Under conventional conditions for normal fibrin aggregation, with and without added calcium, the final turbidity of abnormal fibrin was léss than 10 % of normal fibrin turbidity. The abnormal fibrinogen strongly inhibited clotting of normal fibrinogen. Isoelectric focusing showed an abnormal Aα-chain with greatly increased anodic mobility suggesting that the net electric charge of the Aα-chain is similar to that of the γ-chain. SDS-PAGE as well as reversed phase HPLC of mercap-tolyzed fibrinogen showed normal Bβ- and γ-chains whereas the Aα-chain was degraded. The susceptibility towards degradation appears to be related to the molecular defect in the variant Aα-chain, since normal Aα-chain was not affected under the same conditions. Intact size of Aα-chain was preserved during preparative chromatofocusing of reduced fibrinogen chains. Fibrinogen preparations from the proposita's mother and father contained approximately equal amounts of both normal and abnormal Aα-chain. We conclude that fibrinogen Milano III in the proposita is a new homozygous functionally abnormal variant with a structural defect in the Aochain which is not located in the amino terminus.

EXPRESSION OF PLATELET ALLOANTIGENS ON HUMAN ENDOTHELIAL CELLS AND HEL CELLS

1987 ◽  
Author(s):  
Y Kawai ◽  
R R Montgomery ◽  
K Furihata ◽  
T J Kunicki
Keyword(s):  
Endothelial Cells ◽  
Protein A ◽  
Cell Types ◽  
Membrane Glycoproteins ◽  
Human Endothelial Cells ◽  
Immunoblot Assay ◽  
Sds Page ◽  
Hel Cells ◽  
Erythroleukemia Cell ◽  
Different Cell Types

Analogs of platelet membrane glycoproteins IIb and IIIa (GPIIb-IIIa) have been shown to be synthesized and expressed by human endothelial cells (HEC), a human erythroleukemia cell line (HEL) and various other cells. Previous studies from our laboratory demonstrated that the platelet alloantigen P1A1, is expressed on HEC GPIIIa. Other alloantigen systems, namely, Pen and Bak, are known to be localized on platelet GPIIIa and GPIIb, respectively. Utilizing additional antibodies from patients with PTP specific for Pena, Baka, and Bakb allo-antigens, and isoantibodies (iso-ab) from a patient with Glanzmann's Thrombasthenia (GT), we have studied cultured HEC and HEL cells for expression of epitopes recognized by these antibodies. HEC and HEL cells were meta-bolically labeled with 35S-methionine and lysed in 0.5% TX-100 in the presence of 5mM EDTA. Soluble antigens were immunoprecipitated with these antibodies coupled to Protein A-Sepharose and subjected to SDS-PAGE and fluorography. Anti-Pena and the GT iso-ab reacted with the GPIIb-IIIa complex from both HEC and HEL lysates, but anti-Baka and anti-Bakb failed to immunoprecipitate GPIIb-IIIa analogs from either HEC or HEL. In an immunoblot assay, the GT iso-ab bound to GPIIIa of both HEC and HEL. Anti-Pena failed to react with SDS-denatured proteins. HEL GPIIIa migrates slightly faster than HEC GPIIIa and slightly slower than platelet GPIIIa. These results indicate that the epitopes of platelet GPIIIa recognized by alloantibodies and isoantibodies are shared by GPIIIa analogs of HEC and HEL. GPIIb-associated alloantigens are not expressed by HEC and HEL GPIIb analogs, an observation that is consistent with the decreased structural homology between GPIIb analogs derived from different cell types.

Preliminary Studies on SDS-PAGE and Isoelectric Focusing Identification of Sturgeon Sources of Caviar

1996 ◽  
Vol 61 (3) ◽  
pp. 533-536 ◽  
Author(s):  
I.-C. CHEN ◽  
F.A. CHAPMAN ◽  
C.I. WEI ◽  
S.F. O'KEEFE
Keyword(s):  
Isoelectric Focusing ◽  
Sds Page

scholarly journals Molecular cloning and biochemical characterization of rabbit factor XI

2002 ◽  
Vol 367 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Dipali SINHA ◽  
Mariola MARCINKIEWICZ ◽  
David GAILANI ◽  
Peter N. WALSH
Keyword(s):  
Human Factor ◽  
Biochemical Characterization ◽  
Protein A ◽  
Size Exclusion ◽  
Factor Xi ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Intracellular Process ◽  
And Function

Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process.

scholarly journals A polymer-dependent increase in phosphorylation of beta-tubulin accompanies differentiation of a mouse neuroblastoma cell line.

1985 ◽  
Vol 100 (3) ◽  
pp. 764-774 ◽  
Author(s):  
D L Gard ◽  
M W Kirschner
Keyword(s):  
Neurite Outgrowth ◽  
Isoelectric Focusing ◽  
Developmental Regulation ◽  
Neuroblastoma Cell ◽  
Microtubule Assembly ◽  
Beta Tubulin ◽  
Fourfold Increase ◽  
Cell Extracts ◽  
Mouse Neuroblastoma ◽  
Sds Page

We have examined the phosphorylation of cellular microtubule proteins during differentiation and neurite outgrowth in N115 mouse neuroblastoma cells. N115 differentiation, induced by serum withdrawal, is accompanied by a fourfold increase in phosphorylation of a 54,000-mol-wt protein identified as a specific isoform of beta-tubulin by SDS PAGE, two-dimensional isoelectric focusing/SDS PAGE, and immunoprecipitation with a specific monoclonal antiserum. Isoelectric focusing/SDS PAGE of [35S]methionine-labeled cell extracts revealed that the phosphorylated isoform of beta-tubulin, termed beta 2, is one of three isoforms detected in differentiated N115 cells, and is diminished in amounts in the undifferentiated cells. Taxol, a drug which promotes microtubule assembly, stimulates phosphorylation of beta-tubulin in both differentiated and undifferentiated N115 cells. In contrast, treatment of differentiated cells with either colcemid or nocodazole causes a rapid decrease in beta-tubulin phosphorylation. Thus, the phosphorylation of beta-tubulin in N115 cells is coupled to the levels of cellular microtubules. The observed increase in beta-tubulin phosphorylation during differentiation then reflects developmental regulation of microtubule assembly during neurite outgrowth, rather than developmental regulation of a tubulin kinase activity.

scholarly journals Purification and properties of adenosine 5′-phosphosulphate sulphotransferase from Euglena

1991 ◽  
Vol 274 (2) ◽  
pp. 355-360 ◽  
Author(s):  
J Li ◽  
J A Schiff
Keyword(s):  
Isoelectric Focusing ◽  
Euglena Gracilis ◽  
Deae Cellulose ◽  
Reactive Dye ◽  
Multiple Forms ◽  
Freezing And Thawing ◽  
Disulphide Bonds ◽  
Purification And Properties ◽  
Sds Page ◽  
High Concentrations

Adenosine 5′-phosphosulphate sulphotransferase (APSST) was extracted from Euglena gracilis Klebs var. bacillaris mutant W10BSmL by freezing and thawing and was purified about 10,000-fold (to homogeneity) with 10.5% recovery by (NH4)2SO4 precipitation, Sephadex G-100 chromatography, Reactive Blue-agarose, Reactive Dye-agarose, DEAE-cellulose, preparative isoelectric focusing and non-inactivating SDS/PAGE. The active APSST, with a molecular mass of 102 kDa and multiple forms from pI 5.0 to 5.5, is a tetramer held together by covalent (probably disulphide) bonds. An apparent Km of the purified enzyme for adenosine 5′-phosphosulphate (APS) of 0.1 microM is obtained when dithiothreitol is used as the thiol. The enzyme is stimulated by Mg2+, Ca2+ or Ba2+, and uses almost any thiol; dithiothreitol and dithioerythritol give the highest activity. In the absence of APS, the enzyme is inactivated (and is rendered monomeric) by thiols but is protected from thiol inactivation by AMP, adenosine 5′-phosphoramidate (APA) or adenosine 5′-monosulphate (AMS), which also inhibit APSST activity somewhat. The enzyme resists inactivation by SDS in the absence of thiols; SDS stimulates APSST activity at low concentration, but high concentrations are inhibitory.

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