scholarly journals Degradation of human complement component C4b in the presence of the C4b-binding protein-protein S complex

1983 ◽  
Vol 209 (3) ◽  
pp. 857-863 ◽  
Author(s):  
B Dahlbäck ◽  
B Hildebrand
Keyword(s):  
Binding Sites ◽  
Binding Protein ◽  
Sodium Dodecyl ◽  
Fluid Phase ◽  
Protein S ◽  
Factor I ◽  
Haemolytic Assay ◽  
Dependent Protein ◽  
Molecular Weight Form ◽  
Human Complement Component

Vitamin K-dependent protein S and the higher-molecular-weight form of C4b-binding protein (C4bp-high) interact, forming a 1:1 complex with a KD of approx. 1×10(-7) M [Dahlbäck (1983) Biochem. J. 209, 847-856]. In the present study the effect of protein S on the degradation of C4b by Factor I (C3b inactivator) and C4bp was investigated both in fluid phase and on cell surfaces, with the use of highly purified components. Fluid-phase degradation of C4b was monitored on sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis, and the effect on surface-bound C4b was estimated by haemolytic assay. No effect of protein S could be demonstrated in any of the systems used. Thus, although bound to C4bp, protein S is neither involved in, nor does it affect, the interaction between C4bp and C4b. This indicates that the binding sites on the C4bp molecule for protein S and for C4b are independent and different.

scholarly journals Purification of human vitamin K-dependent protein S and its limited proteolysis by thrombin

1983 ◽  
Vol 209 (3) ◽  
pp. 837-846 ◽  
Author(s):  
B Dahlbäck
Keyword(s):  
Vitamin K ◽  
Gel Electrophoresis ◽  
Binding Protein ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Protein S ◽  
Single Chain ◽  
Free Protein ◽  
Reducing Conditions ◽  
Dependent Protein

Vitamin K-dependent protein S exists in two forms in human plasma, namely as the free protein and in complex with C4b-binding protein [Dahlbäck & Stenflo (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2512-2516]. Now reported is a simple purification procedure for human protein S that includes barium citrate adsorption, DEAE-Sephacel chromatography and chromatography on Blue Sepharose. The yield was approx. 30% relative to the concentration of free protein S in plasma, which was found to be approx. 10 mg/l. Purified protein S migrated as a single-chain band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under non-reducing conditions and as a doublet of Mr approx. 85 000 and 75 000 on reduction. A third band of Mr 16 000 was observed after electrophoresis of 125I-labelled protein S and radioautography of reduced samples. This band appears to be disulphide-linked to the 75 000-Mr chain before reduction. Thrombin converted the 85 000-Mr chain of protein S into a 75 000-Mr chain and an 8000-Mr fragment, the latter again being detectable only by radioautography of reduced samples. The 16 000-Mr fragment was not observed, suggesting its degradation by thrombin. Under non-reducing conditions, no change in apparent molecular weight of thrombin-treated protein S was observed, indicating disulphide linkage of the fragments. Thrombin also affected the mobility of protein S on agarose-gel electrophoresis in the presence of Ca2+, suggesting a decreased affinity to Ca2+ of the cleaved form of protein S as compared with the undegraded molecule. After activation of the complement system in human serum, protein S was found to be a constituent part of the complex formed by C4b-binding protein and component C4b.

scholarly journals Purification of human C4b-binding protein and formation of its complex with vitamin K-dependent protein S

1983 ◽  
Vol 209 (3) ◽  
pp. 847-856 ◽  
Author(s):  
B Dahlbäck
Keyword(s):  
Molecular Weight ◽  
Complex Formation ◽  
Vitamin K ◽  
Gel Electrophoresis ◽  
Binding Protein ◽  
Protein S ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Dependent Protein ◽  
Molecular Weight Form

C4b-binding protein was purified from human plasma in high yield by a simple procedure involving barium citrate adsorption and two subsequent chromatographic steps. Approx. 80% of plasma C4b-binding protein was adsorbed on the barium citrate, presumably because of its complex-formation with vitamin K-dependent protein S. The purified C4b-binding protein had a molecular weight of 570 000, as determined by ultracentrifugation, and was composed of about eight subunits (Mr approx. 70 000). Uncomplexed plasma C4b-binding protein was purified from the supernatant after barium citrate adsorption. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in non-reducing conditions and on agarose-gel electrophoresis it appeared as a doublet, indicating two forms differing slightly from each other in molecular weight and net charge. The protein band with the higher molecular weight in the doublet corresponded to the C4b-binding protein purified from the barium citrate eluate. Complex-formation between protein S and C4b-binding protein was studied in plasma, and in a system with purified components, by an agarose-gel electrophoresis technique. Protein S was found to form a 1:1 complex with the higher-molecular-weight form of C4b-binding protein, whereas the lower-molecular-weight form of C4b-binding protein did not bind protein S. The KD for the C4b-binding protein-protein S interaction in a system with purified components was approx. 0.9×10(-7) M. Rates of association and dissociation at 37 degrees C were low, namely about 1×10(3) M-1 . S-1 and 1.8×10(-4)-4.5×10(-4) S-1 respectively. In human plasma free protein S and free higher-molecular-weight C4b-binding protein were in equilibrium with the C4b-binding protein-protein S complex. Approx. 40% of both proteins existed as free proteins. From equilibrium data in plasma a KD of about 0.7×10(-7) M was calculated for the C4b-binding protein-protein S interaction.

scholarly journals Expression and characterization of a recombinant C4b-binding protein lacking the β-chain

1995 ◽  
Vol 308 (3) ◽  
pp. 795-800 ◽  
Author(s):  
Y Härdig ◽  
P García de Frutos ◽  
B Dahlbäck
Keyword(s):  
Molecular Mass ◽  
Binding Protein ◽  
Expression System ◽  
Activated Protein C ◽  
Protein S ◽  
High Molecular Mass ◽  
Eukaryotic Expression ◽  
Beta Chain ◽  
Factor I ◽  
Dependent Protein

C4b-binding protein (C4BP) is a high-molecular-mass glycoprotein which contains binding sites for complement component C4b, anti-coagulant vitamin K-dependent protein S and serum amyloid P component (SAP). The major form of C4BP in plasma is composed of seven identical alpha-chains and a single beta-chain. We have expressed full-length cDNA for the alpha-chain in a eukaryotic expression system and characterized functional properties of non-beta-chain-containing C4BP. During synthesis, recombinant alpha-chains polymerized into two different high-molecular-mass C4BP forms which were composed of seven or eight alpha-chains. Recombinant C4BP bound C4(H2O) (used instead of C4b) equally as well as native C4BP, functioned equally as well as factor I cofactor in the degradation of C4(H2O) and bound to SAP. In contrast, the recombinant C4BP did not bind protein S and therefore did not inhibit the ability of protein S to function as a cofactor to activated protein C. Tunicamycin treatment of the transfected cells prevented N-linked glycosylation, but did not affect polymerization of the alpha-chains into a high-molecular-mass C4BP. The non-glycosylated C4BP had comparable properties to glycosylated C4BP in several functional assays. These results demonstrate polymerization of C4BP alpha-chains to be independent both of the beta-chain and of the N-linked carbohydrates. Moreover, N-linked carbohydrates and the beta-chain were neither required for the ability of C4BP to bind C4b and to function as factor I cofactor nor for the interaction with SAP.

scholarly journals Vitamin K-dependent protein S is similar to rat androgen-binding protein

1987 ◽  
Vol 243 (1) ◽  
pp. 293-296 ◽  
Author(s):  
M E Baker ◽  
F S French ◽  
D R Joseph
Keyword(s):  
Vitamin K ◽  
Binding Protein ◽  
Protein A ◽  
Serine Proteinase ◽  
Protein S ◽  
Clotting Factors ◽  
The Family ◽  
Androgen Binding Protein ◽  
Dependent Protein ◽  
Androgen Binding

Vitamin K-dependent protein S belongs to the family of clotting factors (e.g. Factors IX and X, and protein C). Unlike the other clotting factors, the C-terminal half (residues 250-634) of protein S is not a serine proteinase. In fact, the function of residues 250-634 of protein S is unknown. By using computer programs designed to detect evolutionary relationships between proteins, we find that this part of protein S is similar to rat androgen-binding protein, a protein produced and secreted by testicular Sertoli cells. The homology between protein S and androgen-binding protein suggests new approaches for elucidating their functions.

Isolation of a Saccharomyces cerevisiae centromere DNA-binding protein, its human homolog, and its possible role as a transcription factor

1987 ◽  
Vol 7 (1) ◽  
pp. 403-409
Author(s):  
R J Bram ◽  
R D Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Binding Sites ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Sodium Dodecyl ◽  
Heat Stable ◽  
Tata Element ◽  
Adenovirus Major Late Promoter ◽  
Major Late Promoter

A protein that binds specifically to Saccharomyces cerevisiae centromere DNA element I was purified on the basis of a nitrocellulose filter-binding assay. This protein, termed centromere-binding protein 1 (CP1), was heat stable and renaturable from sodium dodecyl sulfate (SDS), and assays of eluates from SDS gels indicated a molecular weight of 57,000 to 64,000. An activity with similar specificity and stability was detected in human lymphocyte extracts, and analysis in SDS gels revealed a molecular weight of 39,000 to 49,000. CP1-binding sites occurred not only at centromeres but also near many transcription units, for example, adjacent to binding sites for the GAL4-positive regulatory protein upstream of the GAL2 gene in S. cerevisiae and adjacent to the TATA element of the adenovirus major late promoter. A factor (termed USF) that binds to the latter site and stimulates transcription has been isolated from HeLa cells by others.

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