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 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 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.

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 Plasmid studies ofSalmonella typhimuriumphage type 179 resistant to ampicillin, tetracycline, sulphonamides and trimethoprim

1980 ◽  
Vol 85 (2) ◽  
pp. 293-300 ◽  
Author(s):  
D. M. Anderson
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
New Zealand ◽  
Salmonella Typhimurium ◽  
National Health ◽  
Gel Electrophoresis ◽  
Phage Type ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
K 12

SUMMARYSixteen strains ofSalmonella typhimuriumphage type 179 were referred to the National Health Institute, Wellington, New Zealand, from 1977 to 1979. This phage type had not been observed here before 1977. All strains were resistant to ampicillin, several were also resistant to tetracycline, and several were resistant to ampicillin, tetracycline, sulphafurazele and trimethoprim. All resistances could be transferred toEscherichia coliK 12. Plasmids from these strains and their transconjugants were characterized by agarose gel electrophoresis. It appears that resistance to sulphafurazole and trimethoprim is carried on a plasmid with a molecular weight of 5·2 Mdal and that resistance to ampicillin and tetracycline is carried on a plasmid with a molecular weight of approximately 60 Mdal.

Rabbit Plasma, unlike Its Human Counterpart, Contains no Complex between Protein S and C4b-Binding Protein

1994 ◽  
Vol 71 (04) ◽  
pp. 446-451 ◽  
Author(s):  
Xuhua He ◽  
Björn Dahlbäck
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Complex Formation ◽  
Human Plasma ◽  
High Molecular Weight ◽  
Western Blotting ◽  
Vitamin K ◽  
Protein S ◽  
Rabbit Plasma ◽  
Free Protein

SummaryIn human plasma, the anticoagulant vitamin K-dependent protein S exists in two molecular forms, as free protein and complexed to C4b- binding protein (C4BP), a complement regulatory protein. It has been suggested that rabbit plasma also contains two forms of protein S and that the interaction between protein S and C4BP m rabbits can be modulated by synthetic peptides corresponding to a sequence (residues 605-614) in the carboxy-lerminal part of protein S. In this report, we provide itsulls which challenge the conclusion that rabbit plasma contains the complexed form of protein S. The two forms of protein S in human plasma were separated by gel filtration chromatography on Sephacryl S-300 and the presence of protein S in the various fractions analyzed by Western blotting using a monoclonal antibody (HPS 21) directed against the γ-carboxyglutamic acid rich module of human protein S. This antibody, which was found to cross-react with rabbit protein S on Western blotting, was used in affinity purification of protein S from rabbit plasma as well as of recombinant rabbit protein S. HPS 21 specifically recognized protein S in rabbit plasma and did not cross-react with the other vitamin K-depeudenl plasma proteins. To elucidate whether rabbit plasma contained two forms of protein S, rabbit plasma was subjected to gelfiltration chromatography followed by Western blotting of the fractions with monoclonal antibody HPS 21. Protein S was found only in fractions eluting at a position corresponding to that of free protein S. A radiolabelled trace amount of recombinant rabbit protein S added to rabbit plasma chromatographed as free protein S and no high molecular weight form corresponding to a C4BP-protein S complex was detected. Rabbit protein S had the capacity to bind human C4BP and the addition of human C4BP to rabbit plasma changed the elution profile, of rabbit plasma protein S. After the addition of human C4BP, rabbit plasma protein S quantitatively eluted as a high molecular weight complex, suggesting that all the protein S in rabbit plasma was bound to human C4BP. The anticoagulant activity of human protein S is modulated by the complex formation with C4BP. Our results demonstrate that this function of C4BP and the protein S-C4BP complex formation has not been conserved throughout the evolution even though protein S has a preserved C4BP binding site.

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