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 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.

A Sensitive Peroxidase Staining Immunoblotting Method for Measuring Total Protein S in Human Plasma

1993 ◽  
Vol 69 (04) ◽  
pp. 331-334 ◽  
Author(s):  
Xiangan Li ◽  
Kaoru Hatanaka ◽  
Ling Guo ◽  
Motoo Tsushima ◽  
Yukihiko Kitamura ◽  
...  
Keyword(s):  
Human Plasma ◽  
Total Protein ◽  
Gel Electrophoresis ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Protein S ◽  
Polyacrylamide Gel ◽  
Free Form

SummaryIn plasma, protein S is found in its free form and as a complex with C4b-binding protein. After 125I-protein S was added tonormal human plasma and applied to SDS-8% polyacrylamide gel electrophoresis, the autoradiogram of the gel showed only one single band at free protein S position. Applying this evidence, we have developed a peroxidase staining Western Blotting method to quantitate total protein S in human plasma which consists of sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by blotting to nitrocellulose membrane and a sensitive avidin-biotinylated peroxidase staining method (ABC technique). The measurement of protein S by the immunoblotting was reproducible and the coefficient of variation was 7%. As little as 1 ng of protein S could be detected. C4b-binding protein did not affect the measurement of protein S. Compared to other immunoassays, this peroxidase staining immunoblotting method has the advantage of directly estimating the apparent molecular weight of protein of interest, eliminating nonspecific stain and having high sensitivity without using radioisotope.

LOW PLASMA CONCENTRATIONS OF C4b-BINDING PROTEIN AND VITAMIN K-DEPENDENT PROTEIN S IN PRETERM INFANTS WITH DECREASED FORMATION OF PROTEIN S-C4b-BINDING PROTEIN COMPLEXES

1987 ◽  
Author(s):  
J Malm ◽  
R Bennhagen ◽  
L Holmberg ◽  
B Dahlbäck
Keyword(s):  
Preterm Infants ◽  
Vitamin K ◽  
Total Protein ◽  
Binding Protein ◽  
Plasma Concentrations ◽  
Protein S ◽  
Complement Protein ◽  
Term Infants ◽  
Free Protein ◽  
Adult Level

Protein S is a vitamin K-dependent plasmaprotein functioning as a non-enzymatic cofactor to the activated form of protein C in the degradation of coagulationfactors Va and VIIa. In the circulation approximately 60% of protein S is complexed to the complement protein C4b-binding protein (C4BP). Only the remaining, free fraction exhibits protein Ca cofactor activity.The plasma concentrations of protein S and C4BP were determined in 25 term and 26 preterm infants. Both proteins werequantified with radioimmunoassays. The free, functionally active form of proteinS and the total protein S concentration were determined separately. The level ofC4BP in preterm infants was found to be very low (mean 6% of the adult level). In term infants the level had increased to a mean of 18%. Also the total concentration of protein S was decreased in preterm as well as in term infants; 18% and 31% of the adult level, respectively. Free protein S was the predominant form in plasma representing 83 % of total protein S in preterm and 68 % in term infants. This was probably due to the very low C4BP levels. In adult controls the corresponding value was 34%. The plasma concentration of free protein S in preterm and term infants, when compared to the adult level, was 44% and 66%, respectively. These results demonstrate that although the total protein S concentration in preterm and term infants was very low when compared to adult levels, the difference in the concentration of free, functionally active protein S between infants and adults was less pronounced.

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