Details

Autor(en) / Beteiligte

• ENTWISTLE, RUTH ANN

Titel

A KINETIC STUDY OF FIBRONECTIN AND THE CLQ COMPONENT OF COMPLEMENT (GLYCOPROTEIN, IMMUNE SYSTEM)

Ort / Verlag

ProQuest Dissertations & Theses

Erscheinungsjahr

1986

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• The binding of fibronectin, an adhesive glycoprotein, with the Clq component of the classical complement cascade may possibly modulate the function of this protein in the immune system. Binding of human plasma fibronectin to Clq required Clq immobilized on a solid phase or on an immune complex. To further define the equilibrium constant, K(,d), the dissociation rate constant k(,d), and the nature of the binding reaction was the purpose of this study. Polystyrene tubes were coated with 2-10 (mu)g/ml Clq solutions and the amounts of Clq adsorbed to the solid-phase varied between 1.3 and 3.6 pmoles over a total surface area of 250 mm('2). Fibronectin in concentrations from 0.25 nM to 300 nM reached an equilibrium of binding to Clq after reaction for 2 hours at 37(DEGREES)C in a low ionic strength buffer of 0.015 M NaCl, 10 mM sodium phosphate, and 10 (mu)g/ml bovine serum albumin (BSA) ((mu) = 0.038). The presence of additional salt ions in both the labeled and unlabeled fibronectin preparations resulted in a final ionic strength used in the binding experiments of (mu) = 0.048. For the different Clq coatings used, fibronectin binding exhibited nonlinear Scatchard plots. A minimum of two equilibrium constants were calculated from the data averaging 26 nM and 0.16 nM. The higher affinity constant is more than 100 fold higher than constants obtained with slightly higher ionic strength conditions. Different types of fibronectin-Clq interactions were also indicated by the amounts of bound ('125)I-fibronectin that dissociated over various times and conditions, and in the presence of solution phase fibronectin. Two dissociation rates of fibronectin-Clq complexes were measured as 1.5 x 10('-3) min('-1) and 3.6 x 10('-5) min('-1). The non-linear equilibrium data and dissociation rates may indicate more than one binding site, cooperativity, or different avidity effects in the binding between fibronectin and Clq.