The binding of calcium to human fibronectin.

The binding of calcium to human plasma fibronectin has been measured by equilibrium dialysis at 25 degrees in 0.1 M NaCl 50mM Tris HCL, pH 7.4. Curve fitting of the binding data indicates that fibronectin has two strong calcium binding sites per chain (Mr 220,000), KD = 1.3 mM and approximately 12 weak sites, KD = 2.3 mM. No significant displacement of bound calcium by magnesium was observed at magnesium concentrations up to 1 mM. Calcium binding to a pair of tryptic fragments of fibronectin (Mr congruent to 160,000 and 180,000) that bind to gelatin has also been investigated. These fragments have a single class of calcium binding sites, with 2.2 sites per chain, KD = 1.1 mM. Negligible calcium binding to tryptic fragments derived from other regions of the fibronectin molecule was observed.

Cation binding properties of the multiple subforms of RVV-X, the coagulant protein from Vipera russelli.

The factor X activating enzyme from Russell's viper venom (RVV-X) has been shown to exist in multiple subforms, distinguished from each other by their isoelectric points. The differences in isoelectric points were due, as least in part, to dissimilarities in the respective sialic acid contents of the subforms. No functional difference was, however, discovered between any of the subforms. All of the subforms were found, by equilibrium ultrafiltration, to bind Ca2+ reversibly. At least two equivalent Ca2+ binding sites were observed on each protein molecule (Mr 79 000), with a KD of 50 +/- 15 microM at pH 7.4 and 25 degrees C. A new substrate for RVV-X, which does not bind Ca2+, apoprotein AI from human high-density lipoprotein, was used to show that this reversibly bound Ca2+ was not essential for enzymic activity. All subforms have also been shown, by atomic absorption analysis, to contain nonexchangeable metal ions, to the extent of 1 mol of Ca2+ and 0.7 mol of Zn2+ per mol of protein. No Mn2+ or Mg2+ was detected. This nonexchangeable Ca2+ and Zn2+ could only be removed from the protein by incubation at pH 3.0 or by treatment with 6 M guanidine hydrochloride, conditions under which the protein lost activity irreversibly.

Interaction of calcium with bovine plasma protein C.

The binding of 45Ca2+ to bovine plasma protein C (PC) and to activated bovine plasma protein C (APC) has been examined by equilibrium ultrafiltration at pH 7.4 and 25 degrees C. Under these conditions, PC possesses 16.0 plus or minus 2.0 equivalent Ca2+ binding sites, of average KD (8.7 plus or minus 1.5) x 10(-4) M, and APC contains 9.0 plus or minus 1.0 equivalent Ca2+ binding sites, with an average KD of (4.3 plus or minus 1.1) x 10(-4) M. Both Mn2+ and Sr2+ were capable of ready displacement of Ca2+ from a Ca2+-PC complex, while Mg2+ was less effective in this regard. The alpha-thrombin-catalyzed activation of PC was inhibited by the presence of Ca2+. A kinetic analysis of this effect demonstrated that it was, in large part, due to an increase in the Km of the reaction. Addition of other divalent cations, e.g. Mn2+, Sr2+, and Mg2+, in place of Ca2+ also resulted in inhibition of the alpha-thrombin-catalyzed activation of PC in a manner which paralleled their ability to displace Ca2+ from a Ca2+-PC complex. On the other hand, the activation of PC by the coagulant protein from Russell's Viper venom was augmented by the presence of Ca2+. Other divalent metal ions, such as Sr2+ and Mn2+, in the absence of Ca2+, also weakly stimulated this reaction. Mg2+ was without notable effect.

The binding of calcium to the activation products of bovine factor IX.

Binding isotherms of Ca2+ to the bovine Factor IX activation intermediates and products, i.e. Factor IXalpha, Factor IXa alpha, and Factor IXa beta have been examined. At pH 7.4, Factor IX alpha possesses at least two strong Ca2+ sites, with an average KD of 0.1 mM, and an additional 11 weaker sites, with an average KD of 3.7 mM. Bovine Factor IXa alpha also contains at least two Ca2+ binding sites, with an average KD of 0.1 mM, and an additional 11 weaker sites, with an average KD of 1.3 mM. Factor IXa beta, the ultimate activation product of Factor IX, in the intrinsic system, likewise contains at least two strong Ca2+ sites, of average KD 0.1 mM, as well as seven additional weaker sites, possessing an average KD of 1.0 mM. The Ca2+-binding properties of the above proteins are similar to those of their precursor molecule, Factor IX, which we have earlier shown to possess at least two strong Ca2+ sites, with an average KD of 0.1 mM, and 11 weaker sites, of average KD 1.3 mM (Amphlett, G.W., Byrne, R., and Castellino, F.J. (1978) J. Biol. Chem. 253, 6774-6779). Circular dichroism analysis of all of the above proteins was consistent with the molecules possessing a low alpha-helical content, and a high quantity of beta structure and random coil conformations.

The binding of metal ions to bovine factor IX.

The binding isotherms of Ca2+ and Mn2+ to bovine factor IX have been determined at pH 6.5 and pH 7.4, at 25 degrees C. At pH 7.4, at least 2 strong Ca2+ sites, with an average KDISS of 0.1 +/- 0.02 mM, are found. An additional 10 to 11 weaker Ca2+ binding sites, with an average KDISS of 1.3 +/- 0.2 mM are noted, at high levels of Ca2+. At pH 6.5, again at least 2 strong Ca2+ sites on factor IX are evident, with an average KDISS of 0.11 +/- 0.02 mM; but slightly fewer (7 to 8) weaker sites, with an average KDISS of 1.9 +/- 0.3 mM, are obtained. Qualitatively, the binding of Mn2+ to bovine factor IX appears similar to that of Ca2+. At pH 6.5, approximately 2 strong Mn2+ binding sites, with an average KDISS of 13 +/- 1.5 micrometer, and an additional 5 to 6 weak sites, with an average KDISS of 160 +/- 15 micrometer, are present. Manganese does not completely displace Ca2+; and Ca2+ does not completely displace Mn2+ from their respective binding sites. On the other hand, Tb3+ and Sm3+ readily displace Ca2+, at pH 6.5, from its sites on factor IX. The rate and extent of activation of bovine factor IX, by bovine factor XIa, is dependent on the Ca2+ concentration, up to concentrations of Ca2+ which saturate its effect on the system. Substitution of Sr2+ for Ca2+ leads to approximately 50% of the maximum rate of factor IXa formation, and final yield of factor IXa, in this activation system. Manganese does not substitute for Ca2+ in this activation, but does inhibit the stimulatory effect of Ca2+. Both Tb3+ and Sm3+ are effective inhibitors of Ca2+ in factor IX activation by factor XIa.

The primary structure of troponin T and the interaction with tropomyosin.

1. Eight peptides were separated from the CNBr digest of troponin T from rabbit white skeletal muscle and characterized. 2. By study of the amino acid sequence of the methionine-containing peptides isolated after chymotryptic and tryptic digestion and of the N- and C-terminals of the CNBr peptides, six of the latter were shown to be arranged in the sequence CNB1-CNB2-CNB5-CNB6-CNB8-CNB7. The other two peptides, CNB1' and CNB3, have been shown to be partial digestion products. 3. The CNBr peptides CNB1' and CNB2 contained a common sequence and were the only peptides in CNBr digests of troponin T that formed a complex with tropomyosin as judged by viscometric and electrophoretic studies. 4. It is concluded that tropomyosin interacts with the N-terminal half of the troponin T molecule approximately in the region lying between residues 70 and 160. 5. Electrophoretic evidence indicates that tropomyosin and troponin C interact with troponin T. 6. None of the major CNBr peptides of troponin T isolated formed a complex with troponin C on electrophoresis at pH 8.6.