The relationship between protein-protein and protein-lipid interactions and the immunological properties of bovine prothrombin and several of its fragments.

Isothermal titration calorimetry was used to determine the enthalpy for the calcium-dependent protein conformation change and subsequent interaction of blood clotting factor X and prothrombin with phospholipid vesicles. The effect of vesicle size was also determined. The protein conformation change was accompanied by -12 +/- 1 and -7 to -15 kcal/mol for factor X and prothrombin, respectively. The range of values for prothrombin arose from use of different protein preparations and may be due to non-ideal behavior of this protein when calcium was added. The apparent enthalpy of association (delta H(assoc)) of both factor X and prothrombin with phosphatidylserine (PS)/phosphatidylcholine (PC) large unilamellar vesicles (LUVs, 120 nm diameter) was shown to be near 0 kcal/mol. In comparison, delta H(assoc) for interaction with PS/PC small unilamellar vesicles (SUVs, 40 nm diameter) was -9 +/- 3 and -7 +/- 2 kcal/mol for factor X and prothrombin, respectively. This difference appeared complementary to delta H(assoc) for calcium binding to these vesicles. That is, the interaction of calcium was athermic with SUVs and exothermic with LUVs. While such properties might suggest a considerable difference in the manner of calcium binding to LUVs versus SUVs, little difference in the quantity of calcium bound to SUVs and LUVs was detected by equilibrium dialysis. In any event, the results indicate that protein binding to LUVs was primarily entropy driven whereas binding to SUVs was primarily enthalpy driven. The exothermic process for calcium-dependent factor X or prothrombin binding to SUVs may result from protein-induced changes in the phospholipid packing/calcium interaction, possibly related to changes in how calcium is bound to the phospholipid.

Section: Discussionmentioning

confidence: 99%

Direct Enthalpy Measurements of Factor X and Prothrombin Association with Small and Large Unilamellar Vesicles

Plager¹,

Nelsestuen²

1994

“…For prothrombin it has been observed that the transition precedes prothrombin-phospholipid interactions (Nelsestuen, 1976). In addition, a number of investigators have isolated antibodies that are specific for the calcium-induced conformer of prothrombin (Madar et al, 1982;Tai et al, 1980Tai et al, , 1984. Others have suggested that the observed quenching might be due to calcium-induced self-association of the protein (Jackson et al, 1979).…”

Section: Discussionmentioning

confidence: 99%

“…The first hypothesis suggests that calcium forms an ion bridge between the protein and the phospholipid (Suttie & Jackson, 1977). The second is that calcium binding induces a conformational change that exposes a membrane-binding surface (Rhee et al, 1982;Madar et al, 1982). Gla residues have been shown by a number of investigators to be associated with calcium-mediated protein binding to membranes (Nelsestuen & Suttie, 1972a, 1972bStenflo & Ganrot, 1972;Stenflo, 1972;Esmon et al, 1975).…”

Section: Discussionmentioning

confidence: 99%

Properties of chemically modified protein S: the effect of the conversion of .gamma.-carboxyglutamic acid to .gamma.-methyleneglutamic acid on functional properties

Walker¹

1986

Protein S, the protein cofactor for activated protein C in the proteolytic inactivation of factor Va, was chemically modified with a mixture of morpholine and formaldehyde. This treatment resulted in the conversion of the gamma-carboxyglutamic acid (Gla) residues of this vitamin K dependent protein to gamma-methyleneglutamic acid. With a 10,000-fold molar excess of morpholine and formaldehyde over protein S it was found that between 10 and 11 Gla residues could be modified. The degree of modification was proportional to the concentration of the modifying reagents used. The modification of as few as two residues resulted in the 70% loss of activity. Calcium inhibited the modification of several residues. In the presence of 3.2 mM calcium ion, a derivative with 2.5 residues modified was prepared that appeared to have full activity. Modification of protein S resulted in the alteration of a number of its properties. The quenching of intrinsic fluorescence by calcium decreased. The quenching effect of terbium ions was also decreased. However, the modified protein and the native protein were equivalent when protein-dependent terbium fluorescence was measured. When modified, protein S would no longer bind to phospholipid vesicles. Finally, the ability of protein S to self-associate was decreased by modification. These findings suggest that the gamma-carboxyglutamic acid residues of protein S may play several roles in the maintenance of structure.

“…Consequently, the cross-linking appears to be monitoring a calcium-specific event other than dimerization (Silversmith et al, 1983). Subpopulations of fragment 1 antibodies have been shown to have specificity for metal ion induced structures in fragment 1 (Tai et al, 1980;Madar et al, 1982). Furthermore, these antibodies can be separated into calcium conformation-specific antibodies (Borowski et al, 1986).…”

mentioning

confidence: 99%

Amino-terminal alanine functions in a calcium-specific process essential for membrane binding by prothrombin fragment 1

Welsch¹,

Nelsestuen²

1988

Two acetylation sites on prothrombin fragment 1 (amino-terminal 156 amino acid residues of bovine prothrombin) are essential for the tight calcium and membrane binding functions of the protein; calcium protects both of these sites from acetylation [Welsch, D. J., Pletcher, C. H., & Nelsestuen, G. L. (1988) Biochemistry (first of three papers in this issue)]. The epsilon-amino groups of the lysine residues (positions 3, 11, 44, 57, and 97) were not critical to protein function and were acetylated in the calcium-protected protein. The most reactive of the two essential acetylation sites was identified as amino-terminal alanine. To identify this site, fragment 1 was first acetylated in the presence of calcium to derivatize the nonessential sites. Removal of calcium and partial acetylation with radioactive reagent produced a single major radioactive peptide. Isolation and characterization of this peptide showed that the radioactivity was associated with amino-terminal alanine. In addition, sequence analysis of calcium-protected protein showed the presence of underivatized amino-terminal alanine. Surprisingly, covalent modification with a trinitrophenyl group did not alter membrane binding activity. Thus, the positive charge on the amino terminus did not appear critical to its function. Acetylation of amino-terminal alanine without acetylation of the second essential site produced a fragment 1 derivative which had a high requirement for calcium and which had lost most membrane binding function. However, this protein had only slightly altered affinity for magnesium ion. In agreement with this metal ion selectivity, protection of amino-terminal alanine was calcium specific, and magnesium ion did not protect this site from acetylation.(ABSTRACT TRUNCATED AT 250 WORDS)