The association of coagulation factor Xa and factor Va

The binding of factor Xa to factor Va in the presence of Ca2+ ions and phospholipid is fundamental for the activation of prothrombin to thrombin. Nevertheless, the biochemistry of the intrinsic association between factors Xa and Va is poorly understood. In the present study we have measured the formation of the protein-protein complex in the absence of phospholipid by using analytical ultracentrifugation. Factor Xa or factor Va were respectively modified with a chromophore-peptidyl-chloromethyl ketone or a thiol-specific chromophore, which permitted selective evaluation of the sedimentation of either component by virtue of its unique absorbance properties. Regardless of which protein was labeled, a factor Xa-Va complex (s20,w = 9.8) was formed. The interaction is specific and reversible. In 2 mM Ca2+ and at 20 degrees C, the dissociation constant for the binding of factor Xa to factor Va is 0.8 microM with a 1:1 stoichiometry. The association has multiphasic Ca2+ dependence. At concentrations of Ca2+ below 1 mM or above 2 mM, a weaker protein-protein equilibrium is maintained.     

The interaction of bovine factor V and factor V-derived peptides with phospholipid vesicles

The binding of bovine Factor V, isolated Factor Va, and isolated activation intermediates to single bilayer phospholipid vesicles was studied by light scattering. The vesicles composed of 25% phosphatidylserine and 75% phosphatidylcholine had a mean radius of approximately 163 A as determined by quasi-elastic light scattering. When these vesicles were saturated with Factor V, the radii increased by approximately 120 A in both 0.15 and 1 M NaCl. At saturation, about 35 molecules of Factor V and 141 molecules of Factor Va were bound to each vesicle. Studies of the binding of Factor V and Factor Va at various ionic strengths showed little change in either Kd or n, suggesting that the binding is not electrostatic. The dissociation constants (Kd) and the lipid to protein ratios at saturation, moles/mol (n), obtained by relative light scattering intensities were: Factor V (Kd = 4.3 X 10(-8) M, n = 214); isolated Factor Va (Kd = 1.7 X 10(-7) M, n = 57); component B, Mr = 205,000 (Kd = 1.8 X 10(-7) M, n = 140); component C, Mr = 150,000 (Kd = 7.0 X 10(-7) M, n = 136); component D, Mr = 94,000 (no binding could be demonstrated); component E, Mr = 74,000 (Kd = 3.8 X 10(-7) M, n = 42). The results presented here indicate that the lower Kd exhibited by Factor V compared to Factor Va (components D and E) is primarily due to the interaction present within the component C portion of the molecule which is destroyed when component C is further cleaved to give component D. The interactions responsible for the binding of Factor Va are expressed in component E as well as in its precursor peptide component B. Dissociation of components D and E by the addition of EDTA indicate that component E alone is responsible for the interaction of bovine Factor Va with phospholipid.     

Induced fit activation mechanism of the exceptionally specific serine protease, complement factor D

We have investigated the mechanism by which the complement protease, Factor D, achieves its high specificity for the cleavage of Factor B in complex with C3(H2O). Kinetic experiments showed that Factor B and C3(H2O) associate with a KD of >/=2.5 microM and that Factor D acts on this complex with a second-order rate constant of kcat/KM >/= 2 x 10(6) M-1 s-1, close to the rate of a diffusion-controlled reaction for proteins of this size. In contrast, Factor D, which is a member of the trypsin family of serine proteases, was 10(3)-10(4)-fold less active than trypsin toward both thioester and p-nitroanilide substrates containing an arginine at P1. Furthermore, peptides spanning the Factor B cleavage site were not detectably cleaved by Factor D (kcat/KM /=9 kcal/mol of binding energy to stabilize the transition state for reaction. In support of this, we demonstrate that chemical modification of Factor D at a single lysine residue that is distant from the active site abolishes the activity of the enzyme toward Factor B while not affecting activity toward small synthetic substrates. We propose that Factor D may exemplify a special case of the induced fit mechanism in which the requirement for conformational activation of the enzyme results in a substantial increase in substrate specificity.     

Inhibitor stabilization of human immunodeficiency virus type-2 proteinase dimer formation

We report the first direct observation of the subunit self-association behavior of highly purified recombinant human immunodeficiency virus type-2 (HIV-2) proteinase. Multiple samples of enzyme were subjected to sedimentation equilibrium analytical ultracentrifugation sequentially at 8.8 degrees C and two pH values in the presence and absence of a C2 symmetric, peptidomimetic inhibitor. At both pH values the enzyme exhibited sedimentation equilibrium behavior which fit a monomer-dimer-tetramer model. In the absence of inhibitor, the apparent Kd for dimer formation was less than approximately 100 microM and the apparent Kd for the weaker dimer-tetramer association was greater than approximately 100 microM. In the presence of inhibitor, at either pH, dimer formation was more strongly favored as indicated by a approximately 5-14-fold decrease in the apparent Kd for dimer formation and a approximately 1.2-4-fold increase in the apparent Kd for tetramer formation. The enhanced formation of dimer and decrease in higher order self-associated forms in the presence of an inhibitor is consistent with inhibitor stabilization of an active dimer. The inhibitor-induced stabilization of the dimeric species is consistent with a model for substrate-induced formation of active proteinase dimers in virion assembly.     

Solution studies of the quaternary structure and assembly of human von Willebrand factor

The reversible association of protomers of von Willebrand protein (vWF) was studied in order to analyze the forces and mechanism of vWF polymer assembly. At concentrations of vWF found in plasma (approximately 16 micrograms/mL), disulfide bond reduction with 50 mM 2-mercaptoethanol (2-ME) markedly reduced both vWF activity, as measured by ristocetin-dependent platelet agglutination, and average polymer size (Rh, the mean hydrodynamic radius) in solution, as determined by quasi-elastic light scattering (QLS) and by gel filtration chromatography. With increasing vWF concentration, activity and Rh increased despite reduction of interprotomer disulfide bonds. Changes in temperature after 2-ME treatment produced reversible changes in activity and Rh. Varying the total vWF concentration at any given temperature after 2-ME treatment changed Rh in a consistent and predictable fashion, so that estimates of the dissociation constant for vWF protomer-polymer equilibrium were obtained: Kd5 degrees C = 0.77 micrograms/mL, Kd25 degrees C = 2.4 micrograms/mL, and Kd37 degrees C = 7.7 micrograms/mL, where under the conditions of reduction presented here, the basic protomer of vWF is a dimer. Increasing ionic strength after 2-ME treatment with 1 M KCl did not change Rh, while approximately 100 microM sodium dodecyl sulfate (SDS) or approximately 300 microM sodium deoxycholate (DOC) reduced both Rh and activity compared with those of unreduced polymer. These data show that disulfide bonds are necessary to maintain vWF polymer size and activity at plasma concentrations but that noncovalent forces of association can maintain vWF polymer size and activity at higher concentrations. These forces of association may be important for polymer assembly during intracellular synthesis of vWF.     

Interaction of lanthanide ions with bovine factor X and their use in the affinity chromatography of the venom coagulant protein of Vipera russelli.

The substitution of trivalent lanthanide ions for Ca(II) in the Ca(II)-DEPENDENT ACTIVATION OF BOVINE Factor X by the coagulant protein of Russell's viper venom was studied at pH 6.8. Factor X contains two high affinity metal binding sites which bind Gd(III), Sm(III), and Yb(III) with a Kd of about 4 X 10-7 M and four to six lower affinity metal binding sites which bind Gd(III), Sm(III) with a Kd of about 1.5 X 10-5M. In comparison, 1 mol of Factor X binds 2 mol of Ca(II) with a Kd of 3 X 10-4M and weakly binds many additional Ca(II) ions. No binding of Gd(III) to the venom protein was observed. Dy(III), Yb(III), Tb(III), Gd(III), Eu(III), La(III), AND Nd(III) cannot substitute for Ca(II) in the Ca(II)-dependent activation of Factor X by the venom protein at pH 6.8. Kinetic data consistent with the models of competitive inhibition of Ca(II) by Nd(III) yielded a Ki of 1 to 4 X 10-6M. The substitution of lanthanide ions for Ca(II) to promote protein complex formation of Factor X-metal-venom protein without the activation of Factor X facilitated the purification of the coagulant protein from crude venom by affinity chromatography. Using a column containing Factor X covalently bound to agarose which was equilibrated in 10 mM Nd(III), Tb(III), Gd(III), or La(III), the coagulant protein was purified 10-fold in 40% yield from crude venom and migrated as a single band on gel electrophoresis in sodium dodecyl sulfate. These data suggest that lanthanide ions complete with Ca(II) for the metal binding sites of Factor X and facilitate the formation of a nonproductive ternary complex of venom protein-Factor X-metal. Tb(III) fluorescence, with emission maxima at 490 and 545 nm, is enhanced 10,000-fold in the presence of Factor X. The study of the participation of an energy donor intrinsic to Factor X in energy transfer to Tb(III) may be useful in the characterization of the metal binding sites of Factor X.     

Three-state equilibrium of Escherichia coli trigger factor

Trigger Factor (TF) is the first chaperone that interacts with nascent chains of cytosolic proteins in Escherichia coli. Although its chaperone activity requires association with ribosomes, TF is present in vivo in a 2-3 fold molar excess over ribosomes and a fraction of it is not ribosome-associated after cell lysis. Here we show that TF follows a three-state equilibrium. Size exclusion chromatography, crosslinking and analytical ultracentrifugation revealed that uncomplexed TF dimerizes with an apparent Kd of 18 microM. Dimerization is mediated by the N-terminal ribosome binding domain and the C-terminal domain of TF, whereas the central peptidyl prolyl isomerase (PPlase) and substrate binding domain does not contribute to dimerization. Crosslinking experiments showed that TF is monomeric in its ribosome-associated state. Quantitative analysis of TF binding to ribosomes revealed a dissociation constant for the TF-ribosome complex of approximately 1.2 microM. From these data we estimate that in vivo most of the ribosomes are in complex with monomeric TF. Uncomplexed TF, however, is in a monomer-dimer equilibrium with approximately two thirds of TF existing in a dimeric state.     

Structure and function of recombinant cobra venom factor

Cobra venom factor (CVF) is the complement-activating protein from cobra venom. It is a structural and functional analog of complement component C3. CVF functionally resembles C3b, the activated form of C3. Like C3b, CVF binds factor B, which is subsequently cleaved by factor D to form the bimolecular complex CVF,Bb. CVF,Bb is a C3/C5 convertase that cleaves both complement components C3 and C5. CVF is a three-chain protein that structurally resembles the C3b degradation product C3c, which is unable to form a C3/C5 convertase. Both C3 and CVF are synthesized as single-chain prepro-proteins. This study reports the recombinant expression of pro-CVF in two insect cell expression systems (baculovirus-infected Sf9 Spodoptera frugiperda cells and stably transfected S2 Drosophila melanogaster cells). In both expression systems pro-CVF is synthesized initially as a single-chain pro-CVF molecule that is subsequently proteolytically processed into a two-chain form of pro-CVF that structurally resembles C3. The C3-like form of pro-CVF can be further proteolytically processed into another two-chain form of pro-CVF that structurally resembles C3b. Unexpectedly, all three forms of pro-CVF exhibit functional activity of mature, natural CVF. Recombinant pro-CVF supports the activation of factor B in the presence of factor D and Mg2+ and depletes serum complement activity like natural CVF. The bimolecular convertase pro-CVF,Bb exhibits both C3 cleaving and C5 cleaving activity. The activity of pro-CVF and the resulting C3/C5 convertase is indistinguishable from CVF and the CVF,Bb convertase. The ability to produce active forms of pro-CVF recombinantly ensures the continued availability of an important research reagent for complement depletion because cobra venom as the source for natural CVF will be increasingly difficult to obtain as the Indian cobra is on the list of endangered species. Experimental systems to express pro-CVF recombinantly will also be invaluable for studies to delineate the structure and function relationship of CVF and its differences from C3 as well as to generate human C3 derivatives with CVF-like function for therapeutic complement depletion ("humanized CVF").     

Interaction of clotting factor V heavy chain with prothrombin and prethrombin 1 and role of activated protein C in regulating this interaction: analysis by analytical ultracentrifugation

Changes in the affinity of the heavy subunit of blood coagulation factor Va (Vh) for prothrombin are thought to be important in regulating the rate of thrombin production. Using analytical ultracentrifugation, we have measured the affinity of bovine Vh for prothrombin and for the prethrombin 1 fragment of prothrombin at 23.3 degrees C, pH 7.65, in 50 mM tris(hydroxymethyl)aminomethane, 0.1 M NaCl, 0.1 mM benzamidine, and either 2 mM Ca2+ or 2 mM ethylenediaminetetraacetate (EDTA). Under these conditions a 1:1 complex of Vh with prothrombin is formed that is governed by a dissociation constant (Kd) of 10 microM, regardless of whether the buffer contains Ca2+ or EDTA. An identical Kd is observed when prethrombin 1 is substituted for prothrombin. This indicates that the fragment 1 portion of prothrombin, containing the gamma-carboxyglutamic acid residues, does not influence the association. Substitution of human prethrombin 1 for the bovine molecule also results in a 1:1 Vh-prethrombin 1 complex governed by a slightly weaker Kd (27 microM). Discrete proteolysis of bovine Vh by the anticoagulant activated protein C converts the Vh to a form with little or no affinity for prethrombin 1 (Kd greater than 1 mM), without detectable change in the mass of the Vh.     

Metal binding and metal-induced conformational change of frog bone gamma-carboxyglutamic acid-containing protein

Ca2+ or Cd2+ binding and the conformational change induced by the metal binding in two frog bone Gla-proteins (BGP, termed BGP-1 and BGP-2) were studied by equilibrium dialysis and CD measurement. By CD measurement in the far-ultraviolet region, the alpha-helix content of both apoBGPs was found to be 8%. Binding of both Ca2+ and Cd2+ was accompanied with a change in the CD spectrum, and the alpha-helix content increased to 15 and 25% for BGP-1 and BGP-2, respectively. CD measurement in the near-ultraviolet region indicated that the environment of aromatic amino acid residues in the protein molecule was changed by metal binding. Equilibrium dialysis experiments indicated that each of these two protein binds specifically 2 mol of Ca2+, and nonspecifically an additional 3-4 mol of Ca2+ in 0.02 M Tris-HCl/0.15 M NaCl (pH 7.4), at 4 degrees C. According to the two separate binding sites model, BGP-1 has 1 high-affinity Ca2+ binding site (Kd1 = 0.17 mM) and 1 low-affinity site (Kd2 = 0.29 mM), and BGP-2 contains 1 high-affinity site (Kd1 = 0.14 mM) and 1 low-affinity site (Kd2 = 0.67 mM). In addition, 2 Cd2+ bound to a high-affinity binding site on BGP-1 with Kd1 of 10.4 microM, and 1 Cd2+ bound to a low-affinity binding site with Kd2 of 41.5 microM. On the other hand, BGP-2 had three classes of binding sites and 1 Cd2+ bound to each binding site with Kd1 = 3.6 microM, Kd2 = 16.3 microM, Kd3 = 51.7 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding sites on fibrin(ogen) for guinea pig liver transglutaminase are similar to those of blood coagulation factor XIII. Characterization of the binding of liver transglutaminase to fibrin

The present study represents detailed investigations into the nature of interactions between an intracellular "tissue" transglutaminase and a plasma protein, fibrinogen. We demonstrate a specific, saturable, and reversible binding of transglutaminase to fibrin(ogen). The binding was time- and temperature-dependent, was independent of divalent metal ions, did not require the release of either fibrinopeptide A or B, and was partially inhibited by the presence of sodium chloride or plasma proteins, properties similar to Factor XIII binding to fibrin(ogen). Both Factor XIII and liver transglutaminase also shared similar binding sites on fibrinogen, the A alpha- and the B beta-chains. The binding characteristics of liver transglutaminase were thus similar to Factor XIII binding to fibrin, but there were also important differences. Scatchard analyses of the binding data indicated that the affinity of liver transglutaminase (Kd = 4.17 x 10(-7) M) was at least 40-fold weaker compared with the affinity of Factor XIII to fibrinogen. Consequently, a 20-fold molar excess of Factor XIII a-chains specifically and completely inhibited the binding of liver transglutaminase to des-A-fibrinogen. The association between liver transglutaminase and fibrin(ogen) was also critically controlled by the conformational states of the two proteins. Substances capable of altering the conformation of either transglutaminase (such as guanosine 5'-triphosphate) or of fibrinogen (such as the tetrapeptide Gly-Pro-Arg-Pro and Fragment D) disrupted binding. Excess CaCl2 was able to counteract the effects of guanosine 5'-triphosphate on transglutaminase binding to fibrin. In contrast, Factor XIII binding to fibrin was unaffected by either guanosine 5'-triphosphate, CaCl2, or Gly-Pro-Arg-Pro, suggesting a more stable association between the two proteins. The physiologic implications of transglutaminase-fibrin(ogen) interactions are discussed.     

Ca2+ dependence of the interactions between protein C, thrombin, and the elastase fragment of thrombomodulin. Analysis by ultracentrifugation.

The two-way and three-way interactions among active-site-blocked bovine thrombin, bovine protein C, and the elastase fragment of rabbit thrombomodulin (elTM) were examined by analytical ultracentrifugation at 23.3 degrees C in 100 mM NaCl, 50 mM Tris (pH 7.65), and 1 mM benzamidine, in the presence of 0 to 5 mM calcium chloride. Thrombin and elTM form a tight (Kd less than 10(-8) M) 1:1 complex in the absence of Ca2+ that weakens with the addition of Ca2+ (Kd approximately 4 microM in 5 mM Ca2+). Without Ca2+, thrombin and protein C form a 1:1 complex (Kd approximately 1 microM) and what appears to be a 1:2 thrombin-protein C complex. The Kd for the 1:1 complex weakens over 100-fold in 5 mM CaCl2. Protein C and elTM form a Ca(2+)-independent 1:1 complex (Kd approximately 80 microM). Nearly identical binding to thrombin and elTM is observed when active-site-blocked activated bovine protein C is substituted for protein C. Thrombin inhibited by diisopropyl fluorophosphate and thrombin inhibited by a tripeptide chloromethyl ketone exhibited identical behavior in binding experiments, suggesting that the accessibility of protein C to the substrate recognition cleft of these two forms of thrombin is nearly equal. Human protein C binds with lower affinity than bovine protein C. Ternary mixtures also were examined. Protein C, elTM, and thrombin form a 1:1:1 complex which dissociates with increasing [Ca2+]. In the absence of Ca2+, protein C binds to the elTM-thrombin complex with an apparent Kd approximately 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction between aurovertin and adenine nucleotide binding sites on mitochondrial F1-ATPase and the isolated beta subunit

The effect of aurovertin on the binding parameters of ADP and ATP to native F1 from beef heart mitochondria in the presence of EDTA has been explored. Three exchangeable sites per F1 were titrated by ADP and ATP in the absence or presence of aurovertin. Curvilinear Scatchard plots for the binding of both ADP and ATP were obtained in the absence of aurovertin, indicating one high affinity site (Kd for ADP = 0.6-0.8 microM; Kd for ATP = 0.3-0.5 microM) and two lower affinity sites (Kd for ADP = 8-10 microM; Kd for ATP = 7-10 microM). With a saturating concentration of aurovertin capable of filling the three beta subunits of F1, the curvilinearity of the Scatchard plots was decreased for ATP binding and abolished for ADP binding, indicating homogeneity of ADP binding sites in the F1-aurovertin complex (Kd for ADP = 2 microM). When only the high affinity aurovertin site was occupied, maximal enhancement of the fluorescence of the F1-aurovertin complex was attained with 1 mol of ADP bound per mol of F1 and maximal quenching for 1 mol of ATP bound per mol of F1. When the F1-aurovertin complex was incubated with [3H]ADP followed by [14C]ATP, full fluorescence quenching was attained when ATP had displaced the previously bound ADP. In the case of the isolated beta subunit, both ADP and ATP enhanced the fluorescence of the beta subunit-aurovertin complex. The Kd values for ADP and ATP in the presence of EDTA were 0.6 mM and 3.7 mM, respectively; MgCl2 decreased the Kd values to 0.1 mM for both ADP and ATP. It is postulated that native F1 possesses three equivalent interacting nucleotide binding sites and exists in two conformations which are in equilibrium and recognize either ATP (T conformation) or ADP (D conformation). The negative interactions between the nucleotide binding sites of F1 are strongest in the D conformation. Upon addition of aurovertin, the site-site cooperativity between the beta subunits of F1 is decreased or even abolished.     

Functional role of the noncatalytic subunit of complement C5 convertase

The C5 convertase is a serine protease that consists of two subunits: a catalytic subunit which is bound in a Mg2+-dependent complex to a noncatalytic subunit. To understand the functional role of the noncatalytic subunit, we have determined the C5-cleaving properties of the cobra venom factor-dependent C5 convertase (CVF, Bb) made with CVF purified from the venom of Naja naja (CVFn) and Naja haje (CVFh) and compared them to those for two C3b-dependent C5 convertases (ZymC3b,Bb and C3b,Bb). A comparison of the kinetic parameters indicated that although the four C5 convertases (CVFn,Bb, ZymC3b,Bb, CVFh,Bb, and C3b,Bb) had similar catalytic rate constants (kcat = 0.004-0.012 s-1) they differed 700-fold in their affinity for the substrate as indicated by the Km values (CVFn,Bb = 0.036 microM, ZymC3b,Bb = 1.24 microM, CVFh,Bb = 14.0 microM, and C3b,Bb = 24 microM). Analysis of binding interactions between C5 and the noncatalytic subunits (CVFh or C3b, or CVFn) using the BIAcore, revealed dissociation binding constants (Kd) that were similar to the Km values of the respective enzymes. The kinetic and binding data demonstrate that the binding site for C5 resides in the noncatalytic subunit of the enzyme, the affinity for the substrate is solely determined by the noncatalytic subunit and the catalytic efficiency of the enzyme appears not to be influenced by the nature of this subunit.     

Proteolysis of factor Va by factor Xa and activated protein C

Bovine Factor Va, produced by selective proteolytic cleavage of Factor V by thrombin, consists of a heavy chain (D chain) of Mr = 94,000 and a light chain (E chain) of Mr = 74,000. These peptides are noncovalently associated in the presence of divalent metal ion(s). Each chain is susceptible to proteolysis by activated protein C and by Factor Xa. Sodium dodecyl sulfate electrophoretic analysis indicates that cleavage of the E chain by either activated protein C or Factor Xa yields two major fragments: Mr = 30,000 and Mr = 48,000. Amino acid sequence analysis indicates that the Mr = 30,000 fragments have identical NH2-terminal sequences and that this sequence corresponds to that of intact E chain. The Mr = 48,000 fragments also have identical NH2-terminal sequences, indicating that activated protein C and Factor Xa cleave the E chain at the same position. Sodium dodecyl sulfate electrophoretic analysis indicates that activated protein C cleavage of the D chain yields two products: Mr = 70,000 and Mr = 24,000. Amino acid sequence analysis indicates that the Mr = 70,000 fragment has the same NH2-terminal sequence as intact D chain, whereas the Mr = 24,000 fragment does not. Factor Xa cleavage of the D chain also yields two products: Mr = 56,000 and Mr = 45,000. The Mr = 56,000 fragment corresponds to the NH2-terminal end of the D chain and Factor V. Functional studies have shown that both chains of Factor Va may be entirely cleaved to products by Factor Xa without loss of activity, whereas activated protein C cleavage results in loss of activity. Since activated protein C and Factor Xa cleave the E chain at the same position, the cleavage of the D chain by activated protein C is responsible for the inactivation of Factor Va.     

The binding of Ca2+ ions to pig heart NAD+-isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex.

1. The binding of Ca2+ ions to purified pig heart NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase, freed of contaminating Ca2+ by parvalbumin/polyacrylamide chromatography, has been studied by flow dialysis and by the use of fura-2. 2. For the 2-oxoglutarate dehydrogenase complex, 3.5 mol of Ca2+-binding sites/mol of complex were apparent, with an apparent dissociation constant (Kd value) for Ca2+ of 2.0 microM. These values were little affected by Mg2+ ions, ADP or 2-oxoglutarate. 3. By contrast, binding of Ca2+ to NAD+-isocitrate dehydrogenase (Kd = 14 microM) required ADP, isocitrate and Mg2+ ions. The number of Ca2+-binding sites associated with NAD+-isocitrate dehydrogenase was then 0.9 mol/mol of tetrameric enzyme. 4. The 2-oxoglutarate dehydrogenase complex bound ADP (as ADP3-) to a group of tight-binding sites (Kd = 3.1 microM) with a stoichiometry, 3.3 mol/mol of complex, similar to that for the binding of Ca2+; a variable number of much weaker sites (Kd = 100 microM) for ADP3- was also apparent.     

Insulin-metal ion interactions: the binding of divalent cations to insulin hexamers and tetramers and the assembly of insulin hexamers

An insulin hexamer containing one B10-bound Co(III) ion and one unoccupied B10 site has been synthesized. The properties of the monosubstituted hexamer show that occupancy of only one B10 site by Co3+ is sufficient to stabilize the hexameric form under the conditions of pH and concentration used in these studies. The experimentally determined, second-order rate constants for the binding of Zn2+ and Co2+ to the unoccupied B10 site are consistent with literature rate constants for the rate of association of these divalent metal ions with similar small molecule ligands. These findings indicate that the rate-limiting steps for Zn2+ and Co2+ binding involve the removal of the first aqua ligand. The rate constant for the binding of Cd2+ is significantly lower than the literature values for small molecule chelators, which suggests that some other protein-related process is rate-limiting for Cd2+ binding to the unoccupied, preformed B10 site. The kinetics of the assembly of insulin in the presence of limiting metal ion provides strong evidence indicating that the B13 site of the tetramer species can bind Zn2+, Cd2+, or Ca2+ prior to hexamer formation and that such binding assists hexamer formation. Both the tetramer and the hexamer B13 sites were found to exhibit similar affinities for Zn2+ and Cd2+ (Kd congruent to 9 microM), whereas the tetramer B13 sites bind Ca2+ much more weakly (Kd congruent to 1 mM for tetramer vs 83 microM for hexamer). The second-order rate constants estimated for the association of Zn2+ and Cd2+ to the tetrameric site indicate that the loss of the first inner-sphere aqua ligand is the rate-limiting step for binding.     

Phospholipid-binding properties of bovine factor V and factor Va.

Factor V and factor Va binding to single bilayer phospholipid vesicles was investigated by light-scattering intensity measurements. This technique allows the measurement of free and phospholipid-bound protein concentrations from which equilibrium constants can be obtained. As controls, the Ca2+-dependent phospholipid binding of prothrombin and factor X were also studied. The average values obtained for the dissociation constants (Kd) and lipid to protein ratio at saturation, moles/mole (n), for prothrombin (Kd = 2.3 X 10(-6) M, n = 104) and factor X (Kd = 2.5 X 10(-6) M, n = 46) binding to vesicles containing 25% Folch fraction III and 75% phosphatidylcholine in the presence of 2 mM Ca2+ were in agreement with those reported in the literature. The average factor V and factor Va values for the dissociation constants and lipid to protein ratio at saturation (moles/mole) were Kd = 7.2 X 10(-8) M and n = 270 for factor V and Kd = 4.4 X 10(-7) M and n = 76 for factor Va. In contrast to prothrombin and factor X, factor V and factor Va demonstrated Ca2+-independent lipid binding. In addition, the number of factor V and factor Va molecules bound per vesicle was found to be dependent both on the phosphatidylserine content of the vesicle and the ionic strength of the buffer.     

Stimulation and inhibition of [3H]ryanodine binding to sarcoplasmic reticulum from malignant hyperthermia susceptible pigs

When compared to normal pig sarcoplasmic reticulum (SR), SR from malignant hyperthermia susceptible (MHS) porcine skeletal muscle has been shown to exhibit an increased rate of calcium release, as well as alterations in [3H]ryanodine-binding activity in the presence of microM Ca2+ (Mickelson et al., 1988, J. Biol. Chem. 263, 9310). In the present study, various stimulators (adenine nucleotides and caffeine) and inhibitors (ruthenium red and Mg2+) of the SR calcium release channel were examined for effects on MHS and normal SR [3H]ryanodine binding. The apparent affinity of the MHS SR receptor for ryanodine in the presence of 10 mM ATP (Kd = 6.0 nM) or 10 mM caffeine (Kd = 28 nM) was significantly greater than that of the normal SR (Kd = 8.5 and 65 nM in 10 mM ATP or caffeine, respectively), the Bmax (12-16 pmol/mg) was similar in all cases. The Ca2+(0.5) for inhibition of [3H]ryanodine binding in the presence of 5 mM AMPPNP (238 vs 74 microM for MHS and normal SR, respectively) and the Ca2+(0.5) for stimulation of [3H]ryanodine binding in the presence of 5 mM caffeine (0.049 vs 0.070 microM for MHS and normal SR, respectively) were also significantly different. Furthermore, in the presence of optimal Ca2+, MHS SR [3H]ryanodine binding was more sensitive to caffeine stimulation (C0.5 of 1.7 vs 3.4 mM) and was less sensitive to ruthenium red (C0.5 of 1.9 vs 1.2 microM) or Mg2+ inhibition (C0.5 of 0.34 vs 0.21 mM) than was normal SR. These results further support the hypothesis that differences in the ryanodine/receptor calcium release channel regulatory properties are responsible for the abnormal calcium releasing activity of MHS SR.     

Studies of the role of factor Va in the factor Xa-catalyzed activation of prothrombin, fragment 1.2-prethrombin-2, and dansyl-L-glutamyl-glycyl-L-arginine-meizothrombin in the absence of phospholipid

In order to specifically evaluate the role of Factor Va in the prothrombinase complex, studies of the activation of prothrombin, Fragment 1.2-prethrombin-2, and active-site-blocked meizothrombin were carried out, both in the absence of phospholipid and at concentrations of substrates and Factor Va sufficient to approach saturation in all components. Km values were independent of Factor Va concentrations, whereas kcat (apparent) values approached saturation with respect to Factor Va concentrations. The three respective substrates exhibited the following parameters of kinetics (Km, microM; kcat, s-1 at saturating [Factor Va]): prothrombin (9.0 +/- 0.4; 31 +/- 1); Fragment 1.2-prethrombin-2 (5.4 +/- 0.4; 13 +/- 2); and meizothrombin (3.6 +/- 0.3; 51 +/- 5). Models of kinetics were constructed to interpret the results, and two of these were formally consistent with experimental results. Both models indicated that the variation of kcat(app) with concentrations of Factor Va reflects the formation of a Factor Va-Factor Xa binary complex. Analysis of kinetics indicated Kd values for this interaction of 1.3 +/- 0.1, 3.0 +/- 0.5, and 1.0 +/- 0.1 microM for the three respective substrates. The models differed in the interpretation of Km. One indicated that Km reflects a binary interaction between Factor Xa and prothrombin, whereas the other indicated a binary interaction between Factor Va and prothrombin. Both indicated that two of the three possible binary interactions between the three components would be reflected in Km and kcat values but not the third. To distinguish these models, the binary interactions were studied by extrinsic fluorescence (Va.Xa), light-scattering (Factor Va.prothrombin), and competition kinetics (Xa.II). The first two interactions were detected and were characterized by Kd values of 2.7 +/- 0.1 microM (Va.Xa) and 8.8 +/- 0.8 microM (Factor Va.prothrombin). No active-site-dependent interaction between prothrombin and Factor Xa could be detected in the absence of Factor Va. The results of these studies suggest that Factor Va interacts with both Factor Xa and prothrombin and effectively presents one to the other in the formation of a ternary enzyme-substrate-cofactor complex. In addition, a comparison of the parameters of kinetics of conversion of prothrombin and its intermediates indicates that meizothrombin is the major intermediate of prothrombin activation in the absence, as well as in the presence of phospholipid.