A congenitally abnormal fibrinogen (Vlissingen) with a 6-base deletion in the gamma-chain gene, causing defective calcium binding and impaired fibrin polymerization

A congenitally abnormal fibrinogen (Vlissingen) was isolated from the blood of a young woman suffering from massive pulmonary embolism. Fibrinogen Vlissingen showed an abnormal clotting time with both thrombin and Reptilase. The release of the fibrino-peptides A and B by thrombin was normal, but fibrin polymerization was impaired both in the presence and absence of Ca2+ ions. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed according to Laemmli the gamma-chain of fibrinogen Vlissingen showed two bands, one normal and one having an apparently lower molecular mass of about 1,500 daltons. The previously described protective effect of Ca2+ ions on plasmin degradation of the carboxyl terminus of the gamma-chain of normal fibrinogen was only partially detectable in fibrinogen Vlissingen. In addition the binding of Ca2+ ions was decreased. Fibrinogen Vlissingen bound 2.4 Ca2+ ions per fibrinogen molecule at pH 7.4, whereas normal fibrinogen bound 3.1 Ca2+ ions. At pH 5.8 fibrinogen Vlissingen bound 1.1 Ca2+ ions, whereas normal fibrinogen bound 2.0 Ca2+ ions per molecule fibrinogen in the D-domains, again indicating a structural change in the carboxyl terminus of fibrinogen. The structural defect was determined by sequence analysis of DNA amplified by use of the polymerase chain reaction. Exons VIII, IX, and X of the gamma-chain gene were amplified and the DNA sequence of the amplified fragments was determined. A 6-base deletion was found in 50% of the fragments corresponding to exon VIII, indicating that the patient was heterozygous for the mutation. This deletion codes for amino acids Asn-319 and Asp-320 in the normal fibrinogen gamma-chain. The data indicate that Asn-319 and Asp-320 are crucial for maintaining the integrity of the carboxyl-terminal polymerization sites, the protective effect of Ca2+ ions on plasmin degradation of the carboxyl terminus of the gamma-chain, and the calcium binding domain at the carboxyl terminus of fibrinogen.     

The interaction of human platelet thrombospondin with fibrinogen. Thrombospondin purification and specificity of interaction

Human platelet thrombospondin (TSP) was purified to homogeneity by chromatography on fibrinogen coupled to cyanogen bromide-activated Sepharose. The yield of TSP was 1.3 mg or approximately 22% of that present in platelet-rich plasma as determined by radioimmunoassay. It analyzed on discontinuous sodium dodecyl sulfate gels as a single band having apparent molecular weights of 180,000 and greater than 400,000 under reducing and nonreducing conditions, respectively. Amino acid analysis gave results similar to previously published values. Antibodies raised in rabbits were monospecific as evaluated by radioimmunoassay. In double immunodiffusion tests, these antibodies gave one line of identity against TSP purified by this procedure and TSP purified by published procedures, confirming the identity of the material isolated. The protein possesses no lectin-like activity. The specificity of the TSP-fibrinogen interaction was investigated. TSP binding to fibrinogen-Sepharose occurred in the presence of EDTA, indicating that calcium and magnesium ions are not required for interaction of TSP with fibrinogen. The binding of TSP to fibrinogen-Sepharose was quantitatively blocked by pretreatment with an antibody to the cyanogen bromide cleavage fragment composed of residues 241-476 of the carboxyl-terminal end of the alpha chain of fibrinogen. Antibodies against the D and E domains of fibrinogen had no effect on the binding. Excess fibrinogen (30 mg/ml) added to platelet extract quantitatively inhibited binding of TSP to fibrinogen-Sepharose. TSP preferentially bound to uncross-linked fibrin, suggesting that the TSP-fibrinogen binding site is unavailable in cross-linked fibrin. These results indicate that TSP binds specifically to immobilized fibrinogen or uncross-linked fibrin through determinants present in the carboxyl-terminal portion of the alpha chain and that these interactions do not require calcium or magnesium ions.     

Review of some unusual effects of calcium binding to fibrinogen

Calcium binding curves of human and bovine fibrinogen were obtained by using a calcium sensitive electrode. The two were identical and showed 2 high, 2-3 medium and more than 15 low affinity sites. Differential scanning calorimetry at neutral pH demonstrated the presence of the D and E domains of fibrinogen; however, at pH 3.5 the D-domain was split into two. The presence of the subdomains was demonstrated also by digestion by pepsin at this pH. Combination of digestion of fibrinogen and of its fragments with different enzymes and temperatures identified up to 12 subdomains in the original molecule. Clotting of fibrinogen by thrombin at pH 7.0 was investigated also by differential scanning calorimetry. In the absence of Ca2+ clotting elicited a 40% increase in the enthalpy of thermal denaturation of the D domain of fibrinogen, but the position of the peak increased only by 0.4 degrees C. However, with clotting in the presence of 10(-3) M calcium the former increased by 70-75% and the latter by 11.0 degrees C, while these parameters of the E-domain remained unchanged. Changes of bound calcium during clotting were also measured with the calcium sensitive electrode. These had to be corrected, because the drop in free calcium was partly compensated by release of some calcium that was already bound to fibrinogen. Log of the half time of calcium uptake plotted against log thrombin concentration indicated a first order process with respect to thrombin concentration, moreover, the rate determined corresponded to that of the conformation change measured by calorimetry. The calcium uptake was correlated with release of the fibrinopeptides. Release of fibrinopeptide B follows parallel to binding of calcium and that of fibrinopeptide A is about fourfold faster. Polymerization and formation of thick bundles of fibrin is connected with release of fibrinopeptide A. Clotting with Ancrod, an enzyme that releases only fibrinopeptide A, showed only minimal binding of calcium. The polymerization inhibiting tetrapeptide Gly-Pro-Arg-Pro also depressed binding of calcium. These data suggest that a calcium-binding site must be in the proximity of the site of release of fibrinopeptide B and of a polymerization site.     

Studies on alpha-amylase from a thermophilic bacterium. II. Thermal stability of the thermophilic alpha-amylase.

The effect of pH, mental ions, and denaturing reagents on the thermal stability of thermophilic alpha-amylase [EC 3.2.1.1] were examined. The enzyme was most stable at around pH 9.2, which is coincident with the isoelectric point of the enzyme. The stability of the enzyme was increased by the addition of calcium, strontium, and sodium ions. The addition of calcium ions markedly stabilized the enzyme. The protective effects of calcium and sodium ions were additive. At room temperature, no detectable destruction of the helical structure of the enzyme was observed after incubation for 1 hr in the presence of 1% sodium dodecylsulfate, 8 M urea or 6 M guanidine-HC1. The addition of 8 M urea or 6 M guanidine-HC1 lowered the thermal denaturation temperature of the enzyme. The enzyme contained one atom of tightly bound intrinsic calcium per molecule which could not be removed by electrodialysis unless the enzyme was denatured. The rate constants of inactivation and denaturation reactions in the absence and presence of calcium ions were measured and thermodynamic parameters were determined. The presence of calcium ions caused a remarkable decrease in the activation entropy.     

Physicochemical roles of soluble metal cations in the outer membrane of Escherichia coli K-12

Atomic absorption spectroscopy of isolated native and EDTA-modified (lipopolysaccharide-depleted) outer membrane revealed trace amounts of potassium, manganese, and iron (1.0-7.0 nmol/mg dry weight outer membrane). Sodium, magnesium, and calcium were approximately one order of magnitude more plentiful, but EDTA-modified outer membrane was deficient in calcium. When metal-binding assays were conducted to find the binding capacity of native and EDTA-modified outer membrane, potassium bound poorly compared with sodium. However, there was no difference in the binding of these ions between the OM preparations. In contrast, reduced amounts of magnesium, calcium, manganese, and iron III bound to the EDTA-modified OM. Partitioning of intact cells in a biphasic dextran-polyethyleneglycol system indicated that the reduced lipopolysaccharide content of the EDTA-modified outer membrane increased the hydrophobicity of the cell surface. Exposure of control and EDTA-treated cells to divalent metal salt solutions before phase partitioning also increased cell surface hydrophobicity. Freeze-etching showed that sodium ions had no effect on the membrane fractures observed in control cells, but with EDTA-treated cells, this cation increased the occurrence of small outer membrane fractures (plateaus) which are characteristic of EDTA treatment. Both magnesium and manganese increased the frequency of outer membrane cleavage in control cells, whereas calcium did not. In contrast, all three divalent metallic ions increased the frequency and extent of cleavage in the outer membrane of EDTA-treated cells.     

Effect of calcium and magnesium ions on different stages of genetic transformation in Bacillus subtilis]

Optimal concentrations of magnesium and calcium ions under their combined effect on genetic transformation in Bacillus suttilis are 2 - 10(-2) M and 4 - 10(-2) M respectively. The same concentrations are optimal under the effect of each cation clone. Magnesium ions are efficient during irreversible DNA binding. In the presence of magnesium ions calcium ions stimulate more late stages of transformation. The greatest efficiency of transformation is shown in consecutive effect of magnesium ions at early stages of transformation and of calcium ions at late transformation stages. This suggests that magnesium and calcium ions stimulate the activity of nuclease, taking part at early and late transformation stages.     

Time-resolved fluorescence study of VU-9 calmodulin, an engineered calmodulin possessing a single tryptophan residue

An engineered calmodulin (VU-9 calmodulin), which possesses a single tryptophan residue at position 99 in calcium binding domain III, was studied by time-resolved fluorescence. At least two exponential terms are needed to describe the tryptophan fluorescence decays, either in the presence or in the absence of calcium. The characteristics of the fluorescence decays are strongly dependent upon the number of calcium ions bound per molecule of VU-9 calmodulin until half of the calcium sites are occupied, i.e., three in the absence of magnesium and two in the presence of 5 mM magnesium. A clear time-dependent spectral shift is observed in the presence of calcium. The existence of an isosbestic point in the time-resolved spectra is in agreement with a two-state model. The biexponential analysis of the 340-nm fluorescence decay during calcium titration gives parameters consistent with a two-state model in which tryptophan 99 interconverts between two different conformations, characterized by a different lifetime value, with rates altered by calcium binding. This model explains the decrease in the protein quantum yield induced by calcium binding [Kilhoffer, M. C., Roberts, D. M. Adibi, A. O., Watterson, D. M., & Haiech, J. (1989) Biochemistry (preceding paper in this issue)].     

Metal ion binding in the active site of the junction-resolving enzyme T7 endonuclease I in the presence and in the absence of DNA

Endonuclease I of bacteriophage T7 is a DNA junction-resolving enzyme. We have previously used crystallography to demonstrate the binding of two manganese ions into the active site that is formed by three carboxylate (Glu 20, Asp 55 and Glu 65) and a lysine residue (Lys 67). Endonuclease I is active in the presence of magnesium, manganese, iron (II) and cobalt (II) ions, weakly active in the presence of nickel, copper (II) and zinc ions, and completely inactive in the presence of calcium ions. However, using calorimetry, we have observed the binding of two calcium ions to the free enzyme in a manner very similar to the binding of manganese ions. In the presence of iron (II) ions, we have obtained a cleavage of the continuous strands of a junction bound by endonuclease I, at sites close to (but not identical with) enzyme-induced hydrolysis. The results suggest that this arises from attack by locally generated hydroxyl radicals, arising from iron (II) ions bound into the active site. This therefore provides an indirect way of examining metal ion binding in the enzyme-junction complex. Ion binding in free protein (by calorimetry) and the enzyme-junction complex (iron-induced cleavage) have been studied in series of active-site mutants. Both confirm the importance of the three carboxylate ligands, and the lack of a requirement for Lys67 for the ion binding. Calorimetry points to particularly critical role of Asp55, as mutation completely abolishes all binding of both manganese and calcium ions.     

Clotting of fibrinogen. 1. Scanning calorimetric study of the effect of calcium

The denaturation temperature Td and the enthalpy of thermal denaturation delta Hd of the D nodules of fibrinogen increase 12-13 degrees C and 40%, respectively, when fibrinogen is clotted by thrombin in the presence of 10(-3) M calcium ion. The rate of change of Td and delta Hd is first order in thrombin concentration. In the absence of calcium, little change in Td is observed, but the increase in delta Hd still occurs. The shift in Td as a function of logarithm of calcium concentration is sigmoid, with a half-point at 2.5 X 10(-5) M calcium for human and 6.0 X 10(-5) M calcium for bovine fibrinogens, suggesting that the shift is due to binding of calcium at the high-affinity binding sites of fibrin. The Td of the D nodule of native fibrinogen also increases, but not as much, on addition of calcium. This increase in Td is also sigmoid with log calcium, with a half-point of 1.6 X 10(-3) M calcium for human and 3.2 X 10(-3) M calcium for bovine fibrinogens, and appears to be due to binding of calcium to the low-affinity binding sites of fibrinogen. At calcium concentrations greater than 10(-4) M, traces of factor XIII in the bovine fibrinogen preparation become activated and cause cross-linking of the fibrin gel. But the changes in Td and delta Hd still occur when factor XIIIa is inactivated by iodoacetamide, and the rate of the changes is not altered by addition of large amounts of factor XIIIa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of the fibrinogen receptor on human platelets by epinephrine and the combination of epinephrine and ADP

The capacity of epinephrine alone and the combination of low dose epinephrine and ADP to support the binding of fibrinogen to washed human platelets has been examined, 125I-Fibrinogen was bound to epinephrine-stimulated platelets, but 90 min were required to achieve maximal binding at 22 degrees C in contrast to 20 to 30 min with ADP. The overall rate of interaction appeared to reflect the slow binding of fibrinogen to epinephrine-stimulated platelets as opposed to the rate of stimulation of the cell. Divalent ions were required for binding of fibrinogen to epinephrine-stimulated platelets, and both calcium and magnesium supported binding with a prolonged time course. Fibrinogen binding was maximally supported by 20 to 30 microM epinephrine. The combination of low dose epinephrine (5 microM) and low dose ADP (0.5 microM), which acted synergistically to induce platelet aggregation, supported the rapid (10 min) binding of fibrinogen to platelets. With 4 microM epinephrine, more fibrinogen bound per platelet at all ADP doses than with ADP alone. With all the stimuli, saturable binding of fibrinogen to the platelet was observed, and Scatchard plots were linear, yielding very similar apparent association constants. The number of molecules bound per cell was stimulus-dependent, with 30 microM epinephrine inducing the binding of fewer fibrinogen molecules per cell (mean = 20,400) than 10 microM ADP (mean = 35,900) or the combination of 5 microM epinephrine + 0.5 microM ADP (mean = 43,600). The participation of endogenous ADP in fibrinogen binding to epinephrine-stimulated platelets was suggested since enzymes which remove ADP, apyrase, and creatine phosphate/creatine phosphokinase, and the ADP analogue, 2-chloroadenosine, completely inhibited the binding of fibrinogen to the platelet.     

Dimethyl sulfoxide induces heme oxygenase-1 expression via JNKs and Nrf2 pathways in human umbilical vein endothelial cells

Prothrombin interacts with phosphatidylserine containing platelet membranes via its N-terminal, γ-carboxyglutamate (gla) residue-rich domain. Once bound it is cleaved to form the active protease, thrombin (factor IIa). Human prothrombin was cleaved with cathepsin G in the absence of calcium and magnesium ions. Under these conditions, the gla domain was removed. Phospholipid protected the protein from this proteolytic event, and this suggests that a conformational change may be induced by interaction with phospholipids. Binding of prothrombin to a surface containing 20% phosphatidylserine/80% phosphatidylcholine was detected by surface plasmon resonance, whereas no interaction with gla-domainless prothrombin was observed. Binding of intact prothrombin in the presence of calcium ions showed complex association kinetics, suggesting multiple modes of initial interaction with the surface. The kinetics of the dissociation phase could be fitted to a two-phase, exponential decay. This implies that there are at least two forms of the protein on the surface one of which dissociates tenfold more slowly than the other. Taken together, these data suggest that, on binding to a membrane surface, prothrombin undergoes a conformational change to a form which binds more tightly to the membrane.     

Equilibria in the fibrinogen-fibrin conversion. IX. Effects of calcium ions on the reversible polymerization of fibrin monomer

An investigation was made of the role of calcium ions in the reversible stage of fibrin polymerization, using a direct and relatively simple approach. Purified fibrin monomer in solution (7.5 mg/ml) in 1.0 m NaBr (pH 5.3) was polymerized by raising the pH to 5.7–7.7 by the addition of aliquots of standard NaOH solution and the rate and total extent of proton release during polymerization were measured potentiometrically. In the presence of added CaCl₂ (10⁻⁵-10⁻²m) the rate of proton release was increased and the clotting time was decreased. The profile of equilibrium proton release vs pH of polymerization was also shifted, the maximum being increased and occurring at a lower pH. Sedimentation velocity studies in the intermediate pH range (5.7–6.0) showed that the altered profile of equilibrium proton release was due to a broadening of the pH range of polymerization, and that polymerization remained reversible in the presence of CaCl₂. At pH 5.3, where fibrin is essentially monomeric, addition of CaCl₂ resulted in the release of protons and small increases in sedimentation coefficient and reduced viscosity. Under the same conditions, a similar release of protons was observed from fibrinogen, but there was no effect on its sedimentation coefficient. It was concluded that the proton release at pH 5.3 was due mainly to binding of calcium ions to fibrinogen and fibrin monomer. The effect of CaCl₂ on the sedimentation coefficient of fibrin at pH 5.3 was found to decrease with decreasing protein concentration, indicating that it was the result of a small extent of polymerization, rather than a conformational change. Added MgCl₂ had no effect on fibrin monomer at pH 5.3 and no significant effect on the rate or extent of proton release during polymerization at higher pH, indicating that there are specific binding sites for calcium ions in fibrinogen and fibrin. The observed effects of bound calcium ions on reversible fibrin polymerization are explained most simply in electrostatic terms.     

Influence of synthetic peptide inhibitors on the thermal stability of thermitase, a serine proteinase from Thermoactinomyces vulgaris

The influence of chloromethyl ketones and methyl ketones of N-acylated peptides on the thermal denaturation of thermitase was investigated in the presence and the absence of calcium ions. The chloromethyl ketone derivatives are known to react irreversibly with the enzyme, whereas the corresponding methyl ketones are reversible inhibitors. Both groups of inhibitors offer a broad variety of affinity constants. The irreversible inhibition of thermitase causes a marked stabilization against thermal denaturation. On the other hand, the enzyme stability is not influenced by the binding of reversible inhibitors. The stabilizing effect of calcium ions is not dependent on the inhibitor binding. The importance of bivalent interaction (bridge formation) in the active site region of the enzyme for its thermal stability is discussed.     

Consequences of terbium (111) binding on the conformation and enzymatic activity of Guinea pig liver transglutaminase

Calcium ions are crucial for expression of transglutaminase activity. Although lanthanides have been reported to substitute for calcium in a variety of protein functions, they did not replace the calcium requirement during transglutaminase activity measurements. Furthermore, lanthanides strongly inhibited purified liver transglutaminase activity using either casein or fibrinogen as substrates. Terbium (III) inhibition of transglutaminase-catalyzed putrescine incorporation into casein was not reversed by the presence of 10–200 fold molar excess of calcium ions (Ki for Tb(III)=60 µM). Conformational changes in purified liver transglutaminase upon Tb(III) binding were evident from a biphasic effect of Tb(III) on transglutaminase binding to fibrin. Low concentrations of Tb(III) (1 µM to 10 µM inhibited the binding of transglutaminase to fibrin, whereas higher concentrations (20 µM to 100 µM promoted binding. Conformational changes in purified liver transglutaminase consequent to Tb(III) binding were also demonstrated by fluorescence spectroscopy due to Forster energy transfer. Fluorescence emission was stable to the presence of 200 mM NaCl and 100 mM CaCl₂ only partially quenched emission. Purified liver transglutaminase strongly bound to Tb(III)-Chelating Sepharose beads and binding could not be disrupted by 100 mM CaCl₂ solution. Our data suggest that Tb(III)-induced conformational changes in transglutaminase are responsible for the observed effects on enzyme structure and function. The potential applications of Tb(III)-transglutaminase interactions in elucidating the structure-function relationships of liver transglutaminase are discussed.     

pH and magnesium alter 45calcium binding to platelets at sites other than glycoproteins I or IIb/IIIa

Calcium is a cofactor of human platelet aggregation. Moreover a direct correlation between the ability of platelets to bind this divalent cation and to aggregate has been demonstrated. Since magnesium can substitute for calcium in supporting aggregation, especially in the presence of low calcium concentrations, and platelet aggregation is inhibited at low pH, the present study was designed to examine the effects of magnesium and low pH on 45calcium binding to human platelets, and to determine whether such effects might be associated with calcium binding to glycoproteins I (GPI) or IIb/IIIa (GPIIb/IIIa), the putative fibrinogen receptor. 45Calcium binding to aspirin-treated platelets that had been depleted of surface-associated calcium by brief exposure to EDTA was evaluated. Magnesium (5-10 mM) or a change in hydrogen ion concentration to decrease the pH from 7.5 to 6.0 was found to inhibit the binding of 45calcium to platelets from healthy donors by 34 +/- 6 and 32 +/- 8% (mean +/- SD, n = 13), respectively. Similar results were obtained with platelets incubated with chymotrypsin to selectively remove GPI or platelets from a patient with the Bernard Soulier Syndrome, congenitally deficient in GPI. In contrast, calcium binding to platelets from two patients with thrombasthenia, lacking GPIIb/IIIa, was reduced 49 +/- 6% and 42 +/- 8% (n = 4) by magnesium and hydrogen ions, respectively. This apparently increased inhibition was attributed to the combined effects of an overall decrease (approximately 50%) in calcium binding to thrombasthenic platelets compared with that in control platelets, and a similar absolute reduction in calcium binding in the presence of magnesium and/or hydrogen ions. No additional inhibition of 45calcium binding was noted in the presence of magnesium and at low pH, indicating that magnesium and hydrogen ions may affect the same platelet membrane binding sites. The data suggest that although modulation of platelet aggregation by magnesium and pH is accompanied by changes in platelet-associated calcium, calcium binding to the three major platelet membrane glycoproteins, GPI, IIb, and IIIa is unaffected.     

The binding sites for tRNA on eukaryotic ribosomes.

We have studied the non-enzymic binding of phe-tRNA to ribosomes from rat liver using deacylated tRNA to inhibit binding to the P-site and puromycin (5 x 10-minus3M) to inhibit binding to the A-site. We conclude that at a low concentration of magnesium ions (10mM) phe-tRNA is bound only at the A-site of 80S irbosomes, whereas at a high concentration of magnesium ions (40mM) phe-tRNA is also bound at the P-site. Studies with edeine indicate that, during non-enzymic binding of phe-tRNA, eukaryotic ribosomes (in contrast to prokarotic ribosomes) have the A-site of the 60S subunit and the initiation site of the 40S subunit juxtaposed. This may account for the differences observed, in formation of diphenylalanyl-tRNA and phenylalanyl-puromycin, between phe-tRNA bound non-enzymically to the P-sites of eukaryotic and prokaryotic ribosomes.     

Circular dichroism (CD) studies on yeast enolase: activation by divalent cations

The effect of divalent cations on the near ultraviolet circular dichroism (CD) spectrum of yeast enolase showed that calcium, magnesium, and nickel ions produced identical changes. This was interpreted as indicating that the cations bound to the same sites on the enzyme and produced identical changes in tertiary structure. There was no effect of magnesium ion on the far ultraviolet spectrum. Evidently magnesium ion has no effect on the secondary structure. Substrate bound to the enzyme when the above cations were present although calcium permits no enzymatic activity. The CD spectral difference produced by the substrate was nearly the reverse of that produced by the metal ions. Glycolic acid phosphate, a competitive inhibitor lacking carbon-3, produced no effect, indicating carbon-3 was necessary for the CD spectral changes. The CD and visible absorption spectra of nickel and cobalt bound to various sites on the enzyme showed that the binding sites were octahedral or distorted octahedral in coordination and that the ligands appeared to be oxyligands: water molecules, hydroxyl or carboxyl groups. Examination of the effects of substrate and two compounds thought to be "transition state analogues" showed that these perturbed the "conformational" sites of the enzyme. The "catalytic" and "inhibitory" sites did not appear to be very CD active.     

Evidence of a calcium-induced structural change in the ATP-binding site of the sarcoplasmic-reticulum Ca2+-ATPase using terbium formycin triphosphate as an analogue of Mg-ATP

Terbium ions and terbium formycin triphosphate have been used to investigate the interactions between the cation and nucleotide binding sites of the sarcoplasmic reticulum Ca2+-ATPase. Three classes of Tb3+-binding sites have been found: a first class of low-affinity (Kd = 10 microM) corresponds to magnesium binding sites, located near a tryptophan residue of the protein; a second class of much higher affinity (less than 0.1 microM) corresponds to the calcium transport sites, their occupancy by terbium induces the E1 to E2 conformational change of the Ca2+-ATPase; a third class of sites is revealed by following the fluorescence transfer from formycin triphosphate (FTP) to terbium, evidencing that terbium ions can also bind into the nucleotide binding site at the same time as FTP. Substitution of H2O by D2O shows that Tb-FTP binding to the enzyme nucleotide site is associated with an important dehydration of the terbium ions associated with FTP. Two terbium ions, at least, bind to the Ca2+-ATPase in the close vicinity of FTP when this nucleotide is bound to the ATPase nucleotide site. Addition of calcium quenches the fluorescence signal of the terbium-FTP complex bound to the enzyme. Calcium concentration dependence shows that this effect is associated with the replacement of terbium by calcium in the transport sites, inducing the E2----E1 transconformation when calcium is bound. One interpretation of this fluorescence quenching is that the E1----E2 transition induces an important structural change in the nucleotide site. Another interpretation is that the high-affinity calcium sites are located very close to the Tb-FTP complex bound to the nucleotide site.     

Factor XIII binds to the A alpha- and B beta- chains in the D-domain of fibrinogen: an immunoblotting study

The binding sites in fibrinogen for Factor XIII were localized using an immunoblotting technique. Platelet Factor XIII bound to fibrinogen and to plasmin degradation products of fibrin(ogen) including Fragments: X, D1-D3, and D-dimer, but did not bind to Fragment E. Binding of Platelet Factor XIII was independent of calcium ions but could be inhibited by the presence of 0.5 M NaCl. Binding could also be inhibited by preincubating Factor XIII with a 100-fold molar excess of fibrinogen but not by 100-fold molar excess of Fragment E. Binding of Factor XIII to fibrinogen was specific, since several other proteins tested (ovalbumin, bovine serum albumin, alpha 2-macroglobulin, beta-galactosidase, fructose kinase, lactic dehydrogenase, triose phosphate isomerase, fumarase and pyruvate kinase) did not bind Factor XIII. Furthermore, binding was not observed either when Factor XIII was left out or when antiFactor XIII antiserum was substituted with nonimmune serum. When fibrinogen was reduced prior to electrophoresis, Factor XIII bound to the A alpha and B beta chains of fibrinogen and des A,B fibrinogen, the B beta-chain of Fragment X, but not the gamma-chains. Localization of the Factor XIII binding sites to the carboxy terminal segments of the A alpha and B beta chains in the Fragment D-domain of fibrinogen could have important physiological consequences.     

The binding of calcium to human fibronectin

The binding of calcium to human plasma fibronectin has been measured by equilibrium dialysis at 25° 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 ? 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.