Microcalorimetric investigation of the interaction of calmodulin with seminalplasmin and myosin light chain kinase

Flow microcalorimetric titrations of calmodulin with seminalplasmin at 25 degrees C revealed that the high affinity one-to-one complex in the presence of Ca2+ (Comte, M., Malnoe, A., and Cox, J. A. (1986) Biochem. J. 240, 567-573) is entirely enthalpy-driven (delta H0 = -50 kJ.mol-1; delta S0 = O J.K-1.mol-1; delta Cp0 = O J.K-1.mol-1) and is not influenced by the proton or Mg2+ concentration. The Sr2+- and Cd2+-promoted high affinity complexes are also exothermic for -49 and -45 kJ.mol-1, respectively. The observed low affinity interaction in the absence of divalent ions displays no enthalpy change. No enthalpy changes are observed when calmodulin and seminalplasmin are mixed in the presence of millimolar concentrations of Mg2+, Zn2+, or Mn2+. Enthalpy titrations of the 1:1 calmodulin-seminalplasmin complex with Ca2+ and of partly Ca2+-saturated calmodulin with seminalplasmin revealed that only the species calmodulin.Can greater than or equal to 2 is fully competent for high affinity interaction with seminalplasmin. Binding of the second Ca2+ is strongly enhanced (K2 greater than or equal to 5 X 10(7) M-1) as compared to that in free calmodulin (K2 = 2.6 X 10(5) M-1). This is essentially due to the concomitant strongly exothermic step of isomerization of the calmodulin-seminalplasmin complex from its low to its high affinity form. Binding of the remaining two Ca2+ to the high affinity seminalplasmin-calmodulin complex displays the same affinity constants and endothermic enthalpy change as in free calmodulin. A microcalorimetric study on the complex formation between Ca2+-saturated calmodulin and turkey gizzard myosin light chain kinase revealed that the interaction is strongly exothermic with an important overall gain of order (delta H0 = -85 kJ.mol-1; delta S0 = -122 J.K-1.mol-1) and occurs with significant proton uptake (0.44 H+ per mol at pH 7.5). The observed low affinity interaction (K = 2.2 X 10(5) M-1) in the absence of Ca2+ (Mamar-Bachi, A., and Cox, J. A. (1987) Cell Calcium 8, 473-482) displays neither a change in enthalpy nor in protonation.     

Mode of activation of bovine brain inositol 1,4,5-trisphosphate 3-kinase by calmodulin and calcium.

The effect of Ca2+ and calmodulin (CaM) on the activation of purified bovine brain Ins(1,4,5)P3 kinase was quantified and interpreted according to the model of sequential equilibria generally used for other calmodulin-stimulated systems. Two main conclusions can be drawn. (i) CaM.Ca3 and CaM.Ca4 together are the biologically active species in vitro, as is the case for the great majority of other calmodulin targets. (ii) These species bind in a non-co-operative way to the enzyme with an affinity constant of 8.23 x 10(9) M-1, i.e. approx 10-fold higher than for most calmodulin-activated target enzymes. The dose-response curve of the activation of Ins(1,4,5)P3 kinase by calmodulin is not significantly impaired by melittin and trifluoperazine, whereas under very similar assay conditions the half-maximal activation of bovine brain cyclic AMP phosphodiesterase requires over 30-50-fold higher concentrations of CaM when 1 microM melittin or 20 microM-trifluoperazine is present in the assay medium. Similarly, 1 microM of the anti-calmodulin peptides seminalplasmin and gramicidin S, as well as 20 microM of N-(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide (W7), do not inhibit the activation process. These data suggest that binding and activation of Ins(1,4,5)P3 kinase require surface sites of calmodulin which are different from those involved in the binding of most other target enzymes or of model peptides.     

Microcalorimetric investigation of the interactions in the ternary complex calmodulin-calcium-melittin

Flow microcalorimetric titrations of calmodulin with melittin at 25 degrees C revealed that the formation of the high-affinity one-to-one complex in the presence of Ca2+ (Comte, M., Maulet, Y., and Cox, J. A. (1983) Biochem, J. 209, 269-272) is entirely entropy driven (delta H0 = 30.3 kJ X mol-1; delta S0 = 275 J X K-1 X mol-1). Neither the proton nor the Mg2+ concentrations have any significant effect on the strength of the complex. In the absence of Ca2+, a nonspecific calmodulin-(melittin)n complex is formed; the latter is predominantly entropy driven, accompanied by a significant uptake of protons and fully antagonized by Mg2+. Enthalpy titrations of metal-free calmodulin with Ca2+ in the presence of an equimolar amount of melittin were carried out at pH 7.0 in two buffers of different protonation enthalpy. The enthalpy and proton release profiles indicate that: protons, absorbed by the nonspecific calmodulin-melittin complex, are released upon binding of the first Ca2+; Ca2+ binding to the high-affinity configuration of the calmodulin-melittin complex displays an affinity constant greater than or equal to 10(7) M-1, i.e. 2 orders of magnitude higher than that of free calmodulin; the latter is even more entropy driven (delta H0 = 7.2 kJ X site-1; delta S0 = 158 J X K-1 X site-1) than binding to free calmodulin (delta H0 = 4.7 kJ X site-1; delta S0 = 112 J X K-1 X site-1), thus underlining the importance of hydrophobic forces in the free energy coupling involved in the ternary complex.     

Affinity purification of seminalplasmin and characterization of its interaction with calmodulin.

Bull seminalplasmin antagonizes with high potency and selectivity the activating effect of calmodulin on target enzymes [Gietzen & Galla (1985) Biochem. J. 230, 277-280]. In the present paper we establish that seminalplasmin forms a 1:1, Ca2+-dependent and urea-resistant complex with calmodulin. The dissociation constant equals 1.6 nM. In the absence of Ca2+ a low-affinity complex is formed that is disrupted by 4 M-urea. On the basis of these properties, a fast affinity purification of seminalplasmin was developed. The high specificity of seminalplasmin as a calmodulin antagonist was demonstrated for the multipathway-regulated adenylate cyclase of bovine cerebellum. Far-u.v. c.d. properties are consistent with a random form of seminalplasmin in aqueous solution; 23% alpha-helix is induced on interaction with calmodulin. The fluorescence properties of the single tryptophan residue of seminalplasmin are markedly changed on formation of the complex. These studies allowed us to locate tentatively the peptide segment that interacts with calmodulin, and to ascertain the structural homology between seminalplasmin and other calmodulin-binding peptides. Additional material, showing the inhibition of calmodulin-mediated activation of bovine brain phosphodiesterase by melittin and seminalplasmin and also the near-u.v. spectrum of affinity-purified seminalplasmin, has been deposited as supplement SUP 50135 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1986) 233, 5.     

Trifluoperazine binding to porcine brain calmodulin and skeletal muscle troponin C

Binding of trifluoperazine (TFP), a phenothiazine tranquilizer, to porcine brain calmodulin (CaM) and rabbit skeletal muscle troponin C (Tn C) was measured by an automated high-performance liquid chromatography binding assay using a molecular sieving column; 10 micrograms of either protein per injection is sufficient for determining TFP binding, and results are comparable to those obtained by equilibrium dialysis. Very little binding was observed to either protein in the absence of Ca2+ while in the presence of Ca2+ both proteins bind 4 equiv of TFP. Other characteristics of TFP binding however are different for each protein. For CaM, half-maximal binding occurs at 5.8 microM TFP, the Hill coefficient is 0.82, and the fit of the data to the Scatchard equation is consistent with four independent TFP-binding sites. Binding of one melittin displaces two TFP from CaM. Thus, there are two recognizable classes of TFP-binding sites: those that are displaced by melittin and those that are not. TFP causes an increase in the Ca2+ affinity of CaM, and three Ca2+ must be bound to CaM for TFP binding to occur. The studies also yielded a measure of the intrinsic affinity of three of CaM's Ca2(+)-binding sites that is in agreement with previous reports. For troponin C, half-maximal binding occurs at 16 microM TFP, the Hill coefficient is 1.7, and the data best fit the Adair equation for four binding sites. The measured constants K1, K2, K3, and K4 were 2.5 X 10(4), 6.6 X 10(3), 5.8 X 10(5), and 2.0 X 10(5) M-1, respectively, in 1 mM Ca2+ and were similar when Mg2+ was additionally included. TFP also increases troponin C's Ca2+ affinity, and it is the low-affinity, Ca2(+)-specific binding sites that are affected. These studies yielded a measure of the intrinsic affinity of these Ca2(+)-binding sites that is in agreement with previous measurements.     

Biological activities of bovine cardiac-muscle troponin C C-terminal peptide (residues 84-161).

1. Bovine cardiac-muscle troponin C was digested at cysteine residues 35 and 84, and the C-terminal peptide (residues 84-161) was isolated. 2. The C-terminal peptide contains two Ca2+-binding sites. These sites bind Ca2+ with a binding constant of 2.0 X 10(8) M-1. In the presence of 2 mM-Mg2+ the binding constant for Ca2+ is decreased to 3.7 X 10(7) M-1. The corresponding constants for native troponin C are 5.9 X 10(7) M-1. and 2.9 X 10(7) M-1 respectively. 3. Electrophoretic mobility of the C-terminal peptide is increased in the presence of 0.1 mM-CaCl2 as compared with the mobility in the presence of 2mM-EDTA. The same phenomenon was observed when electrophoresis was performed in the presence of 6 M-urea or 0.1% sodium dodecyl sulphate. 4. When saturated with Ca2+, the C-terminal peptide forms complexes with bovine cardiac-muscle troponin I both in the absence and in the presence of 6 M-urea. This complex is dissociated on removal of Ca2+. 5. The data suggest that the C-terminal peptide of troponin C contains two Ca2+/Mg2+-binding sites and interacts with troponin I. Thus, despite the 30% difference in amino acid composition, the properties of bovine cardiac-muscle troponin C C-terminal peptide are similar to those of rabbit skeletal-muscle troponin C C-terminal peptide.     

The mechanism of Ca2+ regulation of vascular smooth muscle thin filaments by caldesmon and calmodulin

The interactions of vascular smooth muscle caldesmon with actin, tropomyosin, and calmodulin were determined under conditions in which the four proteins can form reconstituted Ca2+-sensitive smooth muscle thin filaments. Caldesmon bound to actin in a complex fashion with high affinity sites (K = 10(7) M-1) saturating at a stoichiometry of 1 per 28 actins, and lower affinity sites at 1 per 7 actins. The affinity of binding was increased in the presence of tropomyosin, and this could be attributed to a direct interaction between caldesmon and tropomyosin which was demonstrated using caldesmon cross-linked to Sepharose. In the presence of tropomyosin, occupancy of the high affinity sites was associated with inhibition of actin-activated myosin MgATPase activity. Caldesmon was found to bind to calmodulin in the presence of Ca2+, with an affinity of 10(6) M-1. The binding of Ca2+ X calmodulin to caldesmon was associated with the neutralization of inhibition of actin-tropomyosin. Ca2+ X calmodulin binding reduced but did not abolish the binding of caldesmon to actin-tropomyosin. From this data we have proposed a model for smooth muscle thin filaments in which Ca2+ regulates activity by converting the inhibited actin-tropomyosin-caldesmon complex to the active complexes, actin-tropomyosin-caldesmon-calmodulin X Ca2+ and actin-tropomyosin.     

Sodium binding sites of gramicidin A: sodium-23 nuclear magnetic resonance study.

In ethanol-water mixtures (90:10), the gramicidin dimer binds Na+ cations at well-defined sites, with a binding constant K = 4 M-1. Partial desolvation of Na+ occurs upon binding, as judged from the magnitude of the quadrupolar coupling constant (1.7 MHz) for bound sodium. The binding sites are identified with the outer sites flanking the channel entrances. The rate constants for binding and release are k+ less than or equal to 2.2 X 10(9) M-1 s-1 and k- less than or equal to 5.5 X 10(8) s-1, respectively.     

The binding of 6-demethylchlortetracycline to 70S, 50S and 30S ribosomal particles: a quantitative study by fluorescence anisotropy

The binding of demeclocycline (6-demethylchlortetracycline) to ribosomes and ribosomal subunits from Escherichia coli was investigated by using the fluorescence anisotropy of the antibiotic to determine the extent of binding. Binding data obtained from 70S and 30S particles differed fundamentally from those obtained from 50S subunits: the first two showed a strong, specific interaction while the third did not. In addition, all three particles possessed weak, unspecific binding sites. Computer-aided least-squares analysis of the data yielded the following numbers of sites and equilibrium constants: for 30S, n1 = 1, K1 = 2.2 X 10(6) M-1, n2 K2 = 0.029 X 10(6) M-1; for 50S, n1 = 0, n2 K2 = 0.035 X 10(6) M-1; for 70S, n1 = 1, K1 = 3.2 X 10(6) M-1, n2 K2 = 0.082 X 10(6) M-1. These data resolve current disagreement in the literature and are a prerequisite for quantitative studies of the mechanism of inhibition by tetracycline of protein biosynthesis.     

Purification, characterization and ion binding properties of human brain S100b protein

Human brain S100b (beta beta) protein was purified using zinc-dependent affinity chromatography on phenyl-Sepharose. The calcium- and zinc-binding properties of the protein were studied by flow dialysis technique and the protein conformation both in the metal-free form and in the presence of Ca2+ or Zn2+ was investigated with ultraviolet spectroscopy, sulfhydryl reactivity and interaction with a hydrophobic fluorescence probe 6-(p-toluidino)naphthalene-2-sulfonic acid (TNS). Flow dialysis measurements of Ca2+ binding to human brain S100b (beta beta) protein revealed six Ca2+-binding sites which we assumed to represent three for each beta monomer, characterized by the macroscopic association constants K1 = 0.44 X 10(5) M-1; K2 = 0.1 X 10(5) M-1 and K3 = 0.08 X 10(5) M-1. In the presence of 120 mM KCl, the affinity of the protein for calcium is drastically reduced. Zinc-binding studies on human S100b protein showed that the protein bound two zinc ions per beta monomer, with macroscopic constants K1 = 4.47 X 10(7) M-1 and K2 = 0.1 X 10(7) M-1. Most of the Zn2+-induced conformational changes occurred after the binding of two zinc ions per mole of S100b protein. These results differ significantly from those for bovine protein and cast doubt on the conservation of the S100 structure during evolution. When calcium binding was studied in the presence of zinc, we noted an increase in the affinity of the protein for calcium, K1 = 4.4 X 10(5) M-1; K2 = 0.57 X 10(5) M-1; K3 = 0.023 X 10(5) M-1. These results indicated that the Ca2+- and Zn2+-binding sites on S100b protein are different and suggest that Zn2+ may regulate Ca2+ binding by increasing the affinity of the protein for calcium.     

Ca2+-calmodulin binding to caldesmon and the caldesmon-actin-tropomyosin complex. Its role in Ca2+ regulation of the activity of synthetic smooth-muscle thin filaments.

We measured the concentration of calmodulin required to reverse inhibition by caldesmon of actin-activated myosin MgATPase activity, in a model smooth-muscle thin-filament system, reconstituted in vitro from purified vascular smooth-muscle actin, tropomyosin and caldesmon. At 37 degrees C in buffer containing 120 mM-KCl, 4 microM-Ca2+-calmodulin produced a half-maximal reversal of caldesmon inhibition, but more than 300 microM-Ca2+-calmodulin was necessary at 25 degrees C in buffer containing 60 mM-KCl. The binding affinity (K) of caldesmon for Ca2+-calmodulin was measured by a fluorescence-polarization method: K = 2.7 x 10(6) M-1 at 25 degrees C (60 mM-KCl); K = 1.4 x 10(6) M-1 at 37 degrees C in 70 mM-KCl-containing buffer; K = 0.35 x 10(6) M-1 at 37 degrees C in 120 mM-KCl- containing buffer (pH 7.0). At 37 degrees C/120 mM-KCl, but not at 25 degrees C/60 mM-KCl, Ca2+-calmodulin bound to caldesmon bound to actin-tropomyosin (K = 2.9 x 10(6) M-1). Ca2+ regulation in this system does not depend on a simple competition between Ca2+-calmodulin and actin for binding to caldesmon. Under conditions (37 degrees C/120 mM-KCl) where physiologically realistic concentrations of calmodulin can Ca2+-regulate synthetic thin filaments, Ca2+-calmodulin reverses caldesmon inhibition of actomyosin ATPase by forming a non-inhibited complex of Ca2+-calmodulin-caldesmon-(actin-tropomyosin).     

Comparison of S100b protein with calmodulin: interactions with melittin and microtubule-associated tau proteins and inhibition of phosphorylation of tau proteins by protein kinase C

To gauge similarities between S100b protein and calmodulin, interactions were observed between S100b and melittin and between S100b and tau, the microtubule-associated proteins. The interaction of melittin with S100b protein in the presence and absence of calcium was studied by fluorescence polarization, UV difference spectroscopy, and sulfhydryl derivatization. Whether calcium was present or not in the solution, melittin and S100b form a complex of molar ratios up to 2:1. Further binding of melittin occurred, but it resulted in precipitation of S100b, as is true of the corresponding case of melittin binding to calmodulin. In the absence of calcium, the interaction of melittin and S100b shielded the tryptophan (Trp) of the former protein and exposed cysteine-84 beta (Cys-84 beta) of the latter protein, leaving the tyrosine-16 beta (Tyr-16 beta) of S100b unaffected. Calcium addition to the complex partially restored the exposure of Trp of melittin and caused changes in the environment of Tyr-16 beta (unlike the environmental changes induced for Tyr-16 beta by calcium in the absence of melittin). The conformational changes induced in S100b by interaction with melittin increased its affinity for calcium and offset the inhibition of calcium binding otherwise observed in the presence of potassium ions. This corroborated the previous finding that S100b affinity for calcium greatly depends on the protein conformation. The phenomena described above are similar to the interactions of melittin with calmodulin and thus suggest that S100b and calmodulin have a common structural domain not only that binds melittin but also that may interact with common target proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence stopped flow analysis of the interaction of virginiamycin components and erythromycin with bacterial ribosomes

The kinetics of the interaction between the 50 S subunits (R) of bacterial ribosomes and the antibiotics virginiamycin S (VS), virginiamycin M (VM), and erythromycin have been studied by stopped flow fluorimetric analysis, based on the enhancement of VS fluorescence upon its binding to the 50 S ribosomal subunit. Virginiamycin components M and S exhibit a synergistic effect in vivo, which is characterized in vitro by a 5- to 10-fold increase of the affinity of ribosomes for VS, and by the loss of the ability of erythromycin to displace VS subsequent to the conformational change (from R to R*) produced by transient contact of ribosomes with VM. Our kinetic studies show that the VM-induced increase of the ribosomal affinity for VS (K*VS = 25 X 10(6) M-1 instead of KVS = 5.5 X 10(6) M-1) is due to a decrease of the dissociation rate constant (k*-VS = 0.008 s-1 instead of 0.04 s-1). The association rate constant remains practically the same (k+VS approximately k*+VS = 2.8 X 10(5) M-1 s-1), irrespective of the presence of VM. VS and erythromycin bind competitively to ribosomes. This effect has been exploited to determine the dissociation rate constant of VS directly by displacement experiments from VS . 50 S complexes, and the association rate constant of erythromycin (k+Ery = 3.2 X 10(5) M-1 S-1) on the basis of competition experiments for binding of free erythromycin and VS to ribosomes. By making use of the change in competition behavior of erythromycin and VS, after interaction of ribosomes with VM, the conformational change induced by VM has been explored. Within the experimentally available concentration region, the catalytic effect of VM has been shown to be coupled to its binding kinetics, and the association rate constant of VM has been determined (k+VM = 1.4 X 10(4) M-1 S-1). Evidence is presented for a low affinity binding of erythromycin (K*Ery approximately 3.3 X 10(4) M-1) to ribosomes altered by contact with VM. A model involving a sequence of 5 reactions has been proposed to explain the replacement of ribosome-bound erythromycin by VS upon contact of 50 S subunits with VM.     

Evidence for four capital and six auxiliary cation-binding sites on calmodulin: divalent cation interactions monitored by direct binding and microcalorimetry

Recently, Mills and Johnson [7] and our group [9] provided evidence that calmodulin contains, in addition to the four Ca2+-binding sites (capital sites), which are essential for drug- and enzyme-binding, a number of divalent cation-binding sites of different ion selectivity (auxiliary sites), which modulate drug-binding as well as the affinity of Ca2+ for the capital sites. In the present study, the number of auxiliary sites and their relationship to the capital sites were determined by equilibrium gel filtration and by flow microcalorimetry with Zn2+ and Mn2+ as selective probes for the auxiliary sites and with Cd2+ as a probe for both types of sites. In the absence of other divalent cations, 6 mol of Zn2+ bind to calmodulin with an identical affinity constant of 2,850 M-1 and a delta H0 of 106 kJ/mol calmodulin. In the presence of millimolar free Ca2+ calmodulin binds, in addition to four Ca2+, six Zn2+ with an affinity constant of 1,200 M-1 and a delta H0 of 47 kJ/mol calmodulin. The Zn2+-Ca2+ antagonism is governed by negative free energy coupling between the capital and auxiliary sites. In contrast, the Zn2+-Mg2+ antagonism follows the rule of straight competition at all six auxiliary sites. Mn2+ also binds exclusively to the auxiliary sites with affinity constants of 800 or 280 M-1 and delta H0 of 45 or 46 kJ/mol calmodulin in the absence and presence of saturating [Ca2+], respectively. Cd2+ binds to the capital sites with an affinity constant of 3.4 10(4) M-1 (delta H = 35 kJ/mol calmodulin) and to the auxiliary sites with ca. 100-fold lower affinity. The Zn2+ much greater than Mn2+ greater than or equal to Cd2+ greater than Mg2+ selectivity of the auxiliary sites corroborates the potencies of these cations in modulating drug binding. The auxiliary site-specific cations are unable to promote high-affinity complex formation between calmodulin and melittin.     

Plasma protein binding interaction of prednisone and prednisolone

The plasma protein binding interaction of prednisone and prednisolone were characterized by equilibrium dialysis. Prednisone and prednisolone are bound equally but weakly to human albumin (affinity constant, K approximately equal to 1 X 10(3) M-1). Transcortin affinity for prednisolone is 10-fold greater (51.3 X 10(6) M-1) than that for prednisone (4.3 X 10(6) M-1). In competition under pharmacologic conditions, prednisolone inhibits prednisone binding to transcortin producing linear binding averaging 55%. Prednisone does not affect prednisolone binding and does not complicate pharmacokinetic studies of the latter.     

Thyroxine-protein interactions. Interaction of thyroxine and triiodothyronine with human thyroxine-binding globulin.

The effect of temperature on the binding of thyroxine and triiodothyronine to thyroxine-binding globulin has been studied by equilibrium dialysis. Inclusion of ovalbumin in the dialysis mixture stabilized thyroxine-binding globulin against losses in binding activity which had been found to occur during equilibrium dialysis. Ovalbumin by itself bound the thyroid hormones very weakly and its binding could be neglected when analyzing the experimental results. At pH 7.4 and 37 degrees in 0.06 M potassium phosphate/0.7 mM EDTA buffer, thyroxine was bound to thyroxine-binding globulin at a single binding site with apparent association constants: at 5 degrees, K = 4.73 +/- 0.38 X 10(10) M-1; at 25 degrees, K = 1.55 +/- 0.17 X 10(10) M-1; and at 37 degrees, K = 9.08 +/- 0.62 X 10(9) M-1. Scatchard plots of the binding data for triiodothyronine indicated that the binding of this compound to thyroxine-binding globulin was more complex than that found for thyroxine. The data for triiodothyronine binding could be fitted by asuming the existence of two different classes of binding sites. At 5 degrees and pH 7.4 nonlinear regression analysis of the data yielded the values n1 = 1.04 +/- 0.10, K1 = 3.35 +/- 0.63 X 10(9) M-1 and n2 = 1.40 +/- 0.08, K2 = 0.69 +/- 0.20 X 10(8) M-1. At 25 degrees, the values for the binding constants were n1 = 1.04 +/- 0.38, K1 = 6.5 +/- 2.8 X 10(8) M-1 and n2 = 0.77 +/- 0.22, K2 = 0.43 +/- 0.62 X 10(8) M-1. At 37 degrees where less curvature was observed, the estimated binding constants were n1 = 1.02 +/- 0.06, K1 = 4.32 +/- 0.59 X 10(8) M-1 and n2K2 = 0.056 +/- 0.012 X 10(8) M-1. When n1 was fixed at 1, the resulting values obtained for the other three binding constants were at 25 degrees, K1 = 6.12 +/- 0.35 X 10(8) M-1, n2 = 0.72 +/- 0.18, K2 = 0.73 +/- 0.36 X 10(8) M-1; and at 37 degrees K1 = 3.80 +/- 0.22 X 10(8) M-1, n2 = 0.44 +/- 0.22, and K2 = 0.43 +/- 0.38 X 10(8) M-1. The thermodynamic values for thyroxine binding to thyroxine-binding globulin at 37 degrees and pH 7.4 were deltaG0 = -14.1 kcal/mole, deltaH0 = -8.96 kcal/mole, and deltaS0 = +16.7 cal degree-1 mole-1. For triiodothyronine at 37 degrees, the thermodynamic values for binding at the primary binding site were deltaG0 = -12.3 kcal/mole, deltaH0 = -11.9 kcal/mole, and deltaS0 = +1.4 cal degree-1 mole-1. Measurement of the pH dependence of binding indicated that both thyroxine and triiodothyronine were bound maximally in the region of physiological pH, pH 6.8 to 7.7.     

Acidity and solubility of gramicidin S in water

The acid-base transformations of the gramicidin S molecule in water were studied. The protonization constants of the antibiotic amino group were calculated by the data of the potentiometric titration and the antibiotic distribution in the system of chloroform-water: K1 1.55 X 10(10), K2 1.38 X 10(6), the logarithm of the distribution coefficient of gramicidin S in the system of chloroform-water (1:1) lg alpha G 4.10. By the same data the constants of water solubility of gramicidin S base (1.02 X 10(7) mol/l), gramicidin S monohydrate (1.06 X 10(-4) mol/l) and gramicidin S dihydrochloride (2.08 X 10(-4) mol/l) were calculated.     

The effect of Mg2+ on the Ca2+-binding properties of non-activated phosphorylase kinase.

The calcium binding properties of non-activated phosphorylase kinase at pH 6.8 have been studied by the gel filtration technique at calcium concentrations from 50 nM to 50 muM. Taking into account the subunit structure alpha4beta4gamma4 the enzyme binds 12 mol Ca2+ per mol with an association constant of 6.0 X 10(7) M-1, 4 mol with an association constant of 1.7 X 10(6) M-1 and 36 mol with a binding constant of 3.9 X 10(4) M-1 at low ionic strength. In buffer of high ionic strength, i.e. 180 mM NH4Cl or 60 mM (NH4)2SO4, only a single set of eight binding sites with a binding constant of 5.5 X 10(7) M-1 is left. In a buffer containing 155 mM NH4Cl and 10 mM MgCl2, the calcium affinity of these sites is reduced to a KCa of 3.0 X 10(6) M-1, indicating competition between Ca2+ and Mg2+. From these measurements, the binding constant of Mg2+ for these sites is calculated to be 1.7 X 10(3) M-1 is left. In a buffer containing 155 mM NH4Cl and 10 mM MgCl2, the calcium affinity of these sites is reduced to a KCa of 3.0 X 10(6) M-1, indicating competition between Ca2+ and Mg2+. from these measurements, the binding constant of Mg2+ for these sites is calculated to be 1.7 X 10(3) M-1. Additionally, 10 mM Mg2+ induces a set of four new Ca2+ binding sites which show positive cooperativity. Their half-saturation constant under the conditions described is 3.5 X 10(5) M-1, and they, too, exhibit competition between Ca2+ and Mg2+. Since this set of sites is induced by Mg2+ a third group of binding sites for the latter metal must be postulated.     

Kinetics of the reactions between streptokinase, plasmin and alpha 2-antiplasmin.

Streptokinase reacts very rapidly with human plasmin (rate constant 5.4 S 10(7) M-1 s-1) forming a 1:1 stoichiometric complex which has a dissociation constant of 5 X 10(-11) M. This plasmin-streptokinase complex is 10(5) times less reactive towards alpha 2-antiplasmin than plasmin, the inhibition rate constant being 1.4 X 10(2) M-1 s-1. The loss of reactivity of the streptokinase-plasmin complex towards alpha 2-antiplasmin is independent of the lysine binding sites in plasmin since low-Mr plasmin, which lacks these sites, and plasmin in which the sites have been blocked by 6-aminohexanoic acid, are both equally unreactive towards alpha 2-antiplasmin on reaction with streptokinase. The plasmin-streptokinase complex binds to Sepharose-lysine and Sepharose-fibrin monomer in the same fashion as free plasmin, showing that the lysine binding sites are fully exposed in the complex. Bovine plasmin is rapidly inhibited by human alpha 2-antiplasmin (k1 = 1.6 X 10(6) M-1 s-1) and similarly loses reactivity towards the inhibitor on complex formation with streptokinase (50% binding at 0.4 microM streptokinase).     

Kinetic studies on [methionine sulphoxide] cytochrome c.

A cytochrome c haem ligand, methionine-80, was photo-oxidized to methionine sulphoxide and the subsequent changes in redox properties and ligand binding were monitored kinetically. Isoelectric focusing of the product showed the presence of a single oxidized species, capable of binding CO when reduced. The binding of CO to the reduced protein was followed in stopped-flow experiments, which revealed the presence of two binding processes, at neutral pH, with rate constants of K+1 = 3.4 X 10(3)M-1-S-1 and k+2 = 5.80 X 10(2)M-1-S-1. When CO was photolytically dissociated from the reduced protein two recombination processes were observed with rates almost identical with those observed in the stopped-flow experiments (k+1 = 3.3 X 10(3)M-1-S-1 and k+2 = 6.0 X 10(2)M-1-S-1). These findings provide evidence of two reduced forms of the protein. The reduction of [methionine sulphoxide]cytochrome c by Cr2+ at neutral pH in stopped-flow experiments showed the presence of a single second-order reduction process (k = 7.2 X 10(3)M-1-S-1, activation energy = 44kJ/mol) and one first-order process. This protein was compared with some other chemically modified cytochromes.