Topographical relationships between the monovalent and divalent cation binding sites of des-1-41-light chain bovine plasma protein C and des-1-41-light chain-activated bovine plasma protein C

The paramagnetic effect of Mn2+ on the longitudinal relaxation rate (T1)-1 of 205Tl+, when both cations are bound to des-1-41-light chain bovine plasma protein C (GDPC) and its activation product, des-1-41-light chain-activated bovine plasma protein C (GDAPC), has been assessed by 205Tl+ NMR spectroscopy. A substantial shortening of the T1 for Tl+ bound to either protein was observed in the presence of Mn2+, an effect not noted upon substitution of Mn2+ with the diamagnetic cation Ca2+, which is known to bind to these proteins in a similar fashion to Mn2+. This paramagnetic effect was employed to estimate distances between the monovalent and divalent cation sites in these proteins, approximately 6.7 +/- 0.2 A with GDPC and 8.3 +/- 0.2 A in GDAPC. These data suggest that a conformational alteration occurs upon activation of GDPC which leads to an increase in the distance between the monovalent and divalent cation sites.     

The effect of divalent cations on the amidolytic activity of bovine plasma activated protein C and des-1-41-light chain activated protein C

The effect of the divalent cations Ca2+ and Mn2+ on the amidolytic activity of bovine plasma activated protein C and a limited chymotryptic digestion product, des-1-41-light chain activated protein C, which lacks all of the gamma-carboxyglutamic acid present in activated protein C, has been examined at 30 degrees C. In each case, the enzymic activities were dependent upon the presence of these cations, which exerted their effects primarily through influence on the kcat of the reaction. For both enzymes (E), the mechanism of the reaction was most consistent with a rapid equilibrium, random addition of substrate (S), and a single cation (A), with substrate hydrolysis occurring only with the ternary complex of S.E.A. The divalent cation site of importance was not associated with gamma-carboxyglutamic acid residues and was found to be independent of the monovalent cation sites, which also function to activate the amidolytic and esterolytic activities of these enzymes.     

The effect of monovalent cations on the pre-steady state reaction kinetics of bovine activated plasma protein C and des-1-41-light chain activated plasma protein C

A pre-steady state kinetic analysis of the stimulation by monovalent cations of the activity of bovine activated protein C (APC) and a proteolytic fragment of APC, des-1-41-light chain activated protein C (GDAPC), toward the substrate, 4-methylumbelliferyl p-guanidinobenzoate, has been undertaken. With the cations Na+ and Cs+, at least two cation sites, or classes of sites, on APC were found to be important to the kinetic effects observed. For GDAPC, with both monovalent cations investigated, a single cation-binding site, or class of sites, of kinetic importance was discovered. The most general mechanism that fits all kinetic data was a rapid equilibrium type, with the cation(s) (A) and substrate (S) binding to the enzyme in a random fashion. Cations were found to be essential activators, and only formation of the EAS or EA2S complex led to product generation. For each enzyme, stimulation of the reaction rates was found to be chiefly due to a dramatic enhancement by monovalent cations of the rate constant (k2) for acylation of the enzyme since the dissociation constant (Ks) for enzyme-substrate interactions was increased in the presence of cations, and the deacylation rate constant (k3) was not affected by these activators.     

The stimulation by monovalent cations of the amidase activity of bovine des-1-41 light chain activated protein C

The kinetic properties of the activation by monovalent cations of the amidolytic activity of bovine des-1-41 light chain activated protein C have been examined. With the cations Cs+, K+, Li+, and Tl+, a single cation site, or class of sites, has been found to be responsible for the stimulation observed, with kinetic Ka values of 98-110, 180-210, 300-310, and 14-16 mM, respectively. The mechanism proposed for participation of these cations in the enzyme reaction involves an ordered addition, with the binding of cation preceding the binding of the amide substrate. On the other hand, the kinetic properties of this same activation by Na+ are consistent with either two cation sites, or classes of sites, of importance. Once again, however, the mechanism of the reaction appears to be of the ordered type, with cation binding occurring prior to substrate binding.     

Estimation of the distance between the divalent cation binding site of des-1-41-light chain-activated bovine plasma protein C and a nitroxide spin label attached to the active-site serine residue.

The paramagnetic effect of Mn2+ on the electron paramagnetic resonance spectrum of a nitroxide spin label covalently attached to the active-site serine residue of des-1-41-light chain bovine plasma-activated protein C, and situated at a distance of approximately 1.2 nm from this amino acid, has been utilized to estimate the distance on the enzyme surface between the single Mn2+ site and the free electron of the spin label. This distance has been found to be approx. 1.12 nm. A significant paramagnetic effect of Mn2+ on the spectrum of this same nitroxide spin label bound to activated protein C (APC) has been found. However, in this case distance calculations are complicated by the existence of a multiplicity of Mn2+ sites on APC. If it is assumed that a single Mn2+ site is responsible for the paramagnetic effect on the spectrum of the spin label, the interelectron distance on APC would be approx. 0.90 nm.     

Topographical relationships among the monovalent cation binding sites of bovine plasma-activated protein C and des(1–41)-light-chain-activated bovine plasma protein C and a nitroxide spin label bound to their active site serine residues

The paramagnetic effect of a spin-labeled sulfonyl fluoride, 4-(2,2,5,5-tetramethylpyrrolidine-1-oxyl)-p-fluorosulfonylbenzamide (p-V), when bound to the active site serine residue of the proteases, bovine plasma-activated protein C (APC) and des(1–41)-light-chain-activated protein C (GDAPC), on the longitudinal relaxation rate (T₁) of Tl⁺ bound to these same proteins has been examined by ²⁰⁵Tl⁺-NMR spectroscopy. The substantial shortening by bound p-V of the T₁ for Tl⁺ has been employed to estimate the distances between Tl⁺ and the unpaired electron on each protein surface. Assuming that a single cation-binding site exists on each enzyme, electron-nuclear distances of 3.4–3.9 Å have been calculated for each protein. This suggests that the removal of 41 amino acid residues and, concomitantly, all γ-carboxyglutamic acid, from the amino-terminal of the light chain of APC, does not significantly affect the protein topography in the region of the molecule probed by this technique.     

Comparison of the binding of Ca2+ and Mn2+ to bovine alpha-lactalbumin and equine lysozyme

The enthalpy change of the binding of Ca2+ and Mn2+ to equine lysozyme was measured at 25 degrees C and pH 7.5 by batch microcalorimetry: delta H degrees Ca2+ = -76 +/- 5 kJ mol-1, delta H degrees Mn2+ = -21 +/- 10 kJ mol-1. Binding constants, log KCa2+ = 6.5 +/- 0.2 and log KMn2+ = 4.1 +/- 0.5, were calculated from the calorimetric data. Therefore, delta S degrees Ca2+ = -131 +/- 20 JK-1 mol-1 and delta S degrees Mn2+ = 8 +/- 44 JK-1 mol-1. Removal of Ca2+ induces small but significant changes in the circular dichroism spectrum, indicating the existence of a partially unfolded apo-conformation, comparable with, but different from, the apo-conformation of bovine alpha-lactalbumin.     

Conductimetric study of the binding of Mn2+ to bovine pancreatic deoxyribonuclease

The binding of Mn2+ on bovine pancreatic deoxyribonuclease has been studied by a conductimetric method. At low ionic strengths, a high-affinity single binding site is demonstrated. The association constant value (K = 1.2 x 10(5) M-1 at pH 8) is high enough to conclude that, in standard experimental conditions for DNA hydrolysis, the reacting species is the DNAase-cation complex. Competitive binding studies in presence of Mg++ and Ca++ show that these cations do not bind on the Mn++ site.     

The binding of Mn2+ and ADP to myosin

The metal ion requirement of myosin-ADP binding was investigated by use of Mn²⁺. Mn²⁺ binds to two sets of noninteracting sites on myosin which are characterized by affinity constants of 10⁶ and 10³, M⁻¹ at 0.016 M KCl concentration. The maximum number of sites is 2 for the high affinity and 20–25 for the low affinity set. Binding of Mn²⁺ to the high affinity sites increases the affinity of ADP binding to myosin. F-actin inhibits ADP binding (Kiely, B., and Martonosi, A., Biochim. Biophys. Acta 172: 158–170 [1969]), but even at F-actin concentrations much higher than that required to saturate the actin binding sites of myosin or its proteolytic fragments, significant ADP binding remained. The actin insensitive portion of ADP binding was inhibited by 10⁻⁴ M inorganic pyrophosphate or ATP. The results are discussed on the basis of a model in which actin and ADP bind to myosin at distinct but interacting sites.     

The binding of activated protein C to factors V and Va

Activated protein C has been derivatized with the active site-directed fluorophore 2-(dimethylamino)-6-naphthalenesulfonylglutamylglycylarginyl chloromethyl ketone (2,6-DEGR-APC). Covalently modified activated protein C has been used to investigate the binding interactions of the protein to factors V and Va in the presence of phospholipid vesicles. The fluorescence polarization of the 6-dimethylaminonaphthalene-2-sulfonyl moiety increased saturably with increasing phospholipid concentrations in the presence or absence of factor V or Va. Differences in the limiting polarization values indicated distinguishable differences in the interactions between 2,6-DEGR-APC and phospholipid in the presence of factor V or Va. The dissociation constant calculated for the 2,6-DEGR-APC/phospholipid interaction (7.3 X 10(-8) M) was not significantly altered by factor V but was decreased to 7 X 10(-9) M in the presence of factor Va. The interaction between 2,6-DEGR-APC and factor V or Va was characterized by a 1:1 stoichiometry. The binding of 2,6-DEGR-APC to factor V or Va in the presence of phospholipid could be reduced in a competitive manner by diisopropylphosphofluoridate-treated activated protein C. An analysis of the displacement curves indicated that the binding of 2,6-DEGR-APC was indistinguishable from the binding of diisopropylphosphofluoridate-treated activated protein C. The interaction between 2,6-DEGR-APC and phospholipid-bound factor Va was further examined using the isolated subunits of factor Va. Fluorescence polarization changes observed with component E of Va (light chain) closely corresponded with the changes observed with factor Va, whereas isolated component D (heavy chain) had little influence on the binding of 2,6-DEGR-APC to phospholipid vesicles. The data presented are consistent with the interpretation that component E of factor Va contains a binding site for activated protein C.     

Interaction of protein inhibitor of activated STAT 2 (PIAS2) with receptor of activated C kinase 1, RACK1

In this study, the evolutionarily conserved intracellular adaptor protein, receptor of activated C kinase 1 (RACK1) was identified as a novel interaction partner of protein inhibitor of activated STAT 2 (PIAS2) using a yeast two-hybrid screening system. The direct interaction and co-localization of RACK1 with PIAS2 was confirmed by immunoprecipitation and immunofluorescence staining analysis, respectively. The 5th to 7th Trp-Asp 40 (5-7 WD40) repeats of RACK1 were identified as the minimal domain required for interaction with PIAS2 by deletion analysis. Furthermore, multiple PIAS2-domains, particularly the 'PINIT' and RLD domains, bind the RACK1 5-7 WD40 domain.     

Protein S is required for bovine platelets to support activated protein C binding and activity

Gel-filtered platelets accelerate activated protein C inactivation of factor Va in a reaction that requires the presence of protein S. With protein S present, specific activated protein C binding to the platelet surface is observed (Kd = 11 +/- 3 nM, 203 +/- 20 sites/platelet). The concentration dependence of the activated protein C-mediated factor Va inactivation is in close agreement with the binding. The observed binding is specific since protein C does not compete with activated protein C. Platelet-bound activated protein C is approximately 8000 times more active than the solution-phase enzyme. Platelet activation with thrombin results in formation of a site capable of accelerating factor Va inactivation by activated protein C in the absence of added protein S. This cell surface site is blocked by the addition of affinity purified antibodies to protein S. We conclude that protein S is required for activated protein C binding to the platelet surface and subsequent rapid factor Va inactivation. Platelet activation leads to the expression of either protein S or an antigenically related protein which can substitute for exogenously added protein S.     

Proteolysis of protein C in pooled normal plasma and purified protein C by activated protein C (APC)

Protein C is a vitamin-K dependent zymogen of the anti-coagulant serine protease activated protein C (APC). In this paper, we report four lines of evidence that APC can activate protein C in pooled normal plasma, and purified protein C. First, the addition of APC to protein C-deficient plasma supplemented with protein C produces a prolongation of the clotting time of plasma that is proportional to the amount of protein C. This behavior was observed with APC from the Chromogenix APC resistance kit (Dia Pharm, Franklin, OH, USA) and from APC derived from the thrombin activation of human protein C (Enzyme Research Laboratories, South Bend, IN, USA). Secondly, using immunoblotting after gel electrophoresis, the disappearance of epitopes for monoclonal antibodies that recognize protein C but not APC indicates a time course for the activation by APC of protein C in pooled normal plasma and protein C purified from plasma. Thirdly, the same time course for the disappearance of protein C specific epitope can be followed using ELISA. Finally, protein C can be activated by APC as indicated by the increase in APC specific synthetic substrate Tryp-Arg-Arg-p nitroaniline hydrolysis. Kinetic data indicate a value of 4.7+/-0.4 mM(-1) s(-1) for the activation of protein C by APC under physiological conditions and in the presence of calcium. These observations document that APC must function not only in the inactivation of activated factors V and VIII, but also in the activation of protein C. This additional action of APC may be important to consider more broadly because of APC in the treatment of sepsis.     

Cooperative binding of Mn2+ and non-cooperative binding of Mg2+ to beta-galactosidase (E. coli).

Binding and activity studies with β-galactosidase at various concentrations of free Mn²⁺ and Mg²⁺ indicate that Mn²⁺ binds and activates β-galactosidase in a highly cooperative manner while Mg²⁺ binds and activates non-cooperatively. When the data are plotted by the Hill method, slopes of 3.4 for Mn²⁺ and of 1.0 for Mg²⁺ are obtained. The rate of lactose utilization when Mg²⁺ is bound is more than twice that when Mn²⁺ is bound.     

Tb3+ binding to bovine prothrombin and bovine prothrombin fragment 1

The binding of Tb3+ to bovine prothrombin and the amino-terminal 156 residues of prothrombin (F-1) was studied. On the basis of various Tb3+ emission properties, three classes of Tb3+-binding sites were described. The first class contained three high affinity sites in the F-1 region. These sites were filled noncooperatively and were saturated with Tb3+ before the other classes of sites started to fill. Ho3+ quenching of Tb3+ emission showed that these sites were in close proximity to one another (estimated distances 6-12 A). The second class of sites contained three lower affinity sites, also in the F-1 region. These sites bound Tb3+ in a stoichiometric manner and saturated prior to metal binding to the final class of sites. The number of protein ligands binding Tb3+ in the high affinity sites decreased as this second set of sites was filled. Ho3+ quenching of Tb3+ emission suggested that these sites were closely spaced and/or close to the first set of sites. The third class of sites contained 4-6 low affinity sites unique to prothrombin (not in the F-1 region). These sites were not studied extensively, but Tb3+ did not appear to bind stoichiometrically and did not saturate these sites in a manner similar to the other two classes of sites. The emission properties of Tb3+ bound to F-1 were different in KCl versus NaCl containing buffer while the emission properties of Tb3+ bound to prothrombin were not. Optimum conditions for studying lanthanide binding to F-1 (i.e. when Tb3+ bound to F-1 showed emission properties similar to Tb3+ bound to prothrombin) were when F-1 experiments were done at low F-1 concentrations in buffer containing 0.1 M KCl.     

Inactivation of human factor VIII by activated protein C: evidence that the factor VIII light chain contains the activated protein C binding site

Factor VIII is represented as a series of heterodimers composed of an 83(81) kDa light chain noncovalently bound to a variable size (93 to 210 kDa) heavy chain. Activated protein C inactivates factor VIII causing several cleavages of the factor VIII heavy chain(s). When factor VIII subunits were dissociated and component heavy and light chains isolated, the heavy chains were no longer a substrate for proteolysis by activated protein C. However, when factor VIII heavy chains were recombined with light chain, the reconstituted factor VIII activity was inactivated by activated protein C. The rate of factor VIII inactivation catalyzed by activated protein C was reduced by the presence of free light chain. The extent of this inhibition was dependent upon the concentration of light chain. Control experiments indicated that this protective effect of free light chain was not the result of inhibition of the activated protein C - lipid interaction. Fluorescence analysis demonstrated binding between the factor VIII light chain, chemically modified with eosin maleimide, and activated protein C, modified at its active site by dansyl-Glu-Gly-Arg chloromethyl ketone. Similar to proteolysis of factor VIII by activated protein C, this binding was dependent upon a lipid surface. Based upon the degree of fluorescence quenching, a spatial distance of 26 A was calculated separating the two fluorophores. These results demonstrate direct binding of activated protein C to the factor VIII light chain and suggest that this binding is an obligate step for activated protein C-catalyzed inactivation of factor VIII.     

Structural analysis of Mg2+ and Ca2+ binding, myristoylation, and dimerization of the neuronal calcium sensor and visinin-like protein 1 (VILIP-1)

Visinin-like protein 1 (VILIP-1) belongs to the neuronal calcium sensor family of Ca(2+)-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca(2+) and Mg(2+) binding, characterize metal-induced conformational changes, and determine structural effects of myristoylation and dimerization. Mg(2+) binds functionally to VILIP-1 at EF3 (ΔH = +1.8 kcal/mol and K(D) = 20 μM). Unmyristoylated VILIP-1 binds two Ca(2+) sequentially at EF2 and EF3 (K(EF3) = 0.1 μM and K(EF2) = 1-4 μM), whereas myristoylated VILIP-1 binds two Ca(2+) with lower affinity (K(D) = 1.2 μM) and positive cooperativity (Hill slope = 1.5). NMR assignments and structural analysis indicate that Ca(2+)-free VILIP-1 contains a sequestered myristoyl group like that of recoverin. NMR resonances of the attached myristate exhibit Ca(2+)-dependent chemical shifts and NOE patterns consistent with Ca(2+)-induced extrusion of the myristate. VILIP-1 forms a dimer in solution independent of Ca(2+) and myristoylation. The dimerization site is composed of residues in EF4 and the loop region between EF3 and EF4, confirmed by mutagenesis. We present the structure of the VILIP-1 dimer and a Ca(2+)-myristoyl switch to provide structural insights into Ca(2+)-induced trafficking of nicotinic acetylcholine receptors.     

The concentration of uncoupling protein correlates with Mg2+ activated mitochondrial binding of GDP

Rats were housed at 4 degrees C for periods of up to 26 days. As little as 2 h of cold exposure caused an increase in the binding of [3H]GDP to mitochondria from brown adipose tissue. Incubation of mitochondria in vitro with 10 mM Mg2+ caused a marked increase in the subsequent binding of GDP to mitochondria from rats housed at 28 degrees C and a smaller increase in that from rats exposed to 4 degrees C for 2 h. Chronic exposure to cold led to an even greater increase in the amount of GDP bound to mitochondria incubated with Mg2+. The time course for the increase in the concentration of uncoupling protein was compared with that for GDP binding to mitochondria with and without Mg2+ treatment. The concentration of uncoupling protein appears to be correlated with the GDP-binding values for mitochondria treated with Mg2+ (r = 0.70) but not with the GDP binding to untreated mitochondria (r = 0.36). Therefore, the binding of GDP to untreated mitochondria may represent thermogenic activity at the time of death, whereas that after treatment with Mg2+ may more closely reflect total thermogenic capacity of the mitochondrion.