Comparative enzymatic properties of avian liver and skeletal muscle D-fructose-1,6-bisphosphate 1-phosphohydrolase.

The enzymatic properties of purified preparations of chicken liver and chicken skeletal muscle fructose bisphosphatases (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) were compared. Both enzymes have an absolute requirement for Mg2+ or Mn2+. The apparent Km for MgCl2 at pH 7.5 was 0.5 mM for the muscle enzyme and 5 mM for the liver enzyme. Fructose bisphosphate inhibited both enzymes. At pH 7.5, the inhibitor constants (Ki) were 0.18 and 1.3 mM for muscle and liver fructose bisphosphatases, respectively. The muscle enzyme was considerably more sensitive to AMP inhibition than the liver enzyme. At pH 7.5 and in the presence of 1 mM MgCl2, 50% inhibition of muscle and liver fructose bisphosphatases occurred at AMP concentrations of 7 X 10(-9) and 1 X 10(-6) M, respectively. EDTA activated both enzymes. The degree of activation was time and concentration dependent. The degree of EDTA activation of both enzymes decreased with increasing MgCl2 concentration. Ca2+ was a potent inhibitor of both liver (Ki, 1 X 10(-4) M) and muscle (Ki, 1 X 10(-5) M) fructose bisphosphatase. This inhibition was reversed by the presence of EDTA. Ca2+ appears to be a competitive inhibitor with regard to Mg2+. There is, however, a positive homeotropic interaction among Mg2+ sites of both enzymes in the presence of Ca2+.     

Guanylate cyclase of isolated bovine retinal rod axonemes.

The guanylate cyclase activity of axoneme--basal apparatus complexes isolated from bovine retinal rods has been investigated. The Mg2+ and Mn2+ complexes of GTP4- serve as substrates. Binding of an additional mole of Mg2+ or Mn2+ per mole of enzyme is required. Among cations which are ineffective are Ca2+, Ni2+, Fe2+, Fe3+, Zn2+, and Co2+. The kinetics are consistent with a mechanism in which binding of Mg2+ or Mn2+ to the enzyme must precede binding of MgGTP or MnGTP. The apparent dissociation constants of the Mg--enzyme complex and the Mn--enzyme complex are 9.5 x 10(-4) and 1.1 x 10(-4) M, respectively. The apparent dissociation constants for binding of MgGTP and MnGTP to the complex of the enzyme with the same metal are 7.9 x 10(-4) and 1.4 x 10(-4) M, respectively. The cyclase activity is maximal and independent of pH between pH 7 and 9. KCl and NaCl are stimulatory, especially at suboptimal concentrations of Mg2+ or Mn2+. Ca2+ and high concentrations of Mg2+ and Mn2+ are inhibitory. Ca2+ inhibition appears to require the binding of 2 mol of Ca2+ per mol of enzyme. The dissociation constant of the Ca2--enzyme complex is estimated to be 1.4 x 10(-6) M2. The axoneme--basal apparatus preparations contain adenylate cyclase activity whose magnitude is 1--10% that of the guanylate cyclase activity.     

Interaction of DNA with divalent metal ions

The interaction between the native DNA macromolecules and Ca2+, Mn2+, Cu2+ ions in solutions of low ionic strength (10(-3) M Na+) is studied using the methods of differential UV spectroscopy and CD spectroscopy. It is shown that the transition metal ions Mn2+ exercise binding to the nitrogen bases of DNA at concentrations approximately 5 x 10(-6) M and form chelates with guanine of N7-Me(2+)-O6 type. Only at high concentrations in solution (5 x 10(-3) M) do Ca2+ ions interact with the nitrogen bases of native DNA. In the process of binding to Ca2+ and Mn2+ the DNA conformation experiences some changes under which the secondary structure of the biopolymer is within the B-form family. The DNA transition to the new conformation is revealed by its binding to Cu2+ ions.     

A male specific hepatic estrogen binding protein: characteristics and binding properties

Mammalian liver is a sex-steroid responsive tissue in that androgen and estrogen receptors are present and mediate differential hepatic hormonal effects. Further, we and others have found a sexual dimorphism in the hepatic cytosolic content of estrogen binding proteins. In addition to the estrogen receptor, the male has a high-capacity (12.0-15.0 pmol/mg protein) estrogen binding protein (MEB) which demonstrates a moderate affinity for estradiol (Kd = 31.0-43.2 nM) if estradiol metabolizing enzymes are first precipitated with protamine sulfate. This protein exhibits a unique specificity for steroidal estrogens: 2-methoxyestriol greater than estradiol greater than estriol = 2-methoxyestradiol greater than 2-hydroxyestradiol greater than estrone greater than 2-methoxyestrone greater than estriol 3-glucuronide greater than 2-hydroxyestrone = 3-methoxyestriol greater than androstanediol greater than dihydrotestosterone greater than testosterone. Other androgens such as androstenedione and methyltrienolone, nonsteroidal estrogens such as diethylstilbestrol, and the antiestrogens tamoxifen and 4-hydroxytamoxifen do not compete for [3H]estradiol ([3H]E2) binding. MEB is a relatively small-molecular-weight protein with a Sr of 20.4 A as determined by gel filtration on Sephadex G-100. The kinetics of [3H]E2 association and dissociation at 4 degrees C are very rapid, with t1/2 values of less than 5 s. Sodium molybdate, generally used to stabilize steroid receptors, inhibits MEB-[3H]estradiol binding activity in cytosol in a time- and dose-dependent manner, an effect not observed with partially purified MEB. Magnesium chloride inhibits binding activity of the Sephadex G-100 MEB pool, an effect reversed by EDTA. Other divalent cations also inhibit binding: Mn2+ greater than Mg2+ greater than Ca2+. Furthermore, EDTA complexes of these cations slightly enhance binding relative to EDTA alone: Ca2+ EDTA greater than Mg2+ EDTA greater than Mn2+ EDTA. These results demonstrate that MEB is a unique sex-steroid binding protein, albeit of unknown function, which is distinct from hepatic steroid receptors.     

Resolution and calcium-binding properties of the two major isoforms of troponin C from crayfish

Crayfish tail muscle troponin C (TnC) has been fractionated into its five components and the Ca2+-binding properties of the two major isoforms (alpha and gamma) determined by equilibrium dialysis. alpha-TnC contains one Ca2+-binding site with a binding constant of 1 x 10(6) M-1 and one Ca2+ site with a binding constant of 1 x 10(4) M-1. In the complex of alpha-TnC with troponin I (TnI) or with TnI and troponin T (TnT), both sites bind Ca2+ with a single affinity constant of 2-4 x 10(6) M-1. gamma-TnC contains two Ca2+-binding sites with a binding constant of 2 x 10(4) M-1. In the gamma-TnC.TnI and gamma-TnC.TnI.TnT complexes, the binding constant of one of the sites is increased to 4-5 x 10(6) M-1, while Ca2+ binding to the second site is hardly affected (KCa = 4-7 x 10(4) M-1). In the presence of 10 mM MgCl2, the two Ca2+-binding sites of both TnC isoforms exhibit a 2-3-fold lower affinity. Assuming competition between Ca2+ and Mg2+ for these sites, their binding constants for Mg2+ were 120-230 M-1. In the absence of Ca2+, however, alpha-TnC and gamma-TnC bind 4-5 mol of Mg2+/mol with a binding constant of 1 x 10(3) M-1. These results suggest that the effect of Mg2+ on Ca2+ binding at the two Ca2+ sites is noncompetitive, i.e. Mg2+ does not bind directly to these sites (Ca2+-specific sites). Since the formation of the complex of crayfish TnI with alpha-TnC or gamma-TnC increases significantly the affinity of one of their two Ca2+-specific sites, I conclude that the binding of Ca2+ to only one site (regulatory Ca2+-specific site) controls the Ca2+-dependent interaction between crayfish TnCs and TnI.     

The binding of divalent cations to myosin.

Centrifuge transport, equilibrium dialysis, and electron paramagnetic resonance studies on the binding of Mn2+ to myosin revealed two sets of noninteracting binding sites which are characterized at low ionic strength (0.016 M KCl) by affinity constants of 10(6) M-1 (Class I) and 10(3) M-1 (Class II), respectively. At 0.6 M KCl concentration, the affinity of Mn2+ for both sets of sites is reduced. The maximum number of binding sites is 2 for the high affinity and 20 to 25 for the low affinity set. Other divalent metal ions displace Mn2+ from the high affinity sites in the following order of effectiveness: Ca greater than Mg = Zn = Co greater than Sr greater than Ni. The inhibitory effects of Mg2+ and Ca2+ upon the Mn2+ binding are competitive with inhibitor constants of 0.75 to 1 mM which is similar to that of the low affinity divalent metal ion binding sites. Exposure of myosin to 37 degrees partially inhibits Mn2+ binding to Class I parallel with inhibition of ATPase activity. The binding of Mn2+ to the high affinity binding sites is not significantly influenced by ADP or PPi, although Mn2+ increases the affinity of ADP binding to myosin at high ionic strength.     

Binding of divalent cations with low density lipoproteins. A study by ESR

The binding of bivalent metal ions Cu2+, Zn2+, Ca2+, Mg2+ to low-density lipoproteins (LDL) was investigated by the ESR technique. The monitoring of ESR spectra of paramagnetic Mn2+ ions in the presence of above-listed cations made it possible to evaluate the dissociation constants of their complexes with LDL. The effective dissociation constant of the complex Mn(2+)-LDL used for calculations was KD = (1.1 +/- 0.4) x 10(-4) M according to literature data. The investigated cations may be classified into two groups: 1) low dissociation constants were characteristic for Cu2+ ions [KD = (1.3 +/- 0.5) x 10(-4) M], which demonstrated a high oxidative ability, and for Zn2+ [KD = (0.95 +/- 0.45) x 10(-4) M] and Mn2+ ions, which could strongly influence the copper-induced LDL oxidation; 2) Ca2+ and Mg2+ were characterized by higher values of KD [(6 +/- 1) x 10(-4) M and (7.5 +/- 1.5) x 10(-4) M, accordingly] and slightly affected the Cu(2+)-induced oxidation of LDL. The results of the present work reinforced our earlier conjecture that cations may influence the process of lipid peroxidation, binding only to particular binding sites on the surface of LDL.     

Evidence for the presence of active and inactive forms of cytosolic triiodothyronine binding protein in rat kidney: cooperative action of Ca2+ in NADPH activation

The effect of NADPH and Ca2+ on 3, 5, 3'-L-triiodothyronine (T3) binding to cytosolic T3 binding protein (CTBP) in rat kidney was investigated in vitro. Extraction of rat kidney cytosol with 10% charcoal at 4 degrees C for 30 min. inactivated specific T3 binding. The decreased T3 binding activity in extracted cytosol could be restored by adding NADPH or Ca2+ to the incubation medium. Each substance increased the capacity for T3 without changes in the affinity for T3. The T3 binding was maximally increased by 25 microM NADPH or by 1.0 mM Ca2+ in the presence of 0.1 mM EDTA. The increase in T3 binding, induced by 25 microM NADPH, was enhanced by adding 0.2-1.0 mM Ca2+ in a concentration dependent-manner. The increase in T3 binding, induced by 1.0 mM Ca2+, was also enhanced by adding 3.125-25.0 microM NADPH. The NADPH-induced increase in T3 binding capacity was amplified by Ca2+ in a multiplicative manner. The results suggested that Ca2+ cooperatively augmented an NADPH function in cytosolic T3 binding.     

The calcium binding properties of phosphorylated and unphosphorylated cardiac and skeletal myosins.

Porcine left ventricular cardiac myosin and rabbit white skeletal myosin were phosphorylated by rabbit skeletal myosin light chain kinase and their Ca2+ binding properties were examined by equilibrium dialysis techniques. No significant effect of phosphorylation on the Ca2+ binding properties of these myosins was observed. Both types of striated muscle myosins bound approximately 2 mol of Ca2+/mol of myosin with similar affinities of 3 x 10(7) M-1. In the presence of 3 x 10(-4) M Mg2+ the myosins bound Ca2+ with a reduced affinity of 3 to 4 x 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the binding sites on myosin, the changes in Ca2+ binding can be accounted for by a Mg2+ affinity of 2.5 to 3.0 x 10(5) M-1.     

Ca2+-dependent proteolysis of the Ah receptor

We previously reported (J. Biol. Chem. (1986) 261, 6352-6465) that the photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, upon incubation with the liver cytosol fraction from C57BL/6 mice, labeled in a 1:1 ratio two peptides that had apparent molecular masses of 95 and 70 kDa and similar proteolytic fragmentation patterns. In the cytosolic fraction of Hepa 1 cells, a cloned murine hepatoma cell line, the product of photoaffinity labeling is almost exclusively a 95-kDa peptide which is rapidly hydrolyzed by a Ca2+-dependent proteinase to a 70-kDa peptide as well as other fragments. Thus, the ligand binding unit of the Ah receptor in C57BL/6 mouse liver and Hepa 1 cell is a 95-kDa peptide, and the 70-kDa fragment is a proteolytic artifact. The Ca2+-dependent proteinase which hydrolyzes the 95-kDa peptide has the properties of calpain II: (i) an absolute requirement for Ca2+, with maximal activity at 0.5 to 1.0 mM Ca2+; (ii) a pH optimum of 7.5 to 8.0; (iii) inhibition by EDTA, iodoacetamide, leupeptin and L-trans-epoxysuccinylleucylamido(4-guanidino)butane, but not by soybean trypsin inhibitor, aprotinin, or phenylmethanesufonyl fluoride. Upon chromatographic separation of the liver cytosol of C57BL/6 mice on DEAE-Sephacel, Ca2+-dependent proteinase activity (using casein or the labeled 95-kDa peptide as substrates) elutes with 0.25 M NaCl, and a specific proteinase inhibitor elutes with 0.15 M NaCl. Ca2+-dependent proteinase activity that hydrolyzes the 95-kDa peptide is found in the liver cytosols of several mammalian species.     

Intracellular Ca2+ homeostasis in the smooth muscle and functional role of calcium pump in the sarcolemma

It has been found that Ca-pump of the smooth muscle sarcolemma has much greater affinity to Ca2+ (Km = 0.5 M) than the system Na-Ca2+ of the exchanger (Km = 40-60 M). The maximal rate of Mg2+, ATP-dependent translocation of Ca2+ is 2-3 times higher than that of Na-dependent. The results of kinetic analysis show that Ca-pump of the smooth muscle sarcolemma is able to compensate the basal diffusion flow of this cation entering into unexcited cells of smooth muscle (5 x 10(-15) mol Ca2+ per 1 cm2 for 1 sec). It can also stationary support the value of Ca2+ concentration in relaxed myocytes on a physiologically significant level (10(-7)-10(-6) M).     

Increase of (Ca2+ +Mg2+)-ATPase activity of renal basolateral membranes by platelet-derived growth factor through a specific receptor

Studies were made on the direct effect of platelet-derived growth factor (PDGF) on the high-affinity (Ca2+ +Mg2+)-ATPase, a membrane bound Ca2+-extrusion pump enzyme of the basolateral membranes (BLM) of canine kidney (Km for free Ca2+ = 1.0 x 10(-7) M, Vmax = 180 nmol Pi/mg/min). At 1 x 10(-7) M free Ca2+, PDGF (10(-10)-10(-8) M) stimulated the enzyme activity significantly. Addition of 5 - 200 microM suramin, a compound that blocks binding of PDGF to its receptors on cell membranes, inhibited the stimulatory effect of PDGF dose-dependently (IC50 = 40 microM). A high affinity specific receptor for PDGF (Kd = 4.4 x 10(-10) M, Bmax = 460 fmol/mg protein) was detected on BLM preparations by radioreceptor assay with 125I-PDGF and unlabelled PDGF. Suramin (10-1000 microM) also inhibited the binding of PDGF to BLM preparations dose-dependently. From these results, it is proposed that PDGF stimulates (Ca2+ +Mg2+)-ATPase activity of kidney BLM preparations by enhancing its affinity for free Ca2+ through a specific receptor.     

Activation of lipid peroxidation in the adrenal cortex by metal ions

The processes of lipid peroxidation have been studied in bovine adrenal cortex in vitro. The lipid peroxidation rate in this tissue is shown to be dependent on the content of metal ions. EDTA, deferroxamine and penicyllamine inhibit spontaneous lipid peroxidation by 25, 50 and 42%, respectively. The ability to activate the process permits arranging metal ions in the following sequence: Fe2+ greater than Fe3+ greater than Cu2+ greater than Mg2+ greater than Mn2+. The maximum activation of lipid peroxidation is observed at Fe2+ and Fe3+ concentrations within the range of 5 x 10(-6) x 10(-4) M.     

Study of catalytically active center of the Ca2+-Mg2+-dependent ATPase of sarcoplasmic reticulum by triplet probe method

It was shown that eosine and erythrosine are competing inhibitors of the Ca2+-Mg2+- dependent ATPase active center of sarcoplasmic reticulum. The eosine and erythrosine inhibition constants are equal to 1.4 x 10(-6) M and 1.1 x 10(-6) M, respectively. Nitroxide radicals of various hydrophobicity and K3Fe(CN)6 were used to compare the constants of triplet states exchange quenching of erythrosine in aqueous solution, in lecithine liposomes and in ATPase active center of sarcoplasmic reticulum. It was established that ATPase binding center was immersed into a liquid phase and was not connected with lipids. Mn2+ and Gd3+-ions, which are competing with Mg2+ and Ca2+ for binding sites in the enzyme active center, diminished the phosphorescence quenching time of eosine at 77K. This means that the ion binding sites are less than 12 A apart from ATP-binding center.     

Interaction of divalent cations with beta-galactosidase (Escherichia coli).

Although the addition of various divalent metals to beta-galactosidase resulted in apparent activation, only Mg2+ and Mn2+ actually did activate. The apparent activation by the other divalent metals was shown to be due to Mg2+ impurities. Calcium did not activate, but experiments suggested that it did bind. Other divalent metals which were studied failed to bind. The dissociation constants for Mg2+ and Mn2+ were 2.8 X 10(-7) and 1.1 X 10(-8) M, respectively, and in each case one ion bound per monomer. These constants corresponded very closely to apparent values which were obtained from activation studies. The apparent binding constant for Ca2+, obtained from competition studies, was 1.5 X 10(-5) M. Data were obtained which showed that Mg2+, Mn2+, and Ca2+ all compete for binding at a single site. Of interest and of possible molecular biological importance was the observation that, while Mg2+ bound noncooperatively (n = 1.0), Mn2+ did so in a highly cooperative manner (n = 3.4). The binding of Mn2+ (as compared to Mg2+) resulted in a twofold drop in the Vmax for the hydrolysis and transgalactosylis reactions of lactose but had little effect on the Vmax of hydrolysis of allolactose, p-nitrophenyl beta-D-galactopyranoside (PNPG), or o-nitrophenyl beta-D-galactopyranoside (ONPG); Km values were not effected differently for any of the substrates by Mn2+ as compared to Mg2+. When very low levels of divalent metal ions were present (0.01 M EDTA added) or when Ca2+ was bound with lactose as the substrate, a greater decrease was observed in the rate of the transgalactosylic reaction than in the rate of the hydrolytic reaction, and the Km values for lactose and ONPG were increased. Of the three divalent metal ions which bound to beta-galactosidase, only Mn2+ had significant stabilizing effects toward denaturing urea and heat conditions.     

Monoclonal antibodies that recognize calcium-dependent structures of human thrombospondin. Characterization and mapping of their epitopes

Monoclonal antibodies (mAbs) raised against reduced and alkylated thrombospondin (TSP) were screened for the ability to react with Ca2+-replete TSP versus EDTA-treated TSP. Two mAbs designated A6.1 and D4.6 were found to react much more strongly with TSP after EDTA treatment. The dissociation constants for these mAbs were measured in 5 mM EDTA and found to be 6 X 10(-10) M for A6.1 and 7 X 10(-9) M for D4.6. Binding to A6.1 was undetectable in the presence of 1 mM Ca2+ while binding of D4.6 occurred with about 100-fold lower affinity. The Ca2+ concentration dependence of A6.1 binding was broad with a midpoint near 50 microM free Ca2+ while that of D4.6 showed a sharp transition below 0.1 microM. Upon dialysis of EDTA-treated TSP into Ca2+ containing buffer, the binding of the mAbs was prevented or decreased, indicating reversibility of the conformational transition induced by the initial removal of Ca2+ . Mg2+ can compete with the Ca2+ binding sites involved in mAb binding, but TSP dialyzed from Ca2+ into Mg2+ binds the two mAbs as well as EDTA-treated TSP, indicating that Mg2+ cannot maintain the Ca2+-replete structure of TSP. The proteolytic fragments of TSP with which the two mAbs react were determined by probing Western blots of digests of TSP with the mAbs. A6.1 reacts with the 70-kDa fragment generated by chymotrypsin in EDTA which contains the interchain disulfide bonds of TSP and the binding site(s) for type V collagen (Mumby, S. M., Raugi, G. J., and Bornstein, P. (1984) J. Cell Biol. 98, 646-652). D4.6 reacts with fragments of 140 and 120 kDa found in digests of Ca2+-replete TSP which are absent from digests in EDTA. Electron microscopy of rotary shadowed, carbon-coated replicas of TSP mAb complexes confirms the Ca2+ sensitivity of mAb binding and has been used to localize the epitopes for both mAbs on the three-dimensional structure of TSP.     

Metal requirements of a diadenosine pyrophosphatase from Bartonella bacilliformis: magnetic resonance and kinetic studies of the role of Mn2+

Recombinant IalA protein from Bartonella bacilliformis is a monomeric adenosine 5'-tetraphospho-5'-adenosine (Ap4A) pyrophosphatase of 170 amino acids that catalyzes the hydrolysis of Ap4A, Ap5A, and Ap6A by attack at the delta-phosphorus, with the departure of ATP as the leaving group [Cartwright et al. (1999) Biochem. Biophys. Res. Commun. 256, 474-479]. When various divalent cations were tested over a 300-fold concentration range, Mg2+, Mn2+, and Zn2+ ions were found to activate the enzyme, while Ca2+ did not. Sigmoidal activation curves were observed with Mn2+ and Mg2+ with Hill coefficients of 3.0 and 1.6 and K0.5 values of 0.9 and 5.3 mM, respectively. The substrate M2+ x Ap4A showed hyperbolic kinetics with Km values of 0.34 mM for both Mn2+ x Ap4A and Mg2+ x Ap4A. Direct Mn2+ binding studies by electron paramagnetic resonance (EPR) and by the enhancement of the longitudinal relaxation rate of water protons revealed two Mn2+ binding sites per molecule of Ap4A pyrophosphatase with dissociation constants of 1.1 mM, comparable to the kinetically determined K0.5 value of Mn2+. The enhancement factor of the longitudinal relaxation rate of water protons due to bound Mn2+ (epsilon b) decreased with increasing site occupancy from a value of 12.9 with one site occupied to 3.3 when both are occupied, indicating site-site interaction between the two enzyme-bound Mn2+ ions. Assuming the decrease in epsilon(b) to result from cross-relaxation between the two bound Mn2+ ions yields an estimated distance of 5.9 +/- 0.4 A between them. The substrate Ap4A binds one Mn2+ (Kd = 0.43 mM) with an epsilon b value of 2.6, consistent with the molecular weight of the Mn2+ x Ap4A complex. Mg2+ binding studies, in competition with Mn2+, reveal two Mg2+ binding sites on the enzyme with Kd values of 8.6 mM and one Mg2+ binding site on Ap4A with a Kd of 3.9 mM, values that are comparable to the K0.5 for Mg2+. Hence, with both Mn2+ and Mg2+, a total of three metal binding sites were found-two on the enzyme and one on the substrate-with dissociation constants comparable to the kinetically determined K0.5 values, suggesting a role in catalysis for three bound divalent cations. Ca2+ does not activate Ap4A pyrophosphatase but inhibits the Mn2+-activated enzyme competitively with a Ki = 1.9 +/- 1.3 mM. Ca2+ binding studies, in competition with Mn2+, revealed two sites on the enzyme with dissociation constants (4.3 +/- 1.3 mM) and one on Ap4A with a dissociation constant of 2.1 mM. These values are similar to its Ki suggesting that inhibition by Ca2+ results from the complete displacement of Mn2+ from the active site. Unlike the homologous MutT pyrophosphohydrolase, which requires only one enzyme-bound divalent cation in an E x M2+ x NTP x M2+ complex for catalytic activity, Ap4A pyrophosphatase requires two enzyme-bound divalent cations that function in an active E x (M2+)2 x Ap4A x M2+ complex.     

Interaction of calcium with bovine plasma protein C

The binding of 45Ca2+ to bovine plasma protein C (PC) and to activated bovine plasma protein C (APC) has been examined by equilibrium ultrafiltration at pH 7.4 and 25 degrees C. Under these conditions, PC possesses 16.0 plus or minus 2.0 equivalent Ca2+ binding sites, of average KD (8.7 plus or minus 1.5) x 10(-4) M, and APC contains 9.0 plus or minus 1.0 equivalent Ca2+ binding sites, with an average KD of (4.3 plus or minus 1.1) x 10(-4) M. Both Mn2+ and Sr2+ were capable of ready displacement of Ca2+ from a Ca2+-PC complex, while Mg2+ was less effective in this regard. The alpha-thrombin-catalyzed activation of PC was inhibited by the presence of Ca2+. A kinetic analysis of this effect demonstrated that it was, in large part, due to an increase in the Km of the reaction. Addition of other divalent cations, e.g. Mn2+, Sr2+, and Mg2+, in place of Ca2+ also resulted in inhibition of the alpha-thrombin-catalyzed activation of PC in a manner which paralleled their ability to displace Ca2+ from a Ca2+-PC complex. On the other hand, the activation of PC by the coagulant protein from Russell's Viper venom was augmented by the presence of Ca2+. Other divalent metal ions, such as Sr2+ and Mn2+, in the absence of Ca2+, also weakly stimulated this reaction. Mg2+ was without notable effect.     

Purification and characterization of myosin from the clonal rat glial cell strain C-6.

A myosin was isolated from the clonal rat glial cell strain C-6 and compared with rat skeletal muscle myosin. After cell extracts were subjected to gel filtration chromatography in the presence of KI and magnesium pyrophosphate the C-6 myosin was rapidly purified by a procedure similar to that used for skeletal muscle myosin. The C-6 myosin resembles muscle myosin both physically and enzymatically. It contains heavy chains of 200,000 daltons and two classes of light chains of 17,000 and 19,000 daltons in approximately equal molar ratios. This myosin forms bipolar thick filaments in 0.1 M KCl and binds reversibly to skeletal muscle F-actin, the binding being inhibited by MgATP. Skeletal muscle F-actin stimulates the C-6 myosin adenosine triphosphatase 2- to 3-fold in the presence of KCl and Mg2+. The action activation of muscle myosin ATPase at low ionic strength is 10-fold greater than that of C-6 myosin. Ca2+ and EDTA stimulated the ATPase activities of both enzymes. When assayed in the presence of 0.6 M KCl and 1 mM EDTA the skeletal muscle myocin ATPase demonstrates substrate saturation while the C-6 myosin enzyme activity is stimulated by ATP concentrations above 2.5 mM.     

Serotonin reuptake inhibitors fluoxetine and citalopram relax intestinal smooth muscle

Selective serotonin reuptake inhibitor antidepressants (SSRIs) exert depressant effects on cardiac myocytes and vascular smooth muscle cells by inhibiting Ca2+ channels. We hypothesized that the SSRIs fluoxetine and citalopram affect the contractile activity of intestinal smooth muscle by interfering with Ca2+ entry and (or) signaling pathways. The effects of fluoxetine and citalopram on contractions of guinea-pig ileum longitudinal muscle-myenteric plexus preparations (LMMP) were compared with the effects of the voltage-operated Ca2+ channel inhibitors nifedipine and diltiazem. In a concentration-dependent manner, nifedipine, diltiazem, fluoxetine, and citalopram elicited relaxation of LMMPs contracted by electrical field stimulation (EC50 values of 4 x 10(-7) M, 1.4 x 10(-6) M, 1.4 x 10(-5), and 6.8 x 10(-6) M, respectively). Nifedipine, diltiazem, fluoxetine, and citalopram also relaxed LMMPs contracted with a depolarizing concentration of KCl (48 mM; EC50 values of 1.8 x 10(-8) M, 1.4 x 10(-7) M, 3.7 x 10(-6) M, and 6.3 x 10(-6), respectively), a response that could be reversed by increasing the extracellular Ca2+ concentration (2.5-30 mM). These data suggest that fluoxetine and citalopram elicit relaxation of intestinal smooth muscle, likely by inhibiting Ca2+ channel(s). This effect may be of clinical importance.