Mn2+ is a native metal ion activator for bacteriophage lambda protein phosphatase

Bacteriophage lambda protein phosphatase (lambdaPP) is a member of a large family of metal-containing phosphoesterases, including purple acid phosphatase, protein serine/threonine phosphatases, 5'-nucleotidase, and DNA repair enzymes such as Mre11. lambdaPP can be activated several-fold by various divalent metal ions, with Mn(2+) and Ni(2+) providing the most significant activation. Despite the extensive characterization of purified lambdaPP in vitro, little is known about the identity and stoichiometry of metal ions used by lambdaPP in vivo. In this report, we describe the use of metal analysis, activity measurements, and whole cell EPR spectroscopy to investigate in vivo metal binding and activation of lambdaPP. Escherichia coli cells overexpressing lambdaPP show a 22.5-fold increase in intracellular Mn concentration and less dramatic changes in the intracellular concentration of other biologically relevant metal ions compared to control cells that do not express lambdaPP. Phosphatase activity assessed using para-nitrophenylphosphate as substrate is increased 850-fold in cells overexpressing lambdaPP, indicating the presence of metal-activated enzyme in cell lysate. EPR spectra of intact cells overexpressing lambdaPP exhibit resonances previously attributed to mononuclear Mn(2+) and dinuclear [(Mn(2+))(2)] species bound to lambdaPP. Spin quantitation of EPR spectra of intact E. coli cells overexpressing lambdaPP indicates the presence of approximately 40 microM mononuclear Mn(2+)-lambdaPP and 60 microM [(Mn(2+))(2)]-lambdaPP. The data suggest that overexpression of lambdaPP results in a mixture of apo-, mononuclear-Mn(2+), and dinuclear-[(Mn(2+))(2)] metalloisoforms and that Mn(2+) is a physiologically relevant activating metal ion in E. coli.     

Identification of the high affinity Mn2+ binding site of bacteriophage lambda phosphoprotein phosphatase: effects of metal ligand mutations on electron paramagnetic resonance spectra and phosphatase activities

Bacteriophage lambda phosphoprotein phosphatase (lambdaPP) has structural similarity to the mammalian Ser/Thr phosphoprotein phosphatases (PPPs) including the immunosuppressant drug target calcineurin. PPPs possess a conserved active site containing a dinuclear metal cluster, with metal ligands provided by a phosphoesterase motif plus two additional histidine residues at the C-terminus. Multiple sequence alignment of lambdaPP with 28 eubacterial and archeal phosphoesterases identified active site residues from the phosphoesterase motif and in many cases 2 additional C-terminal His metal ligands. Most highly similar to lambdaPP are E. coli PrpA and PrpB. Using the crystal structure of lambdaPP [Voegtli, W. C., et al. (2000) Biochemistry 39, 15365-15374] as a structural and active site model for PPPs and related bacterial phosphoesterases, we have studied mutant forms of lambdaPP reconstituted with Mn(2+) by electron paramagnetic resonance (EPR) spectroscopy, Mn(2+) binding analysis, and phosphatase kinetics. Analysis of Mn(2+)-bound active site mutant lambdaPP proteins shows that H22N, N75H, and H186N mutations decrease phosphatase activity but still allow mononuclear Mn(2+) and [(Mn(2+))(2)] binding. The high affinity Mn(2+) binding site is shown to consist of M2 site ligands H186 and Asn75, but not H22 from the M1 site which is ascribed as the lower affinity site.     

Cobalt hexammine inhibition of the hammerhead ribozyme

The effects of Co(NH(3))(6)(3+) on the hammerhead ribozyme are analyzed using several techniques, including activity measurements, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies and thermal denaturation studies. Co(NH(3))(6)(3+) efficiently displaces Mn(2+) bound to the ribozyme with an apparent dissociation constant of K(d app) = 22 +/- 4.2 microM in 500 microM Mn(2+) (0.1 M NaCl). Displacement of Mn(2+) coincides with Co(NH(3))(6)(3+) inhibition of hammerhead activity in 500 microM Mn(2+), reducing the activity of the WT hammerhead by approximately 15-fold with an inhibition constant of K(i) = 30.9 +/- 2.3 microM. A residual 'slow' activity is observed in the presence of Co(NH(3))(6)(3+) and low concentrations of Mn(2+). Under these conditions, a single Mn(2+) ion remains bound and has a low-temperature EPR spectrum identical to that observed previously for the highest affinity Mn(2+) site in the hammerhead ribozyme in 1 M NaCl, tentatively attributed to the A9/G10.1 site [Morrissey, S. R. , Horton, T. E., and DeRose, V. J. (2000) J. Am. Chem. Soc. 122, 3473-3481]. Circular dichroism and thermal denaturation experiments also reveal structural effects that accompany the observed inhibition of cleavage and Mn(2+) displacement induced by addition of Co(NH(3))(6)(3+). Taken together, the data indicate that a high-affinity Co(NH(3))(6)(3+) site is responsible for significant inhibition accompanied by structural changes in the hammerhead ribozyme. In addition, the results support a model in which at least two types of metal sites, one of which requires inner-sphere coordination, support hammerhead activity.     

Determination of the intracellular dissociation constant, K(D), of the fluo-3. Ca(2+) complex in mouse sperm for use in estimating intracellular Ca(2+) concentrations.

In order to calculate the actual, rather than the relative, intracellular Ca(2+) concentration (Ca(2+))(i) in mammalian sperm cells, using fluorescent probes whose fluorescence emission differs between the probe. Ca(2+) complex and free probe, the value of the dissociation constant for the probe. Ca(2+) complex, K(D), is required. Interaction of the probe with cellular components may change the intracellular value of K(D) from that determined in buffered solution. We had previously shown that fluo-3, whose Ca(2+) complex is highly fluorescent whereas free fluo-3 is not, could be used to monitor changes of (Ca(2+))(i) in mouse sperm. In this report, we describe a method for determining K(D) for the fluo-3. Ca(2+) complex in mouse sperm suspended in medium MJB, a medium in which the sperm remain viable, but which contains high Ca(2+). The method involved treating the sperm with ionomycin to provide a plasma membrane Ca(2+) carrier, with nigericin to eliminate pH gradient, and with gramicidin D to eliminate membrane potential, such that (Ca(2+))(i) equilibrates with medium Ca(2+) concentration (Ca(2+))(e), then titrating (Ca(2+))(e) with EGTA in added aliquots to near nil concentration. At EGTA concentrations in excess of total medium Ca(2+), an approximation algorithm was used to calculate (Ca(2+))(e), based on the known K(D) for the EGTA. Ca(2+) complex. The fluorescence of the intracellular fluo-3. Ca(2+) complex, F, decreased with increasing additions of EGTA; (Ca(2+))(i) = (Ca(2+))(e) was plotted as a linear function of F/[F(max) - F]; the slope gives K(D). At 37 degrees C, intracellular K(D) was calculated to be 0.636 +/- 0.018 microM (+/-SEM, n = 8). At 37 degrees C and 20 degrees C, K(D) values in MJB were calculated to be 0.502 +/- 0.022 and 0.578 +/- 0.029 (+/-SEM, n =8 and n = 6), respectively. The higher intracellular K(D) value implies probe interaction with cytosol components, primarily those in the head, as this compartment is the major contributor to sperm fluorescence. Changes in (Ca(2+))(i), monitored with fluo-3 fluorescence, that occur on interaction of capacitated mouse sperm with the zona pellucida and may now be quantified, using 0.636 microM for K(D) of the intracellular fluo-3. Ca(2+) complex.     

Leishmania amazonensis: characterization of an ecto-phosphatase activity

We have characterized a phosphatase activity present on the external surface of Leishmania amazonensis, using intact living parasites. This enzyme hydrolyzes the substrate p-nitrophenylphosphate (p-NPP) at the rate of 25.70+/-1.17 nmol Pi x h(-1) x 10(-7)cells. The dependence on p-NPP concentration shows a normal Michaelis-Menten kinetics for this ecto-phosphatase activity present a V(max) of 31.93+/-3.04 nmol Pi x h(-1) x 10(-7)cells and apparent K(m) of 1.78+/-0.32 mM. Inorganic phosphate inhibited the ecto-phoshatase activity in a dose-dependent manner with the K(i) value of 2.60 mM. Experiments using classical inhibitor of acid phosphatase, such as ammonium molybdate, as well as inhibitors of phosphotyrosine phosphatase, such as sodium orthovanadate and [potassiumbisperoxo(1,10-phenanthroline)oxovanadate(V)] (bpV-PHEN), inhibited the ecto-phosphatase activity, with the K(i) values of 0.33 microM, 0.36 microM and 0.25 microM, respectively. Zinc chloride, another classical phosphotyrosine phosphatase inhibitor, also inhibited the ecto-phosphatase activity in a dose-dependent manner with K(i) 2.62 mM. Zinc inhibition was reversed by incubation with reduced glutathione (GSH) and cysteine, but not serine, showing that cysteine residues are important for enzymatic activity. Promastigote growth in a medium supplemented with 1mM sodium orthovanadate was completely inhibited as compared to the control medium. Taken together, these results suggest that L. amazonensis express a phosphohydrolase ectoenzyme with phosphotyrosine phosphatase activity.     

Structure of the bis divalent cation complex with phosphonoacetohydroxamate at the active site of enolase.

Phosphonoacetohydroxamate (PhAH) is a tight-binding (Ki = 15 pM) inhibitor of enolase that is believed to mimic the aci-carboxylate form of the intermediate carbanion in the reaction [Anderson, V. E., Weiss, P. M., & Cleland, W. W. (1984) Biochemistry 23, 2779]. Electron paramagnetic resonance (EPR) spectroscopy of Mn2+ has been used to map sites of interaction of PhAH with the two divalent cations at the active site of enolase from bakers' yeast. EPR spectra of enolase-PhAH complexes containing two Mn2+ bound at the active site contain multiple fine structure transitions each with a 45-G 55Mn hyperfine spacing that is a characteristic of spin exchange coupled pairs of Mn2+. Magnetically dilute complexes were obtained by preparation of specific Mg2+/Mn2+ hybrid complexes by manipulating the order of addition of the divalent metal species. Thus, Mn2+ was placed in the higher affinity site by addition of 1 equiv of Mn2+ to a solution of enolase and PhAH, followed by addition of 1 equiv of Mg2+. Reversing the order of addition of Mg2+ and Mn2+ placed Mn2+ in the lower affinity site. Regiospecifically 17O-labeled forms of PhAH were prepared, and the binding of the functional groups on PhAH to Mn2+ at the two metal ion sites was determined from the presence or absence of 17O superhyperfine coupling in the EPR signals. The hydroxamate oxygen is a ligand of Mn2+ at the higher affinity site, a phosphonate oxygen is a ligand of Mn2+ at the lower affinity site, and the carbonyl oxygen is a mu-O bridge of the two metal ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Entamoeba histolytica: an ecto-phosphatase activity regulated by oxidation-reduction reactions

In this work, an ecto-phosphatase activity of Entamoeba histolytica was characterized using intact cells. This activity presented the following biochemical characteristics: (i) it hydrolyzes p-NPP with V(max) of 8.00+/-0.22 nmol p-NP x h(-1) x 10(-5) cells and K(m) of 2.68+/-0.25 mM; (ii) it is inhibited by acid phosphatase inhibitors, such as sodium molybdate (K(i)=1.70+/-0.24 microM) and sodium fluoride (K(i)=0.25+/-0.02 mM); (iii) it also showed high sensitivity to phosphotyrosine phosphatase inhibitors, such as sodium orthovanadate (K(i)=1.07+/-0.14 microM), bpV-PHEN (K(i)=0.38+/-0.02 microM) and mpV-PIC (K(i)=0.39+/-0.04 microM). Zn(2+), an oxidizing agent, decreased the enzymatic activity in 50%. DTT and GSH, two reducing agents, enhanced the activity twofold. The non-invasive E. histolytica and free-living E. moshkovskii were less efficient in hydrolyzing p-NPP than the pathogenic E. histolytica suggesting that this enzyme could represent a virulence marker for this cell.     

Divergent effects of the malignant hyperthermia-susceptible Arg(615)-->Cys mutation on the Ca(2+) and Mg(2+) dependence of the RyR1

The sarcoplasmic reticulum (SR) Ca(2+) release channel (RyR1) from malignant hyperthermia-susceptible (MHS) porcine skeletal muscle has a decreased sensitivity to inhibition by Mg(2+). This diminished Mg(2+) inhibition has been attributed to a lower Mg(2+) affinity of the inhibition (I) site. To determine whether alterations in the Ca(2+) and Mg(2+) affinity of the activation (A) site contribute to the altered Mg(2+) inhibition, we estimated the Ca(2+) and Mg(2+) affinities of the A- and I-sites of normal and MHS RyR1. Compared with normal SR, MHS SR required less Ca(2+) to half-maximally activate [(3)H]ryanodine binding (K(A,Ca): MHS = 0.17 +/- 0.01 microM; normal = 0.29 +/- 0.02 microM) and more Ca(2+) to half-maximally inhibit ryanodine binding (K(I,Ca): MHS = 519.3 +/- 48.7 microM; normal = 293.3 +/- 24.2 microM). The apparent Mg(2+) affinity constants of the MHS RyR1 A- and I-sites were approximately twice those of the A- and I-sites of the normal RyR1 (K(A,Mg): MHS = 44.36 +/- 4.54 microM; normal = 21.59 +/- 1.66 microM; K(I,Mg): MHS = 660.8 +/- 53.0 microM; normal = 299.2 +/- 24.5 microM). Thus, the reduced Mg(2+) inhibition of the MHS RyR1 compared with the normal RyR1 is due to both an enhanced selectivity of the MHS RyR1 A-site for Ca(2+) over Mg(2+) and a reduced Mg(2+) affinity of the I-site.     

Fluoride inhibition of bovine spleen purple acid phosphatase: characterization of a ternary enzyme-phosphate-fluoride complex as a model for the active enzyme-substrate-hydroxide complex.

Purple acid phosphatases (PAPs) employ a dinuclear Fe(3+)Fe(2+) or Fe(3+)Zn(2+) center to catalyze the hydrolysis of phosphate monoesters. The interaction of fluoride with bovine spleen purple acid phosphatase (BSPAP) has been studied using a combination of steady-state kinetics and spectroscopic methods. For FeZn-BSPAP, the nature of the inhibition changes from noncompetitive at pH 6.5 (K(i(comp)) approximately K(i(uncomp)) approximately 2 mM) to uncompetitive at pH 5.0 (K(i(uncomp)) = 0.2 mM). The inhibition constant for AlZn-BSPAP at pH 5.0 (K(i) = 3 microM) is approximately 50-70-fold lower than that observed for both FeZn-BSAP and GaZn-BSPAP, suggesting that fluoride binds to the trivalent metal. Fluoride binding to the enzyme-substrate complex was found to be remarkably slow; hence, the kinetics of fluoride binding were studied in some detail for FeZn-, AlZn-, and FeFe-BSPAP at pH 5.0 and for FeZn-BSPAP at pH 6.5. Since the enzyme kinetics studies indicated the formation of a ternary enzyme-substrate-fluoride complex, the binding of fluoride to FeZn-BSPAP was studied using optical and EPR spectroscopies, both in the presence and absence of phosphate. The characteristic optical and EPR spectra of FeZn-BSPAP. F and FeZn-BSPAP.PO(4).F are similar at pH 5.0 and pH 6.5, indicating the formation of similar fluoride complexes at both pHs. A structural model for the ternary enzyme-(substrate/phosphate)-fluoride complexes is proposed that can explain the results from both the spectroscopic and the enzyme kinetics experiments. In this model, fluoride binds to the trivalent metal replacing the water/hydroxide ligand that is essential for the hydrolysis reaction to take place, while phosphate or the phosphate ester coordinates to the divalent metal ion.     

Improved yields for baculovirus-mediated expression of human His(6)-PDK1 and His(6)-PKBbeta/Akt2 and characterization of phospho-specific isoforms for design of inhibitors that stabilize inactive conformations

PDK1 and PKB/Akt have a pleckstrin homology (PH) domain at the C-terminus and N-terminus, respectively, which stabilizes an unphosphorylated, autoinhibited conformation. Binding of the PH domain to a phospholipid second messenger causes relief of autoinhibition, which results in kinase phosphorylation and activation. Baculovirus-mediated expression in Sf9 insect cells of both His(6)-PDK1 and His(6)-PKBbeta/Akt2 were optimized, which significantly improved the yields (5-fold) of the affinity purified enzymes over previously reported values. Isoelectric focusing (IEF) and Western analyses indicated that the apparent V(max)=192+/-13 U/mg and K(m) (PDK-Tide)=55+/-10 microM of purified His(6)-PDK1 results from a mixture of at least three different phospho-specific isoforms (pI values of 6.8, 6.5, and 6.4). A purely unphosphorylated isoform of His(6)-PDK1 (pI=6.8) was generated by treatment with lambda protein phosphatase (lambdaPP), which decreased V(max) to 2.4+/-0.4 U/mg and increased K(m) (PDK-Tide) to 217+/-61 microM. Isoelectric focusing and Western analyses indicated that the apparent V(max)=0.21+/-0.03 U/mg and K(m) (Crosstide)=87+/-30 microM of purified His(6)-PKBbeta/Akt2 results from a mixture of the enzyme monophosphorylated either at Ser-474 ( approximately 90%) or at Thr-309 ( approximately 10%). A purely unphosphorylated isoform of His(6)-PKBbeta/Akt2 (pI=6.4) was generated by treatment with lambdaPP, which decreased V(max) approximately 2-fold. The optimization of high-level production and detailed characterization of purified and lambdaPP-treated His(6)-PDK1 and His(6)-PKBbeta/Akt2 will facilitate detailed structural and kinetic studies aimed at understanding the mechanism of second messenger-induced activation.     

A high-affinity, calmodulin-sensitive (Ca2+ + Mg2+)-ATPase and associated calcium-transport pump in the Ehrlich ascites tumor cell plasma membrane

A unique cytoplast preparation from Ehrlich ascites tumor cells (G. V. Henius, P. C. Laris, and J. D. Woodburn (1979) Exp. Cell. Res. 121, 337-345), highly enriched in plasma membranes, was employed to characterize the high-affinity plasma membrane calcium-extrusion pump and its associated adenosine triphosphatase (ATPase). An ATP-dependent calcium-transport system which had a high affinity for free calcium (K0.5 = 0.040 +/- 0.005 microM) was identified. Two different calcium-stimulated ATPase activities were detected. One had a low (K0.5 = 136 +/- 10 microM) and the other a high (K0.5 = 0.103 +/- 0.077 microM) affinity for free calcium. The high-affinity enzyme appeared to represent the ubiquitous high-affinity plasma membrane (Ca2+ + Mg2+)-ATPase (calcium-stimulated, magnesium-dependent ATPase) seen in normal cells. Both calcium transport and the (Ca2+ + Mg2+)-ATPase were significantly stimulated by the calcium-dependent regulatory protein calmodulin, especially when endogenous activator was removed by treatment with the calcium chelator ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid. Other similarities between calcium transport and the (Ca2+ + Mg2+)-ATPase included an insensitivity to ouabain (0.5 mM), lack of activation by potassium (20 mM), and a requirement for magnesium. These similar properties suggested that the (Ca2+ + Mg2+)-ATPase represents the enzymatic basis of the high-affinity calcium pump. The calcium pump/enzyme system was inhibited by orthovanadate at comparatively high concentrations (calcium transport: K0.5 congruent to 100 microM; (Ca2+ + Mg2+)-ATPase: K0.5 greater than 100 microM). Upon Hill analysis, the tumor cell (Ca2+ + Mg2+)-ATPase failed to exhibit cooperative activation by calcium which is characteristic of the analogous enzyme in the plasma membrane of normal cells.     

Hg(2+) affects the intracellular free Ca(2+) oscillatory pattern and the correlated membrane conductance changes in Mg(2+)-stimulated oocytes of the prawn Palaemon serratus

The impact of mercuric ions (Hg(2+)) on prawn oocytes was examined. Prawn oocytes constitute an unusual system in that they are activated at spawning by seawater Mg(2+), which mediates correlated dynamic changes in intracellular free calcium concentration [(Ca(2+))(i)] and membrane conductance associated with the meiosis resumption. Using a voltage clamp technique and intracellular calcium measurements, we observed that treatment with Hg(2+) (5, 10, and 20 microM) resulted in simultaneous impairments of both (Ca(2+))(i) and membrane current responses to external Mg(2+). Treatment with Hg(2+) also resulted in a gradual dose-dependent slow increase in the baseline level of both (Ca(2+))(i) and membrane conductance, independent of stimulation with external Mg(2+). The effect of Hg(2+) on (Ca(2+))(i) and membrane conductance changes resulted from a block of the signal transduction pathway at some point before the InsP(3) receptor channel involved in Ca(2+) release from the endoplasmic reticulum (ER) stocks. The Hg(2+)-dependent gradual increase in both (Ca(2+))(i) and membrane conductance baseline levels may potentially result from a slow permeabilization of the ER membrane, resulting in Ca(2+) leaking into the cytosol. Indeed, this effect could be blocked with the cell permeable Hg(2+) competitor dithiothreitol, which was able to displace Hg(2+) from its intracellular target regardless of whether external Ca(2+) was present or not.     

Electrochemical studies of the mono-Fe, Fe-Zn, and Fe-Fe metalloisoforms of bacteriophage lambda protein phosphatase

Bacteriophage lambda protein phosphatase (lambdaPP) is a member of a large superfamily of metallophosphoesterases, including serine/threonine protein phosphatases, purple acid phosphatases, 5'-nucleotidase, and DNA repair enzymes such as Mre11. Members of this family share several common characteristics, including a common phosphoesterase motif, secondary structural fold (betaalphabetaalphabeta), and metal ligand environment, and often accommodate a dinuclear metal center. The identity of the active site metals often differs between family members. Despite the extensive spectroscopic studies of several family members, only the standard redox potential of porcine purple acid phosphate (PAP) has been measured. In this report, we investigate the redox properties of another member of this protein family. The standard redox potentials of the mono-Fe, Fe-Zn, and Fe-Fe metalloisoforms of lambdaPP were determined from anaerobic redox titration experiments. Two different S = 5/2, mono-Fe3+ lambdaPP species were identified: the first with an E/D approximately 0.17, g = 8.9 and 4.8, and an Eo' approximately +130 mV; the second with E/D approximately 0.05, g = 6.7, 5.9, and 4.4, and an Eo' approximately +120 mV. The first and second mono-Fe3+ species are thought to represent Fe present in the M2 and M1 sites, respectively. The addition of Zn2+ to mono-Fe3+ lambdaPP results in a decrease in both mono-Fe3+ species and the appearance of a new S = 5/2, Fe(3+)-Zn2+ species (E/D approximately 0.02, g = 5.9, and an Eo' > +175 mV). The Fe-Fe lambdaPP titration revealed an S = 1/2, Fe(3+)-Fe2+ (g < 2) species with an Eo' > +128 mV. These results suggest that the active site of lambdaPP supports a high oxidation potential for both metal sites and may indicate an equally oxidizing active site for other member metallophosphoesterases.     

Mn2+ binding to factor VIII subunits and its effect on cofactor activity

Metal ions, such as Ca2+ and Mn2+, are necessary for the generation of cofactor activity following reconstitution of factor VIII from its isolated light chain (LC) and heavy chain (HC). Titration of EDTA-treated factor VIII with Mn2+ showed saturable binding with high affinity (K(d) = 5.7 +/- 2.1 microM) as detected using a factor Xa generation assay. No significant competition between Ca2+ and Mn2+ for factor VIII binding (K(i) = 4.6 mM) was observed as measured by equilibrium dialysis using 20 microM Ca2+ and 8 microM factor VIII in the presence of 0-1 mM Mn2+. The intersubunit affinity measured by fluorescence energy transfer of an acrylodan-labeled LC (fluorescence donor) and fluorescein-labeled HC (fluorescence acceptor) in the presence of 20 mM Mn2+ (K(d) = 53.0 +/- 17.1 nM) was not significantly different from the affinity value previously obtained in the absence of metal ion (K(d) = 53.8 +/- 14.2 nM). The sensitization of phosphorescence of Tb3+ bound to factor VIII subunits was utilized to detect Mn2+ binding to the subunits. Mn2+ inhibited the phosphorescence of Tb3+ bound to HC and LC, as well as the HC-derived A1 and A2 subunits with a relatively wide range of estimated inhibition constant values (K(i) values = 169-1147 microM), whereas Ca2+ showed no effect on Tb3+ phosphorescence. These results suggest that factor VIII cofactor activity can be generated by Mn2+ binding to site(s) on factor VIII that are different from the high-affinity Ca2+ binding site. However, like Ca2+, Mn2+ did not alter the affinity for HC and LC association. Thus, Mn2+appears to generate factor VIII cofactor activity by a similar mechanism as observed for Ca2+following its association at nonidentical sites on the protein.     

The PPP-family protein phosphatases PrpA and PrpB of Salmonella enterica serovar Typhimurium possess distinct biochemical properties.

Salmonella enterica serovar Typhimurium requires Mn(2+), but only a few Mn(2+)-dependent enzymes have been identified from bacteria. To characterize Mn(2+)-dependent enzymes from serovar Typhimurium, two putative PPP-family protein phosphatase genes were cloned from serovar Typhimurium and named prpA and prpB. Their DNA-derived amino acid sequences showed 61% identity to the corresponding Escherichia coli proteins and 41% identity to each other. Each phosphatase was expressed in E. coli and purified to near electrophoretic homogeneity. Both PrpA and PrpB absolutely required a divalent metal for activity. As with other phosphatases of this class, Mn(2+) had the highest affinity and stimulated the greatest activity. The apparent K(a) of PrpA for Mn(2+) of 65 microM was comparable to that for other bacterial phosphatases, but PrpB had a much higher affinity for Mn(2+) (1.3 microM). The pH optima were pH 6.5 for PrpA and pH 8 for PrpB, while the optimal temperatures were 45 to 55 degrees C for PrpA and 30 to 37 degrees C for PrpB. Each phosphatase could hydrolyze phosphorylated serine, threonine, or tyrosine residues, but their relative specific activities varied with the specific substrate tested. These differences suggest that each phosphatase is used by serovar Typhimurium under different growth or environmental conditions such as temperature or acidity.     

Inhibition of human plasma cholinesterase by malachite green and related triarylmethane dyes: mechanistic implications

The inhibitory effects of the cationic triarylmethane (TAM+) dyes, pararosaniline (PR+), malachite green (MG+), and methyl green (MeG+) on human plasma cholinesterase (BChE) were studied at 25 degrees C in 100 mM Mops, pH 8.0, with butyrylthiocholine as substrate. PR+ and MG+ caused linear mixed inhibition of enzyme activity. The respective inhibitory parameters were K(i) = 1.9 +/- 0.23 microM, alpha = 13 +/- 48, beta = 0 and K(i) = 0.28 +/- 0.037 microM, alpha = 23 +/- 7.4, beta = 0. MeG+ acted as a competitive inhibitor with K(i) = 0.12 +/- 0.017 microM (alpha, infinity, beta, not applicable). The K(i) values were within the same range reported for a number of ChE inhibitors including propidium ion, donepezil, and the phenothiazines, suggesting that TAM+s are active site ligands. On the other hand, the alpha values failed to correlate with values previously reported for a number of ChE inhibitors. It appears that mixed inhibition is the combined result of more than one type of binding and S-I interference. The impact of ligands at the choline-specific and peripheral anionic sites (or, possibly, accessory structural domains) on BChE activity needs to be studied in further detail.     

Calmodulin antagonists differentiate between Ni(2+)- and Mn(2+)-stimulated phosphatase activity of calcineurin.

The interaction of calmodulin antagonists with a phosphoprotein phosphatase, calcineurin, was investigated using para-nitrophenyl phosphate (pNPP) as a substrate. Calmidazolium, a potent calmodulin antagonist, inhibited the Ni(2+)-stimulated calmodulin-independent phosphatase activity to much the same extent as it did the Ca2+/calmodulin-stimulated activity. Other calmodulin antagonists, such as trifluoperazine, thioridazine, and W-7, also inhibited the Ni(2+)-stimulated phosphatase activity. On the other hand, calmidazolium only weakly and partially inhibited the Mn(2+)-stimulated phosphatase activity and the other calmodulin antagonists examined increased the Mn(2+)-stimulated activity, in the absence of calmodulin. With the addition of an equimolar amount, as to the inhibited holoenzyme, of the purified B subunit of calcineurin, the Ni(2+)-stimulated phosphatase activity recovered from 38 to 63% of the control level in the presence of 5 microM calmidazolium. When the amount of additional B subunit was increased, the phosphatase activity recovered to 94% of the control level, thereby implying that calmidazolium inhibits the Ni(2+)-stimulated phosphatase activity by interacting with the B subunit, in the absence of calmodulin. The Mn(2+)-stimulated phosphatase activity also recovered from the inhibition by calmidazolium, but a much larger amount of the B subunit was necessary for the recovery. These results indicate that the Ni(2+)- and Mn(2+)-stimulated activities of calcineurin are differentially affected by calmodulin antagonists and that the B subunit plays a crucial role in the expression of the Ni(2+)-stimulated phosphatase activity.     

Protein tyrosine kinase-dependent modulation of voltage-dependent potassium channels by genistein in rat cardiac ventricular myocytes

Effects of the isoflavone protein tyrosine kinase (PTK) inhibitor genistein on voltage-dependent K(+) currents, i.e., transient outward K(+) current (I(to)), sustained K(+) current (I(ss)), and inward rectifier K(+) current (I(K1)) were studied in rat cardiac ventricular myocytes. It was found that I(to) was reversibly inhibited by genistein in a concentration-dependent manner (IC(50)=28.1 microM), while I(ss) was suppressed by genistein with IC(50) of 18.5 microM. In addition, I(K1) (at -50 mV) was significantly decreased by 36.3+/-4.4% with 25 microM genistein. The inhibition of I(to), I(ss), and I(K1) by genistein was significantly reversed by the application of the protein tyrosine phosphatase inhibitor sodium orthovanadate (1 mM). However, I(to), I(ss), and I(K1) were not affected by the non-isoflavone PTK inhibitor tyrphostin A23 (100 microM) and PP2 (1 microM). These results indicate that activation of I(to), I(ss), and I(K1) channels is modulated by genistein-sensitive PTKs in rat ventricular myocytes.     

Manganese oxidation by modified reaction centers from Rhodobacter sphaeroides

The transfer of an electron from exogenous manganese (II) ions to the bacteriochlorophyll dimer, P, of bacterial reaction centers was characterized for a series of mutants that have P/P(+) midpoint potentials ranging from 585 to 765 mV compared to 505 mV for wild type. Light-induced changes in optical and EPR spectra of the mutants were measured to monitor the disappearance of the oxidized dimer upon electron donation by manganese in the presence of bicarbonate. The extent of electron transfer was strongly dependent upon the P/P(+) midpoint potential. The midpoint potential of the Mn(2+)/Mn(3+) couple was calculated to decrease linearly from 751 to 623 mV as the pH was raised from 8 to 10, indicating the involvement of a proton. The electron donation had a second order rate constant of approximately 9 x 10(4) M(-1) s(-1), determined from the linear increase in rate for Mn(2+) concentrations up to 200 microM. Weak dissociation constants of 100-200 microM were found. Quantitative EPR analysis of the six-line free Mn(2+) signal revealed that up to seven manganese ions were associated with the reaction centers at a 1 mM concentration of manganese. The association and the electron transfer between manganese and the reaction centers could be inhibited by Ca(2+) and Na(+) ions. The ability of reaction centers with high potentials to oxidize manganese suggests that manganese oxidation could have preceded water oxidation in the evolutionary development of photosystem II.