Mechanism of glucagon activation of adenylate cyclase in the presence of Mn2+

For a variety of ligand states, adenylate cyclase activity in the presence of Mn2+ was greater than with Mg2+. Trypsin treatment of intact hepatocytes, under conditions which destroy cell surface glucagon receptors, led to a first order loss of glucagon-stimulated adenylate cyclase activity in isolated membranes assayed in the presence of Mn2+ whether or not GTP (100 microM) was present in the assays. Arrhenius plots of basal activity exhibited a break at around 22 degrees C, those with NaF were linear and those with glucagon +/- GTP (100 microM) were biphasic with a break at around 28 degrees C. It is suggested that Mn2+ perturbs the coupling interaction between the glucagon receptor and catalytic unit of adenylate cyclase at the level of the guanine nucleotide regulatory protein. This appears to take the form of Mn2+ preventing GTP from initiating glucagon's activation of adenylate cyclase through a collision coupling mechanism.     

Effects of Mn2+ on the exchange reaction of phosphoenolpyruvate carboxykinase in the presence of high concentrations of Mg2+

The mitochondrial phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32), purified from chick embryo liver, was synergistically activated by a combination of Mn2+ and Mg2+ in the oxaloacetate ---- H14CO-3 exchange reaction. Increases in the Mg2+ concentration caused decreases in the K0.5 value of Mn2+ in line with the earlier finding that the enzyme was markedly activated by low Mn2+ (microM) plus high Mg2+ (mM). In the presence of 2.5 mM Mg2+, increases in the Mn2+ level first enhanced the activity of phosphoenolpyruvate carboxykinase, and then suppressed it to the maximal velocity shown in the presence of Mn2+ alone. Kinetic studies showed that high Mn2+ inhibited the activity of Mg2+ noncompetitively, and those of GTP and oxaloacetate uncompetitively. The inhibition constant for oxaloacetate (K'i = 550 microM) was lower than that of Mg2+ (Ki = K'i = 860 microM) or GTP (K'i = 1.6 mM), and was nearly equal to the apparent half-maximal inhibition concentration of Mn2+. These results suggested that Mn2+ can play two roles, of activating and suppressing phosphoenolpyruvate carboxykinase activity in the presence of high Mg2+.     

Regulation of ANP-stimulated guanylate cyclase in the presence of Mn2+ in rat lung membranes

The catalytic activity of guanylate cyclase (GCase) coupled to atrial natriuretic peptide (ANP) receptor depends on the metal co-factor, Mn²⁺ or Mg²⁺. ATP synergistically stimulates the ANP-stimulated GCase in the presence of Mg²⁺. We have now shown the ATP regulation of the ANP-stimulated GCase in the presence of Mn²⁺ in rat lung membranes. ANP stimulated the GCase 2.1-fold compared to the control. ATP enhanced both the basal (basal-GCase) and the ANP-stimulated GCase maximally 1.7- and 2.3- fold compared to the control, respectively, at a concentration of 0.1 mM. The stimulation by ATP was smaller in the presence of Mn²⁺ than in the presence of Mg²⁺. The addition of inorganic phosphate to the reaction mixture altered the GCase activities in the presence of Mn²⁺ with or without ANP and/or ATP. In the presence of 10 mM phosphate, ATP dose-dependently stimulated the basal GCase 5-fold compared to the control at a concentration of 1 mM and augmented the ANP-stimulated GCase, which was 4.2-fold compared to the basal-GCase, 5.5-fold compared to the control at a concentration of 0.5 mM. Protein phosphatase inhibitors, okadaic acid (100 nM), H8 (1 M) and staurosporin (1 M), did not alter the activity. Orthovanadate (1 mM), an inorganic phosphate analogue, significantly stimulated both the basal-GCase and the ANP-stimulated GCase, which were inhibited by ATP. It was assumed that phosphate and orthovanadate might interact with the GCase to regulate the activity in the opposite manner. This was the first report that inorganic phosphate and orthovanadate affected the ATP-regulation of the ANP-stimulated GCase in the presence of Mn²⁺.     

Template-dependent variation in the relative fidelity of DNA polymerase I of Escherichia coli in the presence of Mg2+ versus Mn2+

The fidelity of E. coli DNA polymerase I in the presence of Mg2+ vs Mn2+ was examined at many positions along natural DNA templates, by use of an electrophoretic assay of misincorporation. Although there was an overall greater tendency for misincorporation to occur in Mn2+-activated chain elongation, some specific sites on the template were more prone to misincorporation with Mg2+ and others with Mn2+. This sequence-dependent effect was seen in spite of the finding that the relative rate of incorporation of the correct nucleotide at different positions on the template was essentially the same with Mg2+ and Mn2+. In agreement with previous studies, the fidelity of E. coli pol I was higher at activating, than at inhibiting, concentrations of Mg2+. The results reveal new complexities regarding the role of divalent cation in the control of fidelity in DNA synthesis and attest to the dynamic nature of interactions between DNA polymerase, its substrates and divalent metal activator during the course of polymerization on natural templates.     

In silico study of M18 aspartyl amino peptidase (M18AAP) of Plasmodium vivax as an antimalarial drug target

Plasmodium vivax (Pv) is the second most malaria causing pathogen among Plasmodium species. M18 aspartic aminopeptidase (M18AAP) protein is a single gene copy present in Plasmodium. This protein is functional at the terminal stage of hemoglobin degradation of host and completes the hydrolysis process which makes it an important target for new chemotherapeutics. No experimental and structural study on M18AAP protein of P. vivax is reported till today. This paper advocates the application of multiple computational approaches like protein model prediction, ligand-based 3D QSAR study, pharmacophore, structure-based virtual screening and molecular docking simulation for identification of potent lead molecules against the enzyme. The 3D QSAR model was developed using known bioactive compounds against the PvM18AAP protein which statistically signify the k-NN model with q^2 = 0.7654. The study reports a lead molecule from ligand-centric approach with good binding affinity and possessing lowest docking score. The findings will be helpful for in-vivo and in-vitro validations and development of potent anti-malarial molecules against the drug resistant strains of malaria parasite.     

Location of Mn2+ in concanavalin A containing only a Mn2+ ion

The metal-sugar distances in two metallized forms of concanavalin A have been compared by ¹⁹F magnetic resonance techniques. Using relaxation times measured at two different frequencies we have shown that the distance between the Mn²⁺ ion and the bound sugar in concanavalin A containing only Mn²⁺ is essentially identical to that found in concanavalin A containing both Mn²⁺ and Ca²⁺. Our results rule out the possibility that Mn²⁺ activates concanavalin A by binding at the Ca²⁺ site (S2) and would suggest that Mn²⁺ alone can induce an active saccharide binding conformation by binding at the transition metal site (S1).     

Passive skeletal muscle can function as an osmotic engine

Muscles are composite structures. The protein filaments responsible for force production are bundled within fluid-filled cells, and these cells are wrapped in ordered sleeves of fibrous collagen. Recent models suggest that the mechanical interaction between the intracellular fluid and extracellular collagen is essential to force production in passive skeletal muscle, allowing the material stiffness of extracellular collagen to contribute to passive muscle force at physiologically relevant muscle lengths. Such models lead to the prediction, tested here, that expansion of the fluid compartment within muscles should drive forceful muscle shortening, resulting in the production of mechanical work unassociated with contractile activity. We tested this prediction by experimentally increasing the fluid volumes of isolated bullfrog semimembranosus muscles via osmotically hypotonic bathing solutions. Over time, passive muscles bathed in hypotonic solution widened by 16.44 ± 3.66% (mean ± s.d.) as they took on fluid. Concurrently, muscles shortened by 2.13 ± 0.75% along their line of action, displacing a force-regulated servomotor and doing measurable mechanical work. This behaviour contradicts the expectation for an isotropic biological tissue that would lengthen when internally pressurized, suggesting a functional mechanism analogous to that of engineered pneumatic actuators and highlighting the significance of three-dimensional force transmission in skeletal muscle.     

Complete spectra of the far-red chemiluminescence of the oxygenase reaction of Mn2+-activated ribulose-bisphosphate carboxylase/oxygenase establish excited Mn2+ as the source

Chemiluminescence emitted by Mn(2+)-activated ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) while catalyzing oxygenation was analyzed to clarify the source of the emission. Using dual detectors capturing radiation over a wide range of visible and infrared wavelengths, we tested for radiation from singlet O(2) decay and found it to be essentially absent (less than 0.1% of the total luminescence intensity). Spectra were determined between 647 and 885 nm with a very sensitive, charge-coupled detector-based spectrograph to detect differences in the emission spectra between rubiscos from bacterial and higher plant sources. All Mn(2+)-activated rubiscos emitted a broad, smooth spectrum of chemiluminescence, unchanging as the reaction progressed. The spectra from higher plant rubiscos (spinach and both the wild type and an L335V mutant from tobacco), all exhibited maxima at about 800 nm. However, Mn(2+)-activated rubisco from the bacterium, Rhodospirillum rubrum, emitted at shorter wavelengths (760 nm peak), demonstrating host ligand-field influences arising from aminoacyl residue differences and/or conformational changes caused by the absence of small subunits. The findings provide strong evidence that the chemiluminescence arises from an excited state of the active-site Mn(2+) that is produced during oxygenation. We propose that the Mn(2+) becomes excited by a one-electron exchange mechanism of oxygenation that is not available to Mg(2+)-activated rubisco.     

The aminopeptidase from Aeromonas proteolytica can function as an esterase

The aminopeptidase from Aeromonas proteolytica (AAP) can catalyze the hydrolysis of L-leucine ethyl ester ( L-Leu-OEt) with a rate of 96 +/- 5 s-1 and a Km of 700 microM. The observed turnover number for L-Leu-OEt hydrolysis by AAP is similar to that observed for peptide hydrolysis, which is 67 +/- 5 s-1. The k(cat) values for the hydrolysis of L-Leu-OEt and L-leucine- p-nitroanilide ( L- pNA) catalyzed by AAP were determined at different pH values under saturating substrate concentrations. Construction of an Arrhenius plot from the temperature dependence of AAP-catalyzed ester hydrolysis indicates that the rate-limiting step does not change as a function of temperature and is product formation. The activation energy ( Ea) for the activated ES ester complex is 13.7 kJ mol-1, while the enthalpy and entropy of activation at 25 degrees C calculated over the temperature range 298-338 K are 11.2 kJ mol-1 and -175 J K-1 mol-1, respectively. The free energy of activation at 25 degrees C was found to be 63.4 kJ mol-1. The enthalpy of ionization was also measured and was found to be very similar for both peptide and ester substrates, yielding values of 20 kJ mol-1 for L-Leu-OEt and 25 kJ mol-1 for L- pNA. For peptide and L-amino acid ester cleavage reactions catalyzed by AAP, and 6.07, respectively. Proton inventory data suggest that two protons are transferred in the rate-limiting step of ester hydrolysis while only one is transferred in peptide hydrolysis. The combination of these data with the available X-ray crystallographic, kinetic, spectroscopic, and thermodynamic data for AAP provides new insight into the catalytic mechanism of AAP.     

Characterization of extracellular Mn2+-oxidizing activity and isolation of an Mn2+-oxidizing protein from Leptothrix discophora SS-1.

Supernatant fluid from Leptothrix discophora SS-1 cultures possessed high Mn2+-ozidizing activity. Studies of temperature and pH optima, chemical inhibition, and protease sensitivity suggested that the activity may be enzymatic. Kinetic studies of unconcentrated supernatant fluid indicated an apparent Km of 7 microM Mn2+ in the 1 to 200 microM Mn2+ range. The greatest Vmax value observed was 1.4 nmol of Mn2+ oxidized min-1 micrograms of protein-1 in unconcentrated samples. When the supernatant fluid was concentrated on DEAE-cellulose and the activity was eluted with MgSO4, an Mn2+-oxidizing protein was detected in the concentrate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mn2+-oxidizing protein appeared to have a molecular weight of 110,000 in 10% polyacrylamide gels and of 100,000 in 8% gels. Periodic acid-Schiff base staining of overloaded polyacrylamide gels showed that the DEAE-cellulose concentrate contained abundant high-molecular-weight polysaccharides; concurrent staining of the Mn2+-oxidizing band suggested that it too contained carbohydrate components. Isolation of the protein was achieved by subjecting the DEAE-cellulose concentrate to Sephacryl gel filtration in the presence of 1% sodium dodecyl sulfate, followed by preparative electrophoresis and reverse-polarity elution. However, these procedures resulted in loss of a large proportion of the activity, which precluded recovery of the protein in significant quality.     

Mn 2+-stimulatedATPase in rat brain

Divalent cation ATPases were prepared from rat brain synaptic vesicles, synaptosomal plasma membranes, and plasma membranes from the brain stem and sciatic nerve and tested for optimal stimulation by Mn2+, Mg2+, or Ca2+. ATPase in the synaptic vesicle subfraction was optimally stimulated by Mn2+. All plasma membrane preparations were optimally stimulated by Mg2+. Separate Mn2+ and Mg2+ ATPases could not be distinguished by either chemical inactivation or substrate preference criteria. Mn2+ stimulated ATPase in the micromolar range and it is suggested that Mn2+ interaction with ATPase may be of physiological and/or toxicological importance by being related to the cellular metabolism of this element.     

CadA, the Cd2+-ATPase from Listeria monocytogenes, can use Cd2+ as co-substrate

CadA is a membrane protein of the P-type ATPase family which is the major determinant of the resistance to Cd2+ in Listeria monocytogenes. During its catalytic cycle, CadA undergoes auto-phosphorylation from ATP at Asp398, which allows Cd2+ translocation across the membrane. In the reverse mode, Asp398 is phosphorylated from Pi. From the data obtained so far, the CadA catalytic mechanism is similar to that proposed for the sarcoplasmic reticulum Ca2+-ATPase, the model of the P-type ATPase family. We show here that CadA is sensitive to two different ranges of Cd2+ concentration. The 0.1-10 microM range of added CdCl2 corresponds to Cd2+ binding at the transport site of unphosphorylated CadA which induces the reaction of the enzyme with ATP and impairs its reaction with Pi. The 0.1-1 mM range of added CdCl2 could correspond to Cd2+ binding to the transport site accessible from the extracellular medium. In addition, although it is widely accepted that the actual substrate of P-type ATPases is the MgATP complex, we show here that CadA can also perform its cycle in the absence of Mg2+, using CdATP in the place of MgATP at the catalytic site.     

Alpha-synuclein can function as an antioxidant preventing oxidation of unsaturated lipid in vesicles

Alpha-synuclein, a presynaptic protein associated with Parkinson's disease, is found as both soluble cytosolic and membrane-bound forms. Although the function of alpha-synuclein is unknown, several observations suggest that its association with membranes is important. In the present study we investigated the effect of alpha-synuclein on lipid oxidation in membranes containing phospholipids with unsaturated fatty acids. The kinetics of lipid oxidation were monitored by the change in fluorescence intensity of the dye C11-BODIPY. We find that monomeric alpha-synuclein efficiently prevented lipid oxidation, whereas fibrillar alpha-synuclein had no such effect. Our data suggest that the prevention of unsaturated lipid oxidation by alpha-synuclein requires that it bind to the lipid membrane. The antioxidant function of alpha-synuclein is attributed to its facile oxidation via the formation of methionine sulfoxide, as shown by mass spectrometry. These findings suggest that the inhibition of lipid oxidation by alpha-synuclein may be a physiological function of the protein.     

Site binding as a local equilibrium. Mn2+ binding to poly(acrylic acid) as a function of the degree of ionization

Mn2+ binding to poly(acrylic acid) at different degrees of ionization, alpha, has been studied from the frequency dependence of the water protons' relaxation rates T1(-1) and T2(-1). Site binding is treated as an equilibrium with the concentration of free ions at the immediate vicinity (CIV) of the polyion. The CIV is calculated as the solution of the Poisson-Boltzmann equation at the surface of the cylindrical polyion. A single value of K is shown to fit the results at all values of alpha. The amount of site binding is higher than the total amount of condensed divalent counterions predicted for a finite polyion concentration in the presence of monovalent counterions by Manning's theory.     

Na+ and H+ dependent Mn2+ binding to phosphatidylserine vesicles as a test of the Gouy-Chapman-Stern theory

Summary Mn²⁺ binding to phosphatidylserine (PS) vesicles was measured by EPR as a function of [Na⁺] and pH. At nearly physiological monovalent salt concentration the apparent Mn²⁺ affinity (K _a) increased monitonically over the pH range 5.7–8.35, withK _a roughly [H⁺]^–1 above pH 7.3. It was found, moreover, thatK _a fell off more rapidly with added NaCl at pH 6.1 than at pH 7.87. Qualitatively, these results are consistent with two types of Mn²⁺-PS binding: (i) simple adsorption and (ii) adsorption with the release of an amino proton from PS. The existence of Mn²⁺-induced H⁺ displacement from PS was verified through titration measurements, employing a pH electrode.When H⁺ displacement is taken into account, the variation inK _a with [Na⁺] observed at pH 6.1 is found to be in reasonably good agreement with that expected from the Gouy-Chapman-Stern theory of ionic binding to charged surfaces.     

Integrin alphavbeta6 mediates HT29-D4 cell adhesion to MMP-processed fibrinogen in the presence of Mn2+

Mn(2+) was found to induce adhesion of HT29-D4 adenoma carcinoma cells to fibrinogen (Fb). This was independent of the expression of the beta3 integrin subunit and involved endogenous alphavbeta6 but not alphavbeta5 integrin. Thus, addition of Mn(2+) led to a change in integrin alphavbeta6 specificity. Furthermore, Mn(2+) was found to strongly activate the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway in the HT29-D4 cell line. As a MAPK inhibitor strongly reduced the Mn(2+)-induced cell adhesion to Fb, it is suggested that a link between MAPK activation and cell adhesion to Fb exists. Both expression and activity of matrix metalloproteinase-9 (MMP-9) were enhanced by Mn(2+) and this led to Fb processing. MMP inhibitors prevented Mn(2+)-mediated cell adhesion to Fb, leading us to suggest that Mn(2+) promoted convergent changes in integrin alphavbeta6 conformation and Fb structure through activation of ERK/MAPK and MMP-9. Finally, we found that Mn(2+) and activators of the ERK pathway cooperated in HT29-D4 cell adhesion to Fb. Such a process may be involved in bone metastasis of some cancer cells.     

Amino-terminal octapeptides function as recognition signals for the mitochondrial intermediate peptidase.

We have shown previously that cleavage of a number of precursors by the mitochondrial processing peptidase (MPP) requires an intermediate octapeptide (FXXSXXXX) between the MPP cleavage site and the mature protein amino terminus. We show now that these octapeptides, present at the amino termini of the intermediates, direct recognition of these substrates by the mitochondrial intermediate peptidase (MIP), leading to formation of mature proteins. Synthetic peptides, corresponding to the intermediate octapeptides of human ornithine transcarbamylase (OTC) and of Neurospora cytochrome c reductase Fe/S subunit (Fe/S), inhibit the processing activity of purified rat liver MIP in vitro, without affecting MPP activity; this indicates that the octapeptides can be recognized by MIP independent of the presence of the corresponding mature proteins and interact with a site that is crucial for MIP activity. MIP activity is not inhibited by a peptide lacking the amino-terminal hydrophobic residue, while substitution of such a residue by a polar amino acid causes a 10-fold reduction in the efficiency of MIP inhibition. To analyze the requirements for removal of the octapeptide from the intermediate proteins by MIP, artificial intermediates were synthesized and subjected to in vitro processing by purified MIP. The octapeptide can be cleaved by MIP only when the amino-terminal hydrophobic residue is also the amino terminus of the intermediate. Further, when the OTC octapeptide is joined to the mature amino terminus of another twice-cleaved precursor (pFe/S; rat malate dehydrogenase, pMDH), the chimeric intermediate is cleaved by MIP to the corresponding mature-sized protein. When the OTC octapeptide is joined to the mature amino terminus of a once-cleaved precursor (yeast F1-beta-ATPase, pF1-beta), however, this intermediate is not cleaved by MIP; rather, it is processed by MPP to mature-sized F1-beta. Therefore, amino-terminal octapeptides can be cleaved by MIP only within the structural context of twice-cleaved precursors.     

FTIR studies on the bond properties of the aspartyl phosphate moiety of the Ca2+ -ATPase

As part of our work to determine the bond properties of the aspartyl phosphate moiety of the Ca(2+)-ATPase (SERCA1a) phosphoenzymes, we analyzed Morse potentials of the bridging P-O bond as well as C=O bond strengths for the model compound acetyl phosphate and the two phosphoenzyme intermediates Ca(2)E1P and E2P. Reaction-induced infrared difference spectroscopy was used and a carbonyl band of E2P at 1708 cm(-1) in the presence of mM Mg(2+) was tentatively assigned to the carbonyl group of phosphorylated Asp(351) because of its sensitivity to divalent cations. This band is found at 1716 cm(-1) with mM Ca(2+), for Ca(2)E1P at 1717 cm(-1) with Mg(2+), and at 1719 cm(-1) with Ca(2+) and at 1718 cm(-1) for acetyl phosphate in the absence of divalent cations. The similar band positions indicate similar strengths of interaction of the carbonyl oxygen in acetyl phosphate and the two phosphoenzymes. Together with information on the P-O bond strengths, this implies that the bridging oxygen exerts stronger interactions in the phosphoenzymes than in acetyl phosphate.