Remarkable stability of solubilized and delipidated sarcoplasmic reticulum Ca2+-ATPase with tightly bound fluoride and magnesium against detergent-induced denaturation

Conditions were developed in the absence of Ca(2+) for purification, delipidation, and long term stabilization of octaethylene glycol monododecyl ether (C(12)E(8))-solubilized sarcoplasmic reticulum Ca(2+)-ATPase with tightly bound Mg(2+) and F(-), an analog for the phosphoenzyme intermediate without bound Ca(2+). The Ca(2+)-ATPase activity to monitor denaturation was assessed after treatment with 20 mm Ca(2+) to release tightly bound Mg(2+)/F(-). The purification and delipidation was successfully achieved with Reactive Red-agarose affinity chromatography. The solubilized Mg(2+)/F(-)-bound Ca(2+)-ATPase was very rapidly denatured at pH 8, but was perfectly stabilized at pH 6 against denaturation for over 20 days at 4 degrees C even without exogenously added phospholipid and at a high C(12)E(8)/enzyme weight ratio (10:1). The activity was not restored unless the enzyme was treated with 20 mm Ca(2+), showing that tightly bound Mg(2+)/F(-) was not released during the long term incubation. The perfect stability was attained with or without 0.1 mm dithiothreitol, but inactivation occurred with a half-life of 10 days in the presence of 1 mm dithiothreitol, possibly due to reduction of a specific disulfide bond(s). The remarkable stability is likely conferred by intimate gathering of cytoplasmic domains of Ca(2+)-ATPase molecule induced by tight binding of Mg(2+)/F(-). The present study thus reveals an essential property of the Mg(2+)/F(-)/Ca(2+)-ATPase complex, which will likely provide clues to understanding structure of the Ca(2+)-released form of phosphoenzyme intermediate at an atomic level.     

DnaK, hsp73, and their molten globules. Two different ways heat shock proteins respond to heat.

The thermal stability of bovine brain hsp73, Escherichia coli DnaK, and its mutant T199A was studied by a combination of spectroscopic and chromatographic methods. DnaK undergoes a temperature-induced conformational change that leads to the formation of a molten globule at physiologically relevant temperatures (midpoint of the transition, tm, 41 degrees C). Native DnaK binds to a denatured form of alpha-lactalbumin in a temperature-dependent manner with maximum rate at about 40 degrees C. The molten globule of DnaK is unable to bind denatured alpha-lactalbumin but recovers native structure and activity upon cooling. The half-life of the refolding process is 10 min at 35 degrees C. Mg/ATP and Mg/ADP increase the thermal stability of DnaK; in the presence of these nucleotides the tm is shifted to 59 degrees C. Binding of Mg/ATP (but not Mg/ADP or Mg/adenosine 5'-[gamma-thio]triphosphate) causes a conformational change in DnaK as determined by the emission fluorescence spectrum. The DnaK mutant T199A which lacks the threonine residue that is essential for ATP hydrolysis and autophosphorylation activity (McCarty, J. S., and Walker, G. C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9513-9517) shows nearly identical properties to the wild type in the presence or absence of nucleotides. Hsp73 undergoes similar temperature-induced transitions as determined by spectroscopic methods (Palleros, D.R., Welch, W.J., and Fink, A.L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5719-5723); however, contrary to DnaK, the molten globule of hsp73 irreversibly aggregates at temperatures higher than its tm (42 degrees C).     

Thermal stability studies of a globular protein in aqueous poly(ethylene glycol) by (1)H NMR

The reversible folding destabilization of hen lysozyme has been confirmed by a melting temperature (T(m)) decrease in aqueous poly(ethylene glycol) (PEG). The percent denatured, extracted from the histidine 15 C2H (H15 C2H) native and denatured peak areas from 500-MHz one-dimensional proton nuclear magnetic resonance (1D (1)H NMR) spectra in D(2)O, was analyzed through denaturation temperatures at 0% and 20% (w/w) PEG 1000. The lysozyme (3.5 mM) T(m) decreased by 4.2 degrees C and 7.1 degrees C in 20% (w/w) PEG 1000 at pH 3.8 and 3.0, respectively. The T(m) decreased with increasing lysozyme concentration. Additionally, the temperature-induced resonance migrations of 17 protons from 8 residues indicate that the native lysozyme structure undergoes temperature-induced conformational changes. The changes were essentially identical in both 0% and 20% (w/w) PEG 1000 at both pH 3.0 and 3.8. This small, local restructuring of the hydrophobic box region may be a manifestation of temperature-dependent solution hydrophobicity, whereas active-site cleft fluctuations may be due to the inherent active-site flexibility. The lysozyme structure in PEG at 35 degrees C was determined to be essentially native from the (1)H nuclear Overhauser effect spectroscopy (NOESY) fingerprint regions. Additionally, lysozyme chemical shifts, from 1D spectra, in PEG 200, 300, and 1000 at 35 degrees C and various concentrations were essentially identical, further confirming that the conformation remains native in various PEG solutions. (c) 1996 John Wiley & Sons, Inc.     

Neuronal-specific endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration in mixed primary hippocampal culture homogenates

Endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration is crucial for maintenance of neuronal Ca(2+) homeostasis. The use of cell culture in conjunction with modern Ca(2+) imaging techniques has been invaluable in elucidating these mechanisms. While imaging protocols evaluate endoplasmic reticulum Ca(2+) loads, measurement of Mg(2+)/Ca(2+) ATPase activity is indirect, comparing cytosolic Ca(2+) levels in the presence or absence of the Mg(2+)/Ca(2+) ATPase inhibitor thapsigargin. Direct measurement of Mg(2+)/Ca(2+) ATPase by isolation of microsomes is impossible due to the minuscule amounts of protein yielded from cultures used for imaging. In the current study, endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration was measured in mixed homogenates of neurons and glia from primary hippocampal cultures. It was demonstrated that Ca(2+) uptake was mediated by the endoplasmic reticulum Mg(2+)/Ca(2+) ATPase due to its dependence on ATP and Mg(2+), enhancement by oxalate, and inhibition by thapsigargin. It was also shown that neuronal Ca(2+) uptake, mediated by the type 2 sarco(endo)plasmic reticulum Ca(2+) ATPase isoform, could be distinguished from glial Ca(2+) uptake in homogenates composed of neurons and glia. Finally, it was revealed that Ca(2+) uptake was sensitive to incubation on ice, extremely labile in the absence of protease inhibitors, and significantly more stable under storage conditions at -80 degrees C.     

Interaction of the small heat shock protein with molecular mass 25 kDa (hsp25) with actin.

The interaction of heat shock protein with molecular mass 25 kDa (HSP25) and its point mutants S77D + S81D (2D mutant) and S15D + S77D + S81D (3D mutant) with intact and thermally denatured actin was analyzed by means of fluorescence spectroscopy and ultracentrifugation. Wild type HSP25 did not affect the polymerization of intact actin. The HSP25 3D mutant decreased the initial rate without affecting the maximal extent of intact actin polymerization. G-actin heated at 40-45 degrees C was partially denatured, but retained its ability to polymerize. The wild type HSP25 did not affect polymerization of this partially denatured actin. The 3D mutant of HSP25 increased the initial rate of polymerization of partially denatured actin. Heating at more than 55 degrees C induced complete denaturation of G-actin. Completely denatured G-actin cannot polymerize, but it aggregates at increased ionic strength. HSP25 and especially its 2D and 3D mutants effectively prevent salt-induced aggregation of completely denatured actin. It is concluded that the interaction of HSP25 with actin depends on the state of both actin and HSP25. HSP25 predominantly acts as a chaperone and preferentially interacts with thermally unfolded actin, preventing the formation of insoluble aggregates.     

Inhibition of Na+/K(+)-ATPase and Mg(2+)-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na(+)/K(+)-ATPase and Mg(2+)-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu(2+), Zn(2+), Fe(2+) and Co(2+)) and heavy metals (Hg(2+) and Pb(2+)) ions were studied. All investigated metals produced a larger maximum inhibition of Na(+)/K(+)-ATPase than Mg(2+)-ATPase activity. The free concentrations of the key species (inhibitor, MgATP(2-), MeATP(2-)) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu(2+)/Fe(2+) or Hg(2+)/Pb(2+) caused additive inhibition, while Cu(2+)/Zn(2+) or Fe(2+)/Zn(2+) inhibited Na(+)/K(+)-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu(2+)/Fe(2+) or Cu(2+)/Zn(2+) inhibited Mg(2+)-ATPase activity synergistically, while Hg(2+)/Pb(2+) or Fe(2+)/Zn(2+) induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na(+)/K(+)-ATPase activity by reducing the maximum velocities (V(max)) rather than the apparent affinity (Km) for substrate MgATP(2-), implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg(2+)-ATPase activity by Zn(2+), Fe(2+) and Co(2+) as well as kinetic analysis indicated two distinct Mg(2+)-ATPase subtypes activated in the presence of low and high MgATP(2-) concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na(+)/K(+)-ATPase with various potencies. Furthermore, these ligands also reversed Na(+)/K(+)-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg(2+)-induced inhibition was not obtained.     

Nuclear magnetic resonance and calorimetric study of the structure, dynamics, and phase behavior of uranyl ion/dipalmitoylphosphatidylcholine complexes.

The interaction of UO2(2+) with dipalmitoylphosphatidylcholine (DPPC) has been studied as a function of temperature and composition using nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC), and monolayer studies. Computer simulations of the 31P-NMR powder spectra of DPPC dispersions in the presence of various concentrations of UO2(2+) are consistent with the binding stoichiometry of [UO2(2+)]/[DPPC] = 1:4 at [UO2(2+)]/[DPPC] less than 0.3. This complex undergoes a phase transition to the liquid crystalline phase at T'm = 50 +/- 3 degrees C with a breadth delta T'm = 7 +/- 3 degrees C. This broad transition gradually disappears at higher UO2(2+) concentrations, suggesting the presence of yet another UO2(2+)/DPPC complex (or complexes) whose NMR spectra are indistinguishable from those of the 1:4 UO2(2+)/DPPC species. The temperature-dependent 13C powder spectra of 2(1-13C) DPPC dispersions in the presence of 1.2 mol ratio of UO2(2+) show that this higher order complex (complexes) also undergoes a phase transition to the liquid crystalline state at T'm +/- = 58 +/- 3 degrees C with a breadth delta T"m = 15 +/- 5 degrees C. The NMR spectra indicate that exchange among these various UO2(2+)/DPPC complexes is slow. In addition, computer simulations of the 31P-, 13C-, and 2H-NMR powder spectra show that axial diffusion of the DPPC molecules about their long axes is quenched by addition of UO2(2+) and acyl chain isomerization is the dominant motional mode. The isomerization is best described as two-site hopping of the greater than C-D bond at a rate of approximately 10(6) s-1, a motional mode which is expected for a kink diffusion.     

A 2D-IR study of heat- and [(13)C]urea-induced denaturation of sarcoplasmic reticulum Ca(2+)-ATPase

Two-dimensional infrared correlation spectroscopy (2D-IR) was applied to the study of urea- and heat-induced unfolding denaturation of sarcoplasmic reticulum Ca(2+)-ATPase (SR ATPase). Urea at 2-3 M causes reversible loss of SR ATPase activity, while higher concentrations induce irreversible denaturation. Heat-induced denaturation is a non-two-state process, with an "intermediate state" (at t approximately 45 degrees C) characterized by the presence of protein monomers, instead of the native oligomers. 2D-IR reveals that urea denaturation causes loss of the structural transition to the "intermediate state". Whenever the urea effect can be reversed, the transition to the "intermediate state" is re-established.     

Tobacco nectarin I. Purification and characterization as a germin-like, manganese superoxide dismutase implicated in the defensE of floral reproductive tissues

Nectarin I, a protein that accumulates in the nectar of Nicotiana sp. , was determined to contain superoxide dismutase activity by colorimetric and in-gel assays. This activity was found to be remarkably thermostable. Extended incubations at temperatures up to 90 degrees C did not diminish the superoxide dismutase activity of nectarin I. This attribute allowed nectarin I to be purified to homogeneity by heat denaturation of the other nectar proteins. By SDS-polyacrylamide gel electrophoresis, nectarin I appeared as a 29-kDa monomer. If the protein sample was not boiled prior to loading the gel, then nectarin I migrated as 165-kDa oligomeric protein. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the protomer subunit was found to be a 22.5-kDa protein. Purified nectarin I contained 0.5 atoms of manganese/monomer, and the superoxide dismutase activity of nectarin I was not inhibited by either H(2)O(2) or NaCN. Following denaturation, the superoxide dismutase activity was restored after Mn(2+) addition. Addition of Fe(2+), Cu(2+), Zn(2+), and Cu(2+)/Zn(2+) did not restore superoxide dismutase activity. The quaternary structure of the reconstituted enzyme was examined, and only tetrameric and pentameric aggregates were enzymatically active. The reconstituted enzyme was also shown to generate H(2)O(2). Putative nectarin I homologues were found in the nectars of several other plant species.     

Reconstitution of the purified (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B membranes into lipid vesicles and a characterization of the resulting proteoliposomes

The purified (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B membranes was reconstituted with dimyristoylphosphatidylcholine using a cholate solubilization and dialysis procedure. The incorporation of this enzyme into the phospholipid bilayer is accompanied by an enhancement of its specific activity and by a restoration of its lipid phase state-dependent properties which were lost during solubilization and purification from native membranes. Moreover, reconstitution of this ATPase with phospholipid also stabilizes it against cold inactivation at low temperatures (approximately equal to 0 degrees C), oxidative degradation at room temperature, and thermal denaturation at elevated temperatures (approximately equal to 55 degrees C). The elution profile from a Sepharose 4B-CL column indicates that all of the ATPase protein is associated with the phospholipid vesicles and that the Stoke's radius of the proteoliposomes formed is smaller than that of the lipid vesicles formed in the absence of any protein. The latter conclusion is supported by sedimentation velocity measurements and by an electron microscopic examination of negatively stained preparations. The electron microscopic studies demonstrate that sealed vesicles are formed only at low protein-to-lipid ratios. These observations indicate that the Acholeplasma laidlawii B (Na+ + Mg2+)-ATPase has been structurally and functionally reconstituted into lipid vesicles and that the proteoliposomes formed are amenable to studies aimed at the clarification of its proposed role as a sodium ion pump.     

Co2+, Cu2+, and Zn2+ accumulation by cyanobacterium Spirulina platensis

The Spirulina platensis biomass was characterized for its metal accumulation as a function of pH, external metal concentration, equilibrium isotherms, kinetics, effect of co-ions under free (living cells, lyophilized, and oven-dried) and immobilized (Ca-alginate and polyacrylamide gel) conditions. The maximum metal biosorption by S. platensis biomass was observed at pH 6.0 with free and immobilized biomass. The studies on equilibrium isotherm experiments showed highest maximum metal loading by living cells (181.0 +/- 13.1 mg Co(2+)/g, 272.1 +/- 29.4 mg Cu(2+)/g and 250.3 +/- 26.4 mg Zn(2+)/g) followed by lyophilized (79.7 +/- 9.6 mg Co(2+)/g, 250.0 +/- 22.4 mg Cu(2+)/g and 111.2 +/- 9.8 mg Zn(2+)/g) and oven-dried (25.9 +/- 1.9 mg Co(2+)/g, 160.0 +/- 14.2 mg Cu(2+)/g and 35.1 +/- 2.7 mg Zn(2+)/g) biomass of S. platensis on a dry weight basis. The polyacrylamide gel (PAG) immobilization of lyophilized biomass found to be superior over Ca-alginate (Ca-Alg) and did not interfere with the S. platensis biomass biosorption capacity, yielding 25% of metal loading after PAG entrapment. The time-dependent metal biosorption in both the free and immobilized form revealed existence of two phases involving an initial rapid phase (which lasted for 1-2 min) contributing 63-77% of total biosorption, followed by a slower phase that continued for 2 h. The metal elution studies conducted using various reagents showed more than 90% elution with mineral acids, calcium salts, and Na(2)EDTA with free (lyophilized or oven-dried) as well as immobilized biomass. The experiments conducted to examine the suitability of PAG-immobilized S. platensis biomass over multiple cycles of Co(2+), Cu(2+), and Zn(2+) sorption and elution showed that the same PAG cubes can be reused for at least seven cycles with high efficiency.     

Wheat germ phosphoglycerate mutase: evidence for a metalloenzyme

Wheat germ phosphoglycerate mutase, exposed to 3.4 M guanidinium chloride at 22 degrees C and pH 7.8, slowly undergoes time-dependent inactivation which can be fully reversed by adding excess Co2+ or Mn2+ to a 50-fold dilution of the denaturing medium. Titration of the denatured enzyme with either Co2+ or Mn2+ shows that wheat germ mutase preferentially binds Co2+. Assuming 1:1 complexation between metal atom and protein, the apparent dissociation constants (Kd) for E Co2+ and E Mn2+ at 22 degrees C and pH 8.7 are approximately 1.06 and 1.84, respectively. Other metal atoms (e.g., Cr2+, Cu2+, Fe2+, Fe3+, Mg2+, and Ni2+) have no effect in restoring the apoenzyme's catalytic activity. At low concentrations (0.11-0.23 mM) Zn2+ partially restores activity, but promotes protein precipitation at elevated concentrations. Evidence suggests that all bisphosphoglycerate-independent phosphoglycerate mutases require either an intra- or an extramolecular metal atom in order to function. Attempts to characterize wheat germ mutase as a glycoprotein have yielded negative results.     

Effect of pH on stability of anthrax lethal factor: correlation between denaturation and activity

Anthrax is caused by Gram positive bacterium Bacillus anthracis. Pathogenesis is result of production of three protein components, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA in combination with LF (lethal toxin) is lethal to animals, while PA in combination with EF (edema toxin), causes edema. PA, LF, and EF are very thermolabile. Differential scanning calorimetry (DSC) was used to unravel the energetics of LF denaturation as a function of pH ranging from 7.8 to 5.5. Transition temperature (T(m)) of LF was found to be approximately equal to 42 degrees C and onset of denaturation occurs at approximately equal to 30 degrees C. The ratio of calorimetric to van't Hoff's enthalpy was nearly equal to unity at pH 7.0, indicative of presence of single structural domain in LF at pH 7.0, unlike PA which has been structurally observed to consist of 4 domains. It was found by cytotoxicity studies using J774A.1 macrophage like cells that LF was most stable at pH approximately 6.5. This paper reports for the first time the denaturation of LF at different pH values at 37 degrees C and tries to establish a correlation between denaturation and loss of LF activity at different pH values.     

pH and ligand binding modulate the strength of protein-protein interactions in the Ca(2+)-ATPase from sarcoplasmic reticulum membranes.

The Ca(2+)-ATPase from sarcoplasmic reticulum (SR) membranes couples the Ca(2+) transport to ATP hydrolysis through phosphorylation in its cytoplasmic catalytic domain. Interactions between protein domains and the role of monomer-monomer interactions remain unclear. Here, we report a differential scanning calorimetric study of the thermal unfolding of this protein. In the pH range 6-8, thermal unfolding of the Ca(2+)-ATPase in glycogen phosphorylase-free SR membranes shows a major endothermic peak with a critical temperature midpoint ranging between 51 and 55 degrees C, depending on pH, Ca(2+), Mg(2+)-ADP and KCl concentrations. The enthalpy change of the overall unfolding process ranged between 250 and 300 kcal/mol of Ca(2+)-ATPase monomer. Thermal denaturation of the Ca(2+)-ATPase in SR membranes is well fitted to an irreversible process that can be rationalized in terms of a non-two state process, N (native)right harpoon over left harpoon I (intermediate)-->D (denatured). Thermodynamic analysis show that this protein has a compact structure, implying a tight structural interconnection between catalytic and Ca(2+) transport domains. The apparent cooperative unit, defined by the van 't Hoff enthalpy to the overall unfolding enthalpy ratio, increased from 1.1 at pH 6 to 1.8 at pH 8, showing that monomer-monomer interactions are stronger at weakly basic pH than at weakly acidic pH. While micromolar Ca(2+) concentrations had only a weak effect on the cooperativity of the unfolding process, this is clearly increased by millimolar Mg(2+)-ADP. In addition, high ionic strength lowered the apparent cooperative unit to approximately 1.0 in the pH range 6-8. Taken together, these results suggest that protein-protein interactions are altered by variables that modulate the catalytic activity of this enzyme.     

I. Study of protein aggregation due to heat denaturation: A structural approach using circular dichroism spectroscopy, nuclear magnetic resonance, and static light scattering

The objective of this study was to investigate the relationship between oxidized RNase A protein structure and the occurrence of protein aggregation using several spectroscopic techniques. Circular dichroism spectroscopy (CD) measurements taken at small temperature intervals were used to determine the protein's melting temperature, Tm, of approximately 65 degrees C in deionized water. A more detailed examination of the protein structure was undertaken at several temperatures around Tm using near- and far-UV CD and one-dimensional nuclear magnetic resonance (NMR) measurements. These measurements revealed the presence of folded structures at 55 degrees C and below, while denatured structures appeared at 65 degrees C and above. Concurrent static light scattering (SLS) measurements, employed to detect the presence of RNase A aggregates, showed that RNase A aggregation was observed at 65 degrees C and above, when much of the protein was denatured. Subsequent NMR time-course data demonstrated that aggregates forming at 75 degrees C and pH 7.8 were indeed derived from heat-denatured protein. However, aggregation was also detected at 55 degrees C when the spectroscopic data suggested the protein was present predominantly in the folded configuration. In contrast, heat denaturation did not lead to RNase A aggregation in a very acidic environment. We attribute this phenomenon to the effect of charge-charge repulsion between the highly protonated RNase A molecules in very acidic pH.     

Pressure effect on the temperature-induced unfolding and tendency to aggregate of myoglobin

This work demonstrates that pressure-induced partially unfolded states play a very important role in the aggregation of proteins. The high-pressure unfolding of horse heart metmyoglobin results in an intermediate form that shows a strong tendency to aggregate after pressure release. These aggregates are similar to those that are usually observed upon temperature denaturation. Infrared spectra in the amide I region indicate the formation of an intermolecular antiparallel beta-sheet stabilized by hydrogen bonding. The formation of the aggregates is temperature-dependent. Below 30 degrees C, no aggregation is taking place as seen from the infrared spectra. At 45 and 60 degrees C, two types of aggregates are formed: one that can be dissociated by moderate pressures and one that is pressure-insensitive. When precompressed at 5 degrees C, temperature-induced aggregation takes place at lower temperature (38 degrees C) than without pressure pretreatment (74 degrees C).     

Microcalorimetric study of iodized and noniodized cells and C-phycocyanin of Spirulina platensis

It was shown that eight stages of transition are observed in the heating process of Spirulina platensis cells in temperature range 5-140 degrees C. The first stage covers the temperature range 5-53 degrees C with maximum approximately 45 degrees C. The heat evolved in this temperature range is equal to 380 +/- 20 J/g of dry biomass, it does not change at scanning rate lower than 0.083 degrees C/min and belongs, mainly, to cell respiration in a stationary regime, in the dark. It was shown that endotherm approximately 66 degrees C belongs to denaturation of C-phycocyanin which denaturates in solutions with Td = 64.2 degrees C, deltaHd = 34.7 +/- 2.1 J/g and for it deltaHd(cal)/deltaH(V.H) is equal to 10.8 +/- 1.2. The endotherms with Td equal to 58 and 88 degrees C are connected with denaturation of phycobilisome proteins and endotherm with Td = 48 degrees C and deltaHd = 4.2J/g of dry biomass-with denaturation of protein which, apparently, is connected with cell respiration.     

The 42-amino acid insert in the FMN domain of neuronal nitric-oxide synthase exerts control over Ca(2+)/calmodulin-dependent electron transfer.

The neuronal and endothelial nitric-oxide synthases (nNOS and eNOS) differ from inducible NOS in their dependence on the intracellular Ca(2+) concentration. Both nNOS and eNOS are activated by the reversible binding of calmodulin (CaM) in the presence of Ca(2+), whereas inducible NOS binds CaM irreversibly. One major divergence in the close sequence similarity between the NOS isoforms is a 40-50-amino acid insert in the middle of the FMN-binding domains of nNOS and eNOS. It has previously been proposed that this insert forms an autoinhibitory domain designed to destabilize CaM binding and increase its Ca(2+) dependence. To examine the importance of the insert we constructed two deletion mutants designed to remove the bulk of it from nNOS. Both mutants (Delta40 and Delta42) retained maximal NO synthesis activity at lower concentrations of free Ca(2+) than the wild type enzyme. They were also found to retain 30% of their activity in the absence of Ca(2+)/CaM, indicating that the insert plays an important role in disabling the enzyme when the physiological Ca(2+) concentration is low. Reduction of nNOS heme by NADPH under rigorous anaerobic conditions was found to occur in the wild type enzyme only in the presence of Ca(2+)/CaM. However, reduction of heme in the Delta40 mutant occurred spontaneously on addition of NADPH in the absence of Ca(2+)/CaM. This suggests that the insert regulates activity by inhibiting electron transfer from FMN to heme in the absence of Ca(2+)/CaM and by destabilizing CaM binding at low Ca(2+) concentrations, consistent with its role as an autoinhibitory domain.     

Methanococcus jannaschii ORF mj0608 codes for a class C inorganic pyrophosphatase protected by Co(2+) or Mn(2+) ions against fluoride inhibition

Openreading frame mj0608 of the Methanococcus jannaschii genome, recognized by its sequence similarity to that of the gene coding for class C inorganic pyrophosphatase in Bacillus subtilis, was cloned and over-expressed in Escherichia coli. The protein was purified and characterized by SDS-PAGE, M(r), and N-terminal sequence. Under suitable conditions it catalyzed the specific hydrolysis of PPi at about 600 micromol x min(-1) x mg(-1) at 25 degrees C, and at 8000 micromol x min(-1) x mg(-1) at 85 degrees C. Therefore this protein is a specific inorganic pyrophosphatase. The activities of Mg(2+), Mn(2+), Co(2+), and Zn(2+) ions as cofactors for hydrolysis of PPi were compared at pH 7.5 and 9.0. Unlike the class C pyrophosphatase of B. subtilis, this enzyme required no prior activation by low concentrations of Mn(2+) or Co(2+) ions. However, prior exposure to these ions afforded striking protection against inhibition by sodium fluoride, to which the enzyme was otherwise very sensitive.     

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.