Lipid composition modulates ATP hydrolysis and calcium phosphate mineral propagation by TNAP-harboring proteoliposomes.

Bone biomineralization is mediated by a special class of extracellular vesicles, named matrix vesicles (MVs), released by osteogenic cells. The MV membrane is enriched in sphingomyelin (SM), cholesterol (Chol) and tissue non-specific alkaline phosphatase (TNAP) compared with the parent cells' plasma membrane. TNAP is an ATP phosphohydrolase bound to cell and MV membranes via a glycosylphosphatidylinositol (GPI) anchor. Previous studies have shown that the lipid microenvironment influences the catalytic activity of enzymes incorporated into lipid bilayers. However, there is a lack of information about how the lipid microenvironment controls the ability of MV membrane-bound enzymes to induce mineral precipitation. Herein, we used TNAP-harboring proteoliposomes made of either pure dimyristoylphosphatidylcholine (DMPC) or DMPC mixed with either Chol, SM or both of them as MV biomimetic systems to evaluate how the composition modulates the lipid microenvironment and, in turn, TNAP incorporation into the lipid bilayer by means of calorimetry. These results were correlated with the proteoliposomes' catalytic activity and ability to induce the precipitation of amorphous calcium phosphate (ACP) in vitro. DMPC:SM proteoliposomes displayed the highest efficiency of mineral propagation, apparent affinity for ATP and substrate hydrolysis efficiency, which correlated with their highest degree of membrane organization (highest ΔH), among the tested proteoliposomes. Results obtained from turbidimetry and Fourier transformed infrared (FTIR) spectroscopy showed that the tested proteoliposomes induced ACP precipitation with the order DMPC:SM>DMPC:Chol:SM≈DMPC:Chol>DMPC which correlated with the lipid organization and the presence of SM in the proteoliposome membrane. Our study arises important insights regarding the physical properties and role of lipid organization in MV-mediated mineralization.

Cholesterol Regulates the Incorporation and Catalytic Activity of Tissue-Nonspecific Alkaline Phosphatase in DPPC Monolayers.

Matrix vesicles (MVs) are a special class of extracellular vesicles that drive bone and dentin mineralization by providing the essential enzymes and ions for the nucleation and propagation of mineral crystals. Tissue-nonspecific alkaline phosphatase (TNAP) is an integral protein of MV membrane and participates in biomineralization by hydrolyzing extracellular pyrophosphate (PPi), a strong mineralization inhibitor, and forming inorganic phosphate (Pi), necessary for the growth of mineral crystals inside MVs and their propagation once released in the extracellular matrix. MV membrane is enriched in cholesterol (CHOL), which influences the incorporation and activity of integral proteins in biologic membranes; however, how CHOL controls the incorporation and activity of TNAP in MV membrane has not yet been elucidated. In the present study, Langmuir monolayers were used as a MV membrane biomimetic model to assess how CHOL affects TNAP incorporation and activity. Surface pressure-area (π-A) isotherms of binary dipalmitoilphosphatidylcholine (DPPC)/CHOL monolayers showed that TNAP incorporation increases with CHOL concentration. Infrared spectroscopy showed that CHOL influences the conformation and orientation of the enzyme. Optical-fluorescence micrographs of the monolayers revealed the tendency of TNAP to incorporate into CHOL-rich microdomains. These data suggest that TNAP penetrates more efficiently and occupies a higher surface area into monolayers with a lower CHOL concentration due to the higher membrane fluidity. However, the quantity of enzyme transferred to solid supports as well as the enzymatic activity were higher using monolayers with a higher CHOL concentration due to increased rigidity that changes the enzyme orientation at the air-solid interface. These data provide new insights regarding the interfacial behavior of TNAP and CHOL in MVs and shed light on the biochemical and biophysical processes occurring in the MV membrane during biomineralization at the molecular level.

Topographical and mechanical properties of liposome surfaces harboring Na,K-ATPase by means of atomic force microscopy.

In this study, we obtained unprecedented AFM images of the Na,K-ATPase (NKA) pump after being reconstituted into DPPC and DPPC:DPPE liposomes. The mechanical properties observed in the phase images were associated with protrusions correlated to NKA microdomains, which are the darker areas seen in the AFM phase images. Protrusions in the DPPC-NKA proteoliposomes ranged from 38 to 115 nm, with 74 ± 21 nm diameter and 2.1 ± 1.4 nm height. DPPC:DPPE-NKA proteoliposomes showed protrusions from 21 to 78 nm, with 38 ± 16 nm diameter and 0.7 ± 0.5 nm height. We have estimated the presence of annular lipids in the microdomains considering that the areas of the protrusions should contain αß oligomers and annular phospholipids. For DPPC-NKA proteoliposomes, we hypothesize that 40 phospholipids surround an (αß)2 dimer and 46 phospholipids are present for the DPPC:DPPE-NKA proteoliposomes in an αß monomer. Catalytic activity measurements of both lipid compositions of proteoliposomes harboring NKA provide strong evidence regarding the protein orientation in the biomembrane. AFM data suggest that DPPC-NKA proteoliposomes are also rightside-out protein orientated, where the protrusions have an average height of 2.1 nm, while for DPPC:DPPE-NKA proteoliposomes, the majority of the protein reconstituted should be inside-out orientated, where the protrusions' average height is 0.5 nm. This result corroborates with the enzymatic analysis, where 61% and 91% of the enzymatic activity was recovered, respectively. Thus, a new application of AFM as a tool for the determination of topological features of protrusions in proteoliposomes has been brought to the scientific community, in addition to revealing the distinct catalytic orientation of enzymes present in the biomembranes model.

A Xanthomonas citri subsp citri hypothetical protein related to virulence contains a non-functional HD domain and is implicated in flagellar motility.

Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp citri (Xac), severely affects most economically important citrus varieties worldwide. A previous study showed that disruption of the ORF XAC1201 from the Xac 306 strain by transposon Tn5 decreased bacterium virulence in the Rangpur lime host (Citrus limonia L. Osbeck). However, little is known regarding the possible function of the hypothetical protein XAC1201 and how it affects the virulence of Xac 306. Here, we confirmed that disruption of ORF XAC1201 reduces Xac 306 virulence in two different hosts, delaying the onset of typical symptoms. In silico analysis suggested that XAC1201 interacts with the flagellar proteins FliM and FliL, known to be an important factor for virulence. In fact, motility assays revealed that the XAC1201 mutant has a significant difference in motility compared to the wild-type Xac 306. Also, a 3-D structure model revealed modified cofactor binding sites and suggested that XAC1201 has a non-functional HD domain. This hypothesis was confirmed by enzymatic assays performed in purified, XAC1201 recombinant protein expressed in Escherichia coli, which revealed no significant activities previously associated with HD domains for the tested substrates. Thus, the role of the XAC1201 protein in Xac 306 virulence seems to be related to flagellar motility, although a non-classic role for the HD domain cannot be dismissed.

Effects of GPI-anchored TNAP on the dynamic structure of model membranes.

Tissue-nonspecific alkaline phosphatase (TNAP) plays a crucial role during skeletal mineralization, and TNAP deficiency leads to the soft bone disease hypophosphatasia. TNAP is anchored to the external surface of the plasma membranes by means of a GPI (glycosylphosphatidylinositol) anchor. Membrane-anchored and solubilized TNAP displays different kinetic properties against physiological substrates, indicating that membrane anchoring influences the enzyme function. Here, we used Electron Spin Resonance (ESR) measurements along with spin labeled phospholipids to probe the possible dynamic changes prompted by the interaction of GPI-anchored TNAP with model membranes. The goal was to systematically analyze the ESR data in terms of line shape changes and of alterations in parameters such as rotational diffusion rates and order parameters obtained from non-linear least-squares simulations of the ESR spectra of probes incorporated into DPPC liposomes and proteoliposomes. Overall, the presence of TNAP increased the dynamics and decreased the ordering in the three distinct regions probed by the spin labeled lipids DOPTC (headgroup), and 5- and 16-PCSL (acyl chains). The largest change was observed for 16-PCSL, thus suggesting that GPI-anchored TNAP can give rise to long reaching modifications that could influence membrane processes halfway through the bilayer.

Disrupting membrane raft domains by alkylphospholipids.

Using phase contrast and fluorescence microscopy we study the influence of the alkylphospholipid, ALP, 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate, ODPC, in giant unilamellar vesicles, GUVs, composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), brain sphingomyelin (SM) and cholesterol (Chol). The results show that adding 100µM ODPC (below CMC) to the outer solution of GUVs promotes DOPC membrane disruption over a period of 1h of continuous observation. On the other hand, the presence of SM and Chol in homogeneous fluid lipid bilayers protects the membrane from disruption. Interestingly, by adding 100µM ODPC to GUVs containing DOPC:SM:Chol (1:1:1), which display liquid ordered (Lo)-liquid disordered (Ld) phase coexistence, the domains rapidly disappear in less than 1min of ODPC contact with the membrane. The lipids are subsequently redistributed to liquid domains within a time course of 14-18min, reflecting that the homogenous phase was not thermodynamically stable, followed by rupture of the GUVs. A similar mechanism of action is also observed for perifosine, although to a larger extent. Therefore, the initial stage of lipid raft disruption by both ODPC and perifosine, and maybe other ALPS, by promoting lipid mixing, may be correlated with their toxicity upon neoplastic cells, since selective (dis)association of essential proteins within lipid raft microdomains must take place in the plasma membrane.

Interaction of 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate with mimetic membranes and cytotoxic effect on leukemic cells.

10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9 microM for 72 h of treatment for inhibition of proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor cells at concentrations up to 25 microM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein endothelial cells (HUVEC) was 63.4 and 60.7 microM, respectively. The critical micellar concentration (CMC) of ODPC was 200 microM. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition temperature (T(c)) of 41.5 degrees C and an enthalpy (H) variation of 7.3 kcal mol(-1). The presence of 25 microM ODPC decreased T(c) and H to 39.3 degrees C and 4.7 kcal mol(-1), respectively. ODPC at 250 microM destabilized the liposomes (36.3 degrees C, 0.46 kcal mol(-1)). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on membrane composition.

Labaditin, a cyclic peptide with rich biotechnological potential: preliminary toxicological studies and structural changes in water and lipid membrane environment.

Cyclic peptides isolated from the plants of the Euphorbiaceae family have been largely studied due to their rigid conformation, which is considered significant for biologic activity. The peptide Labaditin (L(0)) and its open chain analogs (L(1)) were synthesized by the solid-phase peptide synthesis technique (Fmoc/tBu), and purified to elucidate its interaction with membrane models. A shift in λ(max) emission and Stern-Volmer constants values indicate that both tryptophans migrate to a more apolar environment, with L(1) decreasing less than L(0). A circular dichroism (CD) study revealed that L(0) was kept unstructured in aqueous media as much as in the presence of dipalmitoilphosphatidylcholine liposomes. The thermodynamic studies by differential calorimetry (DSC) show a ΔH increase (50 and 18 kcal/mol, for L(0) and L(1), respectively) with peptide concentrations, which is indicative of lipids associating with peptides, resulting in the inability of the lipids to participate in the main transition. Therefore, all CD, DSC, and fluorescence data suggest a greater L(0) membrane insertion. A probable mechanism for Labaditin interaction is based initially on the hydrophobic interaction of the peptide with the lipid membrane, conformational change, peptide adsorption on the lipid surface, and internalization process. Peptide's antibacterial effect was also evaluated and revealed that only L(0) showed reduction in viability in Gram-positive bacteria while no effects to the Gram-negative.

Lipid-mediated growth of SrCO3/CaCO3 hybrid films as bioactive coatings for Ti surfaces.

SrCO3 is frequently used as Sr2+ source in ceramic cements, but its application as bioactive coating for metallic implants has not been explored yet. Aiming at rapid osteointegration and because of the well-known Sr2+ effects on bone metabolism, researchers have sought to design Sr2+-containing biomaterials. In this context, developing simple techniques to prepare Sr2+-based coatings is a must nowadays. Here, we describe the use of a bioinspired lipid-mediated approach to grow SrCO3 hybrid films on Ti surfaces at room temperature. To obtain these coatings, we applied the Langmuir-Blodgett technique to deposit phospholipid films with high degree of organization on Ti. In this way, we expected that controlled SrCO3 crystal growth could be templated by the array of nucleation points arising from electrostatic interaction between Sr2+ and the phospholipid polar heads. To control surface composition and the amount of Sr2+ released from the coatings, we also promoted CaCO3 co-precipitation in the hybrid films. We characterized the hybrid coatings in terms of morphology, chemical structure, wettability, and ability to release Sr2+ upon immersion in biological medium. In vitro osteoblast culture on mixed SrCO3/CaCO3 films revealed that the osteogenic response depended on surface composition, as indicated by alkaline phosphatase activity overexpression, which is an early indicator of osteoblast differentiation. Results showed that the mixed SrCO3/CaCO3 hybrid film created a synergic environment for osteoblasts, and that proper Sr2+ release associated with a Ca2+-rich environment might have optimized the Sr2+ anabolic effect. In conclusion, we have proposed a bioinspired and versatile technique to grow hybrid films that can control surface composition and Sr2+ release. Our results open an opportunity to explore the use of SrCO3-based coatings for rapid metallic implant osteointegration.

Interface-driven Sr-morin complexation at Langmuir monolayers for bioactive coating design.

Flavonoid-metal complexes are widely studied because of their interesting luminescent behavior and biological activity. Despite the extensive exploration of flavonoid-metal coordination processes in solution, the formation of complexes using the flavonoid molecule inserted in a lipid membrane has been little investigated. This effect could provide important insight into the biological activity of flavonoids at lipid membranes and could represent an attractive strategy to design supramolecular structures. Here, we studied the complexation between Sr2+ and morin inserted in an octadecylphosphonic acid (OPA) Langmuir monolayer. This is a relevant system due to the synergism imposed by the association of the Sr2+ ability to control bone formation/resorption with the morin antioxidative effect. Morin incorporation into the OPA monolayers and further Sr2+ complexation were monitored by surface pressure isotherms. Electronic absorption spectroscopy and fluorescence techniques showed Sr-morin complexation both in solution and at the air-liquid interface. Although morin complexation has been described to occur only at basic pH, the specific thermodynamic properties at the air-liquid interface drove metal complexation. LB films were deposited on Ti surfaces, and the resulting OPA/Sr-morin coatings exhibited high surface free energy and increase on its polar component. This optimized surface feature supported further serum protein adsorption and osteoblast growth and differentiation, indicating that these lipid-based coatings are promising for bioactive coating design. This study paves the way for the use of this lipid-based coating in the design of implants for faster osteointegration. Moreover, flavonoid-metal complexation at membranes could also help to shed light on the biological role played by flavonoids.

Recognition of alpha-helix transmembrane domains with an amphipathy scale generated by molecular dynamics using only the primary sequence of proteins.

We recently developed an amphipathy scale, elaborated from molecular dynamics data that can be used for the identification of hydrophobic or hydrophilic regions in proteins. This amphipathy scale reflects side chain/water molecule interaction energies. We have now used this amphipathy scale to find candidates for transmembrane segments, by examining a large sample of membrane proteins with alpha-helix segments. The candidates were selected based on an amphipathy coefficient value range and the minimum number of residues in a segment. We compared our results with the transmembrane segments previously identified in the PDB_TM database by the TMDET algorithm. We expected that the hydrophobic segments would be identified using only the primary structures of the proteins and the amphipathy scale. However, some of these hydrophobic segments may pertain to hydrophobic pockets not included in transmembrane regions. We found that our amphipathy scale could identify alpha-helix transmembrane regions with a probability of success of 76% when all segments were included and 90% when all membrane proteins were included.

Effect of the presence of cholesterol in the interfacial microenvironment on the modulation of the alkaline phosphatase activity during in vitro mineralization.

Mineralization of the skeleton starts within cell-derived matrix vesicles (MVs); then, minerals propagate to the extracellular collagenous matrix. Tissue-nonspecific alkaline phosphatase (TNAP) degrades inorganic pyrophosphate (PPi), a potent inhibitor of mineralization, and contributes Pi (Phosphate) from ATP to initiate mineralization. Compared to the plasma membrane, MVs are rich in Cholesterol (Chol) (∼32%) and TNAP, but how Chol influences TNAP activity remains unclear. We have reconstituted TNAP in liposomes of dipalmitoylphosphatidylcholine (DPPC) or dioleoylphosphatidylcholine (DOPC) combined with Chol or its derivatives Cholestenone (Achol) and Ergosterol (Ergo). DPPC plus 36% sterols in liposome increased the catalytic activity of TNAP toward ATP. The presence of Chol also increased the propagation of minerals by 3.4-fold. The catalytic efficiency of TNAP toward ATP was fourfold lower in DOPC proteoliposomes as compared to DPPC proteoliposomes. DOPC proteoliposomes also increased biomineralization by 2.8-fold as compared to DPPC proteoliposomes. TNAP catalyzed the hydrolysis of ATP more efficiently in the case of the proteoliposome consisting of DOPC with 36% Chol. The same behavior emerged with Achol and Ergo. The organization of the lipid and the structure of the sterol influenced the surface tension (γ), the TNAP phosphohydrolytic activity in the monolayer, and the TNAP catalytic efficiency in the bilayers. Membranes in the Lα phase (Achol) provided better kinetic parameters as compared to membranes in the Lo phase (Chol and Ergo). In conclusion, the physical properties and the lateral organization of lipids in proteoliposomes are crucial to control mineral propagation mediated by TNAP activity during mineralization.

Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation.

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 +/- 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.

Estrogen and phenol red free medium for osteoblast culture: study of the mineralization ability.

To design an estrogen and phenol red free medium for cell culture and check its effectiveness and safety on osteoblast growth it is necessary to maintain the estrogen receptors free for tests. For this purpose, we tested some modifications of the traditional culture media: estrogen depleted fetal bovine serum; estrogen charcoal stripped fetal bovine serum and phenol red free α-MEM. The aim of this work is to examine the effects of its depletion in the proliferation, differentiation, and toxicity of mesenchymal stromal cells differentiated into osteoblasts to obtain an effective interference free culture medium for in vitro studies, focused on non-previously studied estrogen receptors. We performed viability tests using the following techniques: MTT, alkaline phosphatase specific activity, formation of mineralized matrix by Alizarin technique and analysis of SEM/EDX of mineralized nodules. The results showed that the culture media with estrogen free α-MEM + phenol red free α-MEM did not impact viability, alkaline phosphatase activity and mineralization of the osteoblasts culture compared to control. In addition, its nodules possess Ca/P ratio similar to hydroxyapatite nodules on the 14th and 21st day. In conclusion, the modified culture medium with phenol red free α-MEM with estrogen depleted fetal bovine serum can be safely used in experiments where the estrogen receptors need to be free.

Allosteric modulation by ATP, calcium and magnesium ions of rat osseous plate alkaline phosphatase.

Alkaline phosphatase from rat osseous plate is allosterically modulated by ATP, calcium and magnesium at pH 7.5. At pH 9.4, the hydrolysis of ATP and PNPP follows Michaelis-Menten kinetics with K0.5 values of 154 microM and 42 microM, respectively. However, at pH 7.5 both substrates exhibit more complex saturation curves, while only ATP exhibited site-site interactions. Ca(2+)-ATP and Mg(2+)-ATP were effective substrates for the enzyme, while the specific activity of the enzyme for the hydrolysis of ATP at pH 7.5 was 800-900 U/mg and was independent of the ion species. ATP, but not PNPP, was hydrolyzed slowly in the absence of metal ions with a specific activity of 140 U/mg. These data demonstrate that in vitro and at pH 7.5 rat osseous plate alkaline phosphatase is an active calcium or magnesium-activated ATPase.

Kinetic characterization of a membrane-specific ATPase from rat osseous plate and its possible significance on endochodral ossification.

Treatment with phosphatidylinositol-specific phospholipase C of rat osseous plate membranes released up to 90-95% of alkaline phosphatase, but a specific ATPase activity (optimum pH = 7.5) remained bound to the membrane. The hydrolysis of ATP by this ATPase was negligible in the absence of magnesium or calcium ions. However, at millimolar concentrations of magnesium and calcium ions, the membrane-specific ATPase activity increased to about 560-600 U/mg, exhibiting two classes of ATP-hydrolysing sites, and site-site interactions. GTP, UTP, ITP, and CTP were also hydrolyzed by the membrane-specific ATPase. Oligomycin, ouabain, bafilomycin A1, thapsigargin, omeprazole, ethacrynic acid and EDTA slightly affected membrane-specific ATPase activity, while vanadate produced a 18% inhibition. The membrane-specific ATPase activity was insensitive to theophylline, but was inhibited 40% by levamisole. These data suggested that the membrane-specific ATPase activity present in osseous plate membranes, and alkaline phosphatase, were different proteins.

Alkaline phosphatase from rat osseous plates: purification and biochemical characterization of a soluble form.

A soluble form of an alkaline phosphatase obtained from rat osseous plates was purified 204-fold with a yield of 24.3%. The purified enzyme showed a single protein band of Mr 80,000 on SDS-PAGE and an apparent molecular weight of 163,000 by gel filtration on Sephacryl S-300 suggesting a dimeric structure for the soluble enzyme. The specific activity of the enzyme at pH 9.4 in the presence of 2 mM MgCl2 was 19,027 U/mg and the hydrolysis of p-nitrophenyl phosphate (K0.5 = 92 microM) showed positive cooperativity (n = 1.5). The purified enzyme showed a broad substrate specificity, however, ATP, bis(p-nitrophenyl) phosphate and pyrophosphate were among the less hydrolyzed substrates assayed. Surprisingly the enzyme was not stimulated by cobalt and manganese ions, in contrast with a 20-25% stimulation observed for magnesium and calcium ions. Zinc ions exerted a strong inhibition on p-nitrophenylphosphatase activity of the enzyme. This paper provides a simple experimental procedure for the isolation of a soluble form of alkaline phosphatase which is induced by demineralized bone matrix during endochondral ossification.

Interaction of cyclic and linear Labaditin peptides with anionic and zwitterionic micelles.

Conformational changes of the cyclic (Lo) peptide Labaditin (VWTVWGTIAG) and its linear analogue (L1) promoted by presence of anionic sodium dodecyl sulfate (SDS) and zwitterionic L-α-Lysophosphatidylcholine (LPC) micelles were investigated. Results from λ(max) blue-shift of tryptophan fluorescence emission combined with Stern-Volmer constants values and molecular dynamics (MD) simulations indicated that L1 interacts with SDS micelles to a higher extent than does Lo. Further, the MD simulation demonstrated that both Lo and L1 interact similarly with LPC micelles, being preferentially located at the micelle/water interface. The peptide-micelle interaction elicits conformational changes in the peptides. Lo undergoes limited modifications and presents unordered structure in both LPC and SDS micelles. On the other hand, L1 displays a random-coil structure in aqueous medium, pH 7.0, and it acquires a ß-structure upon interaction with SDS and LPC, albeit with structural differences in each medium.

Allosteric modulation of pyrophosphatase activity of rat osseous plate alkaline phosphatase by magnesium ions.

Pyrophosphatase activity of rat osseous plate alkaline phosphatase was studied at different concentrations of calcium and magnesium ions, with the aim of characterizing the modulation of enzyme activity by these metals. In the absence of metal ions, the enzyme hydrolysed pyrophosphate following "Michaelian" kinetics with a specific activity of 36.7 U/mg and K0.5 = 88 microM. In the presence of low concentrations (0.1 mM) of magnesium (or calcium) ions, the enzyme also exhibited "Michaelian" kinetics for the hydrolysis of pyrophosphate, but a significant increase in specific activity (123 U/mg) was observed, K(m) values remained almost unchanged. Quite different behavior occurred in the presence of 2 mM magnesium (or calcium) ions. In addition to low-affinity sites (K0.5-40 and 90 microM, for magnesium and calcium, respectively), high-affinity sites were also observed with K0.5 values 100-fold lower. The high-affinity sites observed in the presence of calcium ions represented about 10% of those observed for magnesium ions. This was correlated with the fact that only magnesium ions triggered conformational changes yielding a fully active enzyme. These results suggested that the enzyme could hydrolyse pyrophosphate, even at physiological concentrations (4 microM), since magnesium concentrations are high enough to trigger conformational changes increasing the enzyme activity. A model, suggesting the involvement of magnesium ions in the hydrolysis of pyrophosphate by rat osseous plate alkaline phosphatase is proposed.

Conidial alkaline phosphatase from Neurospora crassa.

An alkaline phosphatase was purified from conidia of a Neurospora crassa wild type strain. The M(r) of the purified native enzyme was estimated as ca 145,000 and 110,000 by gel filtration, in the presence and absence of magnesium ions, respectively. A single polypeptide band of M(r) 36,000 was detected by SDS-PAGE, suggesting that the native enzyme was a tetramer of apparently identical subunits. Conidial alkaline phosphatase was an acidic protein (pl = 4.0 +/- 0.1), with 40% carbohydrate content. Optimal pH was affected by substrate concentration and magnesium ions. Low concentrations of calcium ions (0.1 mM) had slight stimulatory effects, but in excess (5 mM) caused protein aggregates with decreased activity. The enzyme specificity against different substrates was compared with those reported for constitutive or Pi-repressible alkaline phosphatases produced by N. crassa. The results suggested that the conidial alkaline phosphatase represented a different class among other such enzymes synthesized by this organism.