Rat Osseous Plate Alkaline Phosphatase Research Articles

Rat osseous plate alkaline phosphatase as Langmuir monolayer—An infrared study at the air–water interface

A glycosylphosphatidylinositol (GPI)-anchored enzyme (rat osseous plate alkaline phosphatase—OAP) was studied as monolayer (pure and mixed with lipids) at the air–water interface. Surface pressure and surface potential–area isotherms showed that the enzyme forms a stable monolayer and exhibits a liquid-expanded state even at surface pressure as high as 30 mN m −1. Isotherms for mixed dimyristoylphosphatidic acid (DMPA)–OAP monolayer showed the absence of a liquid-expanded/liquid-condensed phase transition as observed for pure DMPA monolayer. In both cases, pure or mixed monolayer, the enzyme preserves its native conformation under compression at the air–water interface as observed from in situ p-polarized light Fourier transform-infrared reflection–absorption spectroscopic (FT-IRRAS) measurements. Changes in orientation and conformation of the enzyme due to the presence or absence of DMPA, as well as due to the surface compression, are discussed.

Influence of the glycosylphosphatidylinositol anchor in the morphology and roughness of Langmuir–Blodgett films of phospholipids containing alkaline phosphatases

The morphology of phospholipid Langmuir–Blodgett films containing a glycosylphosphatidylinositol-anchored rat osseous plate alkaline phosphatase was studied by atomic force microscopy. Two forms of the enzyme were used in this study: an anchor-containing detergent-solubilized form, and a phospholipase-solubilized form, without the hydrophobic moiety. Direct measurements of catalytic activity in enzyme/phospholipid mixed films, and nanogravimetric estimations by quartz crystal microbalance confirmed the adsorption of both forms of alkaline phosphatase. Atomic force microscopy analysis showed that both enzyme forms originated aggregates when adsorbed on phospholipid Langmuir–Blodgett films. However, the phospholipase-solubilized form showed a higher roughness when compared to the detergent solubilized alkaline phosphatase. A model suggesting different alignments of the major axis of the ellipsoid polypeptide moiety relative to the film surface is proposed. This model, supported by surface density and catalytic activity data, might explain the difference of roughness observed for films containing each enzyme form adsorbed onto a phospholipid-modified solid support.

Adsorption kinetics and dilatational rheological studies for the soluble and anchored forms of alkaline phosphatase at the air/water interface

This work presents equilibrium and dynamic aspects for the adsorption at the air/liquid interface of two rat osseous plate alkaline phosphatase forms: DSAP, solubilized by a surfactant, C12E9, and containing a glycosylphosphatidylinositol (GPI) anchor; and PLSAP, resulting from phospholipase-C cleavage of the hydrophobic portion of the GPI anchor. Dynamic surface tension, gammadyn, and surface elasticity modulus, epsilon, were determined for PLSAP, DSAP and pure C12E9 solutions using harmonic oscillation and axisymmetric drop shape analysis Adsorption kinetics studies revealed that DSAP adsorbs thirty times faster than PLSAP, presenting a minimum in the curve. For DSAP/ C12E9 mixed system, e increases with concentration and a maximum appears at the critical aggregation concentration (CAC). For PLSAP, a continuous decreasing with concentration for gammadyn and epsilon was observed. For pure C12E9 solution, the elasticity modulus increases with concentration and epsilon values are higher when compared to the mixed system. A model based on the influence of the GPI anchor is proposed.

Surface density as a significant parameter for the enzymatic activity of two forms of alkaline phosphatase immobilized on phospholipid Langmuir–Blodgett films

Rat osseous plate alkaline phosphatase, a glycosylphosphatidylinositol (GPI)-anchored phosphomonohydrolase, was immobilized on Langmuir–Blodgett (LB) films. Enzyme solubilization either with polyoxyethylene-9-lauryl ether or with a glycosylphosphatidylinositol-specific phospholipase C resulted in a GPI-anchor-containing and a GPI-anchor-depleted form, respectively. Both forms were adsorbed on dimyristoylphosphatidic acid LB films and restricted to the outermost layer. The surface density and enzyme activity were determined using a quartz crystal microbalance and p-nitrophenylphosphatase activity, respectively. The detergent-solubilized form was co-spread with dimyristoylphosphatidic acid on the air/water interface and transferred to solid supports, providing an enzyme maximum surface density of 530 ng/cm 2. Maximal phosphohydrolytic activity, corresponding to 43% of that observed in homogeneous medium, was obtained at a surface density of 179 ng/cm 2. The phospholipase C-solubilized form was adsorbed directly from solution, reaching a maximum surface density of 1541 ng/cm 2, although the phosphomonohydrolase activity was 10 times lower than that obtained for the anchor-containing form. The combined analysis of surface density and enzymatic activity suggests that the alignment of the protein molecules on the LB lipid films induced by the glycosylphosphatidylinositol anchor facilitates the access of the substrate to the active site. This access is hampered by increasing enzyme surface densities and depends on a specific orientation of the adsorbed enzyme.

Adsorption of detergent-solubilized and phospholipase C-solubilized alkaline phosphatase at air/liquid interfaces

To investigate the influence of a hydrophobic anchor on protein adsorption, equilibrium and dynamic aspects of the adsorption of two different solubilized forms of rat osseous plate alkaline phosphatase on Langmuir monolayers of dimyristoylphosphatidic acid (DMPA) were studied. Surface pressure and surface potential measurements at air/liquid interfaces were carried out using the detergent-solubilized form (DSAP) of alkaline phosphatase, which holds a glycosylphosphatidylinositol (GPI) hydrophobic anchor, and the glycosylphosphatidylinositol-specific phospholipase C-solubilized form (PLSAP), lacking the GPI anchor. Similar surface transitions observed for both DMPA and DMPA/PLSAP mixed monolayers indicate that the presence of PLSAP does not promote significant changes in surface packing of the DMPA monolayer. However, PLSAP interacts with the polar portion of the phospholipid even at high lateral compression. The presence of the GPI anchor increases the adsorption of DSAP at a plain air/liquid interface and also enables the penetration of the protein into the DMPA monolayers. The penetration is dependent on both time and surface pressure. Up to 20 mN/m, the surface pressure increases smoothly indicating a diffusion followed by an adsorption process. Above 20 mN/m, after a fast increase, the surface pressure slowly decays to equilibrium values quite close to the initial surface pressures. The results indicate that the molecular packing of the lipid layer drives the enzyme adsorption to the interface either through the GPI anchor or by the polypeptide moiety.

Rat osseous plate alkaline phosphatase: effect of neutral protease digestion on the hydrolysis of pyrophosphate and nitrophenylphosphate.

Collagenase treatment, commonly used to prepare alkaline phosphatase-rich matrix vesicles from epiphyseal cartilage growth plates, seems to affect the integrity of this membrane-bound enzyme. Alkaline phosphatase-rich rat osseous plates were incubated with 1,000 U/mL collagenase for 3 h, at 37 degrees C and after purification on Sepharose 4B, kinetic studies were performed using nitrophenylphosphate and pyrophosphate as substrates. The optimum apparent pH for the hydrolysis of p-nitrophenylphosphate and pyrophosphate increased from 9.4 to 10.25 and from 8.0 to 9.0, respectively, as a consequence ofcollagenase treatment. In the absence of Mg2+ ions, the enzyme hydrolyzed PNPP with KM = 322.5 +/- 15.3 microM and V = 965.2 +/- 45.8 U/mg, while in the presence of 2 mM Mg2+ ions, V increased 66%. Cobalt (K0.5 = 5.3 +/- 0.3 microM) and manganese (K0.5 = 0.72 +/- 0.03 microM) ions stimulated the PNPPase activity of the collagenase-treated enzyme, but with a lower apparent affinity when compared with that of not-treated enzyme. In the absence of Mg2+ ions pyrophosphate was hydrolyzed according to Michaelis-Menten kinetics (KM = 105.1 +/- 6.3 microM and V = 64.9 +/- 3.9 U/mg), but site-site interactions (nH = 1.2) were observed in the presence of 2 mM Mg2+ ions (V = 110.8 +/- 5.5 U/mg; K0.5 = 42.7 +/- 2.0 microM). To our knowledge this is the first report showing significant alterations on phosphohydrolytic activity and metal binding properties of bone alkaline phosphatase due to associated neutral proteases in collagenase preparations often used for the isolation of matrix vesicles.

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 K 0.5=88 μM. 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 ( K 0.5=40 and 90 μM, for magnesium and calcium, respectively), high-affinity sites were also observed with K 0.5 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 μM), 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.

Effect of calcium ions on rat osseous plate alkaline phosphatase activity

Rat osseous plate alkaline phosphatase is a metalloenzyme with two binding sites for Zn 2+ (sites I and III) and one for Mg 2+ (site II). This enzyme is stimulated synergistically by Zn 2+ and Mg 2+ (Ciancaglini et al., 1992) and also by Mn 2+ (Leone et al., 1995) and Co 2+ (Ciancaglini et al., 1995). This study was aimed to investigate the modulation of enzyme activity by Ca 2+. In the absence of Zn 2+ and Mg 2+, Ca 2+ had no effects on the activity of Chelex-treated, Polidocanol-solubilized enzyme. However, in the presence of 10 μM MgCl 2, increasing concentration of Ca 2+ were inhibitory, suggesting the displacement of Mg 2+ from the magnesium-reconstituted enzyme. For calcium-reconstituted enzyme, Zn 2+ concentrations up to 0.1 μM were stimulatory, increasing specific activity from 130 U/mg to about 240 U/mg with a K 0.5 = 8.5 nM. Above 0.1 μM Zn 2+ exerted a strong inhibotory effect and concentrations of Ca 2+ up to 1 mM were not enough to counteract this inhibition, indicating that Ca 2+ was easily displaced by Zn 2+. At fixed concentrations of Ca 2+, increasing concentrations of Mg 2+ increased the enzyme specific activity from 472 U/mg to about 547 U/mg, but K 0.5 values were significantly affected (from 4.4 μM to 38.0 μM). The synergistic effects observed for the activity of Ca 2+ plus magnesium-reconstituted enzyme, suggested that these two ions bind to the different sites. A model to explain the effect of Ca 2+ on the activity of the enzyme is presented.

Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification.

Alkaline phosphatase activity was released up to 100% from the membrane by incubating the rat osseous plate membrane-bound enzyme with phosphatidylinositol-specific phospholipase C. The molecular weight of the released enzyme was 145,000 on Sephacryl S-300 gel filtration and 66,000 on PAGE-SDS, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyse PNPP, ATP and pyrophosphate. The hydrolysis of ATP and PNPP by phosphatidylinositol-specific phospholipase C-released enzyme exhibited 'Michaelian' kinetics with K0.5 = 70 and 979 microM, respectively. For pyrophosphate, K0.5 was 128 microM and site-site interactions were observed (n = 1.4). Magnesium ions were stimulatory (K0.5 = 1.5 mM) and zinc ions were a powerful noncompetitive inhibitor (Ki = 6.2 microM) of phosphatidylinositol-specific phospholipase C-released enzyme. Phosphatidylinositol-specific phospholipase C-released alkaline phosphatase was relatively stable at 40 degrees C. However, with increasing temperature from 40-60 degrees C, the enzyme was inactivated rapidly following first order kinetics and thermal inactivation constants varied from 5.08 x 10(-4) min-1 to 0.684 min-1. Treatment of phosphatydilinositol-specific phospholipase C-released alkaline phosphatase with Chellex 100 depleted to 5% its original PNPPase activity. Magnesium (K0.5 = 29.5 microM), manganese (K0.5 = 5 microM) and cobalt ions (K0.5 = 10.1 microM) restored the activity of Chelex-treated enzyme, demonstrating its metalloenzyme nature. The stimulation of Chelex-treated enzyme by calcium ions (K0.5 = 653 microM) was less effective (only 26%) and occurred with site-site interactions (n = 0.7). Zinc ions had no stimulatory effects. The possibility that the soluble form of the enzyme, detected during endochondral ossification, would arise by the hydrolysis of the Pl-anchored form of osseous plate alkaline phosphatase is discussed.

Mechanism of action of cobalt ions on rat osseous plate alkaline phosphatase

Polidocanol-solubilized osseous plate alkaline phosphatase was modulated by cobalt ions in a similar way as by magnesium ions. For concentrations up to 1 μM, the Chelex-treated enzyme was stimulated by cobalt ions, showing K d = 6.0 μM, V = 977.5 U/mg, and site-site interactions ( n = 2.5). Cobalt-enzyme was highly unstable at 37°C, following a biphasic inactivation process with inactivation constants of about 0.0625 and 0.0015 min−1. Cobalt ions stimulated the enzyme synergistically in the presence of magnesium ions ( K d = 5.0 μM; V = 883.0 U/mg) or in the presence of zinc ions ( K d =75.0 μM; V= 1102 U/mg). A steady-state kinetic model for the modulation of enzyme activity by cobalt ions is proposed.

Rat osseous plate alkaline phosphatase: mechanism of action of manganese ions.

Polidocanol-solubilized osseous plate alkaline phosphatase was modulated by manganese ions in a similar way as by zinc ions. For concentrations up to 1.0 nM, the enzyme was stimulated by manganese ions, showing site-site interactions (n = 2.2). However, larger concentrations (> 0.1 microns) were inhibitory. Manganese ions could play the role of zinc ions stimulating the enzyme synergistically in the presence of magnesium ions (Kd = 7.2 microns; V = 1005.5 U mg-1). Manganese ions could also play the role of magnesium ions, stimulating the enzyme synergistically in the presence of zinc ions (Kd = 2.2 microns; V = 1036.7 U mg-1). However, manganese ions could not substitute for zinc and magnesium at the same time since ion assymetry is necessary for full activity of the enzyme. A steady-state kinetic model for the modulation of enzyme activity by manganese ions is proposed.

Phosphodiesterase activity is a novel property of alkaline phosphatase from osseous plate

Phosphodiesterase activity is a novel property of the still-enigmatic alkaline phosphatase from osseous plate. Bis-(p-nitrophenyl) phosphate was hydrolysed at both pH 7.5 and 9.4 with an apparent dissociation constant (K0.5) of 1.9 mM and 3.9 mM respectively. The hydrolysis of p-nitrophenyl-5'-thymidine phosphate followed hyberbolic kinetics with a K0.5 of 500 microM. For p-nitrophenyl phenylphosphonate, site-site interactions [Hill coefficient (h) = 1.3] were observed in the range between 0.2 and 100 microM, and K0.5 was 32.8 mM. The hydrolysis of cyclic AMP by the enzyme followed more complex kinetics, showing site-site interactions (h = 1.7) and K0.5 = 300 microM for high-affinity sites. The low-affinity sites, representing 85% of total activity, also showed site-site interactions (h = 3.8) and a K0.5 of about 22 mM. ATP and cyclic AMP were competitive inhibitors of bis-(p-nitrophenyl) phosphatase activity of the enzyme and Ki values (25 mM and 0.6 mM for cyclic AMP and ATP respectively) very close to those of the K0.5 (22 mM and 0.7 mM for cyclic AMP and ATP respectively), determined by direct assay, indicated that a single catalytic site was responsible for the hydrolysis of both substrates. Non-denaturing PAGE of detergent-solubilized enzyme showed coincident bands on the gel for phosphomonohydrolase and phosphodiesterase activities. Additional evidence for a single catalytic site was the similar pKa values (8.5 and 9.7) found for the two ionizing groups participating in the hydrolysis of bis-(p-nitrophenyl) phosphate and p-nitrophenyl phosphate. The alkaline apparent pH optima, the requirement for bivalent metal ions and the inhibition by methylxanthines, amrinone and amiloride demonstrated that rat osseous-plate alkaline phosphatase was a type I phosphodiesterase. Considering that there is still confusion as to which is the physiological substrate for the enzyme, the present results describing a novel property for this enzyme could be of relevance in understanding the mineralization process.

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 K 0.5 values of 154 μM and 42 μM, 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.

Phosphotransferase activity associated with rat osseous plate alkaline phosphatase: a possible role in biomineralization

1. 1. Alkaline phosphatase from rat osseous plate catalyzed the transfer of phosphate from p-nitrophenylphosphate to glycerol, ethanolamines. Tris, glucose and l-amino-l-methyl-2-propanol, in a wide range of pH. Serine did not stimulate phosphotransferase activity of the enzyme. 2. 2. The best phosphotransferase acceptors were diethanolamine and glycerol while glucose was the poorest phosphotransferase acceptor used. 3. 3. Diethanolamine and glycerol affected both V M and K M of p-nitrophenylphosphate hydrolysis with activation constants ( K A) of 0.25 and 0.85 M, respectively. 4. 4. A kinetic model was proposed for the phosphotransferase reaction observed with alkaline phosphatase from rat osseous plates.

Effect of ph on the modulation of rat osseous plate alkaline phosphatase by metal ions

1. 1. Metal ions other than zinc and magnesium were effective in modulating the activity of rat osseous plate alkaline phosphatase. 2. 2. Increasing pH had remarkable effects on the modulation of rat osseous plate alkaline phosphatase. 3. 3. The modulation of enzyme activity by zinc, manganese and cobalt ions was slightly affected by pH variations. 4. 4. Zinc ions were stimulatory for the enzyme at very low concentrations (50 nM). Above 50 nM zinc ions inhibited the enzyme by displacing magnesium ions. 5. 5. Calcium ions were inhibitors of alkaline phosphatase ( K d = 10 μM) whereas manganese ( K d = 1.3 μM) and cobalt ( K d = 0.2 μM) ions were stimulatory in the pH range 8.0–10.0.