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F A Leone

Publications and source records attributed to F A Leone.

At least 37 records · Page 2Linked to original sources
PubMed1995

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.

Alkaline Phosphatase↗
PubMed1995

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 microM, the Chelex-treated enzyme was stimulated by cobalt ions, showing Kd = 6.0 microM, V = 977.5 U/mg, and site-site interactions (n = 2.5). Cobalt-enzyme was highly unstable at 37 degrees 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 (Kd = 5.0 microM; V = 883.0 U/mg) or in the presence of zinc ions (Kd = 75.0 microM; V = 1102 U/mg). A steady-state kinetic model for the modulation of enzyme activity by cobalt ions is proposed.

Alkaline Phosphatase↗
PubMed1995

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.

Alkaline Phosphatase↗
PubMed1994

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.

Adenosine Triphosphate↗
PubMed1994

Osseous plate alkaline phosphatase is anchored by GPI.

Alkaline phosphatase activity was released up to 100% from the membrane by using 0.1 U of phosphatidylinositol-specific phospholipase C from B. thuringiensis. The M(r) of solubilized enzyme was 145,000 by Sephacryl S-300 gel filtration and 66,000 by SDS-PAGE, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyze p-nitrophenyl phosphate (PNPP) (264.3 mumol min-1 mg-1),ATP (42.0 mumol min-1 mg-1) and pyrophosphate (28.4 mumol min-1 mg-1). The hydrolysis of ATP and PNPP by solubilized 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 (Kd = 1.5 mM) but zinc ions were powerful non-competitive inhibitors (Kd = 6.2 microM) of solubilized enzyme. Treatment of solubilized alkaline phosphatase with Chellex 100 reduced the original PNPPase activity to 5%. Cobalt (K0.5 = 10.1 microM), magnesium (K0.5 = 29.5 microM) and manganese ions (K0.5 = 5 microM) restored the activity of the apoenzyme with positive cooperativity, suggesting that phosphatidylinositol-specific phospholipase C-solubilized alkaline phosphatase is a metalloenzyme. The stimulation of the apoenzyme by calcium ions (K0.5 = 653 microM) was lower than that observed for the other ions (26%) and exhibited site-site interactions (n = 0.7). Zinc ions had no effect on the apoenzyme of the solubilized enzyme.

Alkaline Phosphatase↗
PubMed1993

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.

4-Nitrophenylphosphatase↗
PubMed1993

Kinetic properties of osseous plate alkaline phosphatase from diabetic rats.

1. Increased levels of bone alkaline phosphatase activity were observed in diabetic rats. These animals exhibited impaired bone development without concomitant alterations of the sequence of cellular transformations. 2. Alkaline phosphatase activity was delayed in diabetic rats but the kinetic parameters for the hydrolysis of p-Nitrophenylphosphate (PNPP) were virtually the same observed for controls (N = 1.2 and K0.5 = 43 microM). 3. Alkaline phosphatase from diabetic rats had a better affinity (K0.5 = 38 microM) for magnesium ions than controls (K0.5 = 91 microM). 4. Zinc ions affected alkaline phosphatase activity from control and diabetic rats in the same way (K0.5 = 10 microM).

4-Nitrophenylphosphatase↗
PubMed1992

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

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

Alkaline Phosphatase↗
PubMed1992

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

1. Metal ions other than zinc and magnesium were effective in modulating the activity of rat osseous plate alkaline phosphatase. 2. Increasing pH had remarkable effects on the modulation of rat osseous plate alkaline phosphatase. 3. The modulation of enzyme activity by zinc, manganese and cobalt ions was slightly affected by pH variations. 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. Calcium ions were inhibitors of alkaline phosphatase (Kd = 10 microM) whereas manganese (Kd = 1.3 microM) and cobalt (Kd = 0.2 microM) ions were stimulatory in the pH range 8.0-10.0.

Alkaline Phosphatase↗
PubMed1992

Polyoxyethylene 9-lauryl ether-solubilized alkaline phosphatase: synergistic stimulation by zinc and magnesium ions.

1. Polidocanol-solubilized apoalkaline phosphatase could be stimulated either by zinc ions (Kd = 8.5 nM) or by magnesium ions alone (Kd = 3.8 microM). 2. Zinc and magnesium ions had synergistic effects on Polidocanol-solubilized apoalkaline phosphatase, leading to a fully active enzyme (700-800 U/mg). 3. Zinc ions inhibited non-competitively the Polidocanol-solubilized apoenzyme (Ki = 7.1 microM) by displacing magnesium ions from their binding sites. 4. A model for the action of zinc and magnesium ions on the modulation of the enzyme activity is proposed.

Alkaline Phosphatase↗
PubMed1991

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.

Alkaline Phosphatase↗
PubMed1990

Effect of membrane moiety and magnesium ions on the inhibition of matrix-induced alkaline phosphatase by zinc ions.

1. The inhibition of matrix-induced alkaline phosphatase by zinc ions is due to the displacement of magnesium ions from its binding site. 2. Binding of magnesium ions to alkaline phosphatase induces conformational changes which activate the enzyme. 3. Binding of zinc ions to alkaline phosphatase induces conformational changes which impair the catalytic action of the enzyme. 4. The inhibition of the enzyme by zinc ions is affected by membrane environment and magnesium ions.

Alkaline Phosphatase↗
PubMed1990

Kinetic properties of mitochondrial ATPase during isoproterenol-induced cardiomyopathy.

1. The kinetic characteristics of the ATP hydrolysis by membrane-bound and Triton X-100 solubilized mitochondrial ATPase, during the isoproterenol-induced cardiomyopathy, were investigated. 2. An increase in the inhibitory action of the oligomycin, a decrease in the affinity of the ATP binding sites and an increase of both activation energy and rate of thermal inactivation were observed for mitochondrial ATPase. 3. The possibility that the changes described are related to the modifications of the active configuration of mitochondrial ATPase, during the isoproterenol-induced cardiomyopathy, is discussed.

Adenosine Triphosphatases↗
PubMed1990

Solubilization of membrane-bound matrix-induced alkaline phosphatase with polyoxyethylene 9-lauryl ether (polidocanol): purification and metalloenzyme properties.

1. Matrix-induced alkaline phosphatase prepared from rat osseous plate was solubilized with polidocanol and purified on a Sephacryl S-300 column. 2. Purified solubilized alkaline phosphatase has a molecular weight of ca 115,000 and bind one magnesium and two zinc ions. At least 110 detergent molecules are bound to each enzyme molecule. 3. Solubilization and purification procedures did not destroy the ability of the enzyme to hydrolyze adenosine-5'-triphosphate, p-nitrophenylphosphate, pyrophosphate and bis p-nitrophenylphosphate. 4. Magnesium, manganese and cobalt ions are stimulators of PNPPase activity of solubilized enzyme whereas calcium and zinc ions are inhibitors.

4-Nitrophenylphosphatase↗
PubMed1989

Effect of Zn(II) and Mg(II) on phosphohydrolytic activity of rat matrix-induced alkaline phosphatase.

Rat matrix-induced alkaline phosphatase is an enzyme which requires magnesium and zinc ions for its maximal activity. Two Zn(II) ions and one Mg(II) ion are bound to each subunit of native dimeric enzyme. The presence of magnesium ion (10-100 microM) or zinc ion (7-20 nM) alone is sufficient to stimulate apoenzyme activity. However maximal activity (264 U/mg) requires the presence of both ions. Binding of Zn(II) ions to the Mg(II) binding site causes a strong inhibition of the apoenzyme while the binding of Mg(II) on Zn(II) binding site is not sufficient to stimulate PNPPase activity of the apoenzyme. Binding of both ions to the enzyme molecule did not change the apparent dissociation constant for PNPP hydrolysis.

Alkaline Phosphatase↗
PubMed1987

Triton X-100 solubilized bone matrix-induced alkaline phosphatase.

1. Solubilized and membrane-bound alkaline phosphatase showed Michaelis-Menten behavior in a wide range of different substrate concentrations. 2. Membrane-bound alkaline phosphatase has a molecular weight of 130,000 and its minimum active configuration comprises two identical subunits of about 65,000. 3. The two forms of the enzyme behave similarly with respect to NaCl, urea and guanidine HCl. 4. Catalytic groups have pK values of about 8.5 and 9.7 for both membrane-bound and solubilized enzyme.

Alkaline Phosphatase↗