During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix by promoting the formation of hydroxyapatite seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Here, we have studied phosphosubstrate catalysis by osteoblast-derived MVs at physiologic pH, analyzing the hydrolysis of ATP, ADP, and PPi by isolated wild-type (WT) as well as TNAP-, NPP1- and PHOSPHO1-deficient MVs. Comparison of the catalytic efficiencies identified ATP as the main substrate hydrolyzed by WT MVs. The lack of TNAP had the most pronounced effect on the hydrolysis of all physiologic substrates. The lack of PHOSPHO1 affected ATP hydrolysis via a secondary reduction in the levels of TNAP in PHOSPHO1-deficient MVs. The lack of NPP1 did not significantly affect the kinetic parameters of hydrolysis when compared with WT MVs for any of the substrates. We conclude that TNAP is the enzyme that hydrolyzes both ATP and PPi in the MV compartment. NPP1 does not have a major role in PPi generation from ATP at the level of MVs, in contrast to its accepted role on the surface of the osteoblasts and chondrocytes, but rather acts as a phosphatase in the absence of TNAP. © 2010 American Society for Bone and Mineral Research

Recombinant alkaline phosphatases are becoming promising protein therapeutics to prevent skeletal mineralization defects, inflammatory bowel diseases, and treat acute kidney injury. By substituting the flexible crown domain of human intestinal alkaline phosphatase (IAP) with that of the human placental isozyme (PLAP) we generated a chimeric enzyme (ChimAP) that retains the structural folding of IAP, but displays greatly increased stability, active site Zn²⁺ binding, increased transphosphorylation, a higher turnover number and narrower substrate specificity, with comparable selectivity for bacterial lipopolysaccharide (LPS), than the parent IAP isozyme. ChimAP shows promise as a protein therapeutic for indications such as inflammatory bowel diseases, gut dysbioses and acute kidney injury.

Objective: To evaluate the inactivation of Aggregatibacter actinomycetemcomitans ( A. actinomycetemcomitans ), responsible for causing aggressive periodontitis, using photodynamic therapy (PDT) by rose bengal (RB) as a model of a reactive oxygen species (ROS) generator, in planktonic and biofilm cultures. Materials and Methods: A. actinomycetemcomitans was grown in planktonic and biofilm cultures using tryptic soy broth medium. The sensibility (dark toxicity) to RB was determined, and its ideal concentration for PDT was established. Concentrations in the range from 0.01 to 50.0 μmol L ⁻¹ RB, with different light potencies and incubation times, were used. An odontological resin photopolymerizer that emits the adequate wavelength for absorption of the RB dye was applied. Bacterial viability was determined by colony- forming units (CFU). Results : RB photosensitizer dye in concentrations up to 0.1 μmol L ⁻¹ did not show toxicity per se toward A. actinomycetemcomitans cells. In a PDT study with photoirradiation (1 min) at 0.1 μmol L ⁻¹ , a 55% reduction of A. actinomycetemcomitans viability was obtained in planktonic cultures. Preincubation (30 min) of the bacteria with the dye resulted in a 90% reduction of its viability. It is important to note that, for dye concentrations up to 1 μmol L ⁻¹ , in the same experimental conditions, no death effect on gingival fibroblasts was observed. The A. actinomycetemcomitans biofilm was not affected by RB or light alone. After PDT, the reduction in the biofilm (about 45%) is significantly dependant on RB concentration and irradiation time when this dye was used as a ROS generator. Conclusion: Photodynamic therapy-generated ROS inactivates A. actinomycetemcomitans both in planktonic and biofilm cultures, even in small concentrations of the photosensitizing agent, and it does not cause damage to fibroblast cells under the same conditions.

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.