A comparative analysis of strategies for isolation of matrix vesicles.

Matrix vesicles (MVs) are extracellular organelles involved in the initial steps of mineralization. MVs are isolated by two methods. The first isolation method of MVs starts with collagenase digestion of osseous tissues, followed by two differential centrifugations.

Phosphodiesterase activity of alkaline phosphatase in ATP-initiated Ca(2+) and phosphate deposition in isolated chicken matrix vesicles.

Inorganic pyrophosphate is a potent inhibitor of bone mineralization by preventing the seeding of calcium-phosphate complexes. Plasma cell membrane glycoprotein-1 and tissue nonspecific alkaline phosphatase were reported to be antagonistic regulators of mineralization toward inorganic pyrophosphate formation (by plasma cell membrane glycoprotein-1) and degradation (by tissue nonspecific alkaline phosphatase) under physiological conditions. In addition, they possess broad overlapping enzymatic functions.

Distinct structure and activity recoveries reveal differences in metal binding between mammalian and Escherichia coli alkaline phosphatases.

The amino acids involved in the coordination of two Zn2+ ions and one Mg2+ ion in the active site are well conserved from EAP (Escherichia coli alkaline phosphatase) to BIAP (bovine intestinal alkaline phosphatase), whereas most of their surrounding residues are different. To verify the consequences of this heterology on their specific activities, we compared the activity and structure recoveries of the metal-free forms (apo) of EAP and of BIAP. In the present study, we found that although the sensitivities of EAP and BIAP to ions remained similar, significant differences in dimeric structure stability of apo-enzymes were observed between EAP and BIAP, as well as in the kinetics of their activity and secondary structure recoveries.

Interactions of caged-ATP and photoreleased ATP with alkaline phosphatase.

Photolytic release of ATP from inactive P(3)-[1-(2-nitrophenyl)]ethyl ester of ATP (NPE-caged ATP) provides a means to reveal molecular interactions between nucleotide and enzyme by using infrared spectroscopy. Reaction-induced infrared difference spectra of bovine intestinal alkaline phosphatase (BIAP) and of NPE-caged ATP revealed small structural alterations on the peptide backbone affecting one or two amino-acid residues. After photorelease of ATP, the substrate could be hydrolyzed sequentially by the enzyme producing three Pi, adenosine, and the photoproduct nitrosoacetophenone.

Phosphate binding in the active site of alkaline phosphatase and the interactions of 2-nitrosoacetophenone with alkaline phosphatase-induced small structural changes.

To monitor structural changes during the binding of Pi to the active site of mammalian alkaline phosphatase in water medium, reaction-induced infrared spectroscopy was used. The interaction of Pi with alkaline phosphatase was triggered by a photorelease of ATP from the inactive P(3)-[1-(2-nitrophenyl)]ethyl ester of ATP. After photorelease, ATP was sequentially hydrolyzed by alkaline phosphatase giving rise to adenosine and three Pi.