Proteoliposomes Harboring Alkaline Phosphatase and Nucleotide Pyrophosphatase as Matrix Vesicle Biomimetics

• Published in: The Journal of biological chemistry Vol. 285; no. 10; pp. 7598 - 7609
• Main Authors: Simão, Ana Maria S., Yadav, Manisha C., Narisawa, Sonoko, Bolean, Mayte, Pizauro, Joao Martins, Hoylaerts, Marc F., Ciancaglini, Pietro, Millán, José Luis
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 05.03.2010; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphate - metabolism; Alkaline Phosphatase - genetics; Alkaline Phosphatase - metabolism; Animals; Biomimetics; Calcification, Physiologic - physiology; Calcium/ATPase; Catalysis; Cell/Compartmentation; Cells, Cultured; Enzymes/ATPases; Glycosylphosphatidylinositols - metabolism; Humans; Lipids - chemistry; Membrane Biology; Membrane/Enzymes; Membrane/Reconstitution; Methods/Liposomes; Mice; Osteoblasts - cytology; Osteoblasts - physiology; Polidocanol; Polyethylene Glycols - chemistry; Proteolipids - chemistry; Proteolipids - metabolism; Pyrophosphatases - genetics; Pyrophosphatases - metabolism; Rats; Subcellular Organelles/Vesicles; Subcellular Organelles/Vesicles; Membrane/Enzymes; Cell/Compartmentation; Membrane/Reconstitution; Calcium/ATPase; Enzymes/ATPases; Methods/Liposomes
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.079830

We have established a proteoliposome system as an osteoblast-derived matrix vesicle (MV) biomimetic to facilitate the study of the interplay of tissue-nonspecific alkaline phosphatase (TNAP) and NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1) during catalysis of biomineralization substrates. First, we studied the incorporation of TNAP into liposomes of various lipid compositions (i.e. in pure dipalmitoyl phosphatidylcholine (DPPC), DPPC/dipalmitoyl phosphatidylserine (9:1 and 8:2), and DPPC/dioctadecyl-dimethylammonium bromide (9:1 and 8:2) mixtures. TNAP reconstitution proved virtually complete in DPPC liposomes. Next, proteoliposomes containing either recombinant TNAP, recombinant NPP1, or both together were reconstituted in DPPC, and the hydrolysis of ATP, ADP, AMP, pyridoxal-5′-phosphate (PLP), p-nitrophenyl phosphate, p-nitrophenylthymidine 5′-monophosphate, and PPi by these proteoliposomes was studied at physiological pH. p-Nitrophenylthymidine 5′-monophosphate and PLP were exclusively hydrolyzed by NPP1-containing and TNAP-containing proteoliposomes, respectively. In contrast, ATP, ADP, AMP, PLP, p-nitrophenyl phosphate, and PPi were hydrolyzed by TNAP-, NPP1-, and TNAP plus NPP1-containing proteoliposomes. NPP1 plus TNAP additively hydrolyzed ATP, but TNAP appeared more active in AMP formation than NPP1. Hydrolysis of PPi by TNAP-, and TNAP plus NPP1-containing proteoliposomes occurred with catalytic efficiencies and mild cooperativity, effects comparable with those manifested by murine osteoblast-derived MVs. The reconstitution of TNAP and NPP1 into proteoliposome membranes generates a phospholipid microenvironment that allows the kinetic study of phosphosubstrate catabolism in a manner that recapitulates the native MV microenvironment.