Alkaline phosphatase reconstituted in liposomes: study of the effect of the lipid microenvironment on the pyrophosphatase and ATPase activity of the enzyme

Abstract

The process of biomineralization consists in the accumulation of mineral constituted primarily by phosphate and calcium ions that becomes a calcium phosphate salt, and this structure becomes hydroxyapatite. This process is mediated by osteoblasts, cells responsible for the beginning of the biomineralization process, mediated by the liberation of matrix vesicles (MVs). These vesicles appear by budding from the cells surfaces and are secreted into the specific location of the beginning of biomineralization in the bone matrix. MVs contain high concentrations of Ca2+ ions and inorganic phosphate (Pi), providing a microenvironment appropriate for the initial formation and propagation of the hydroxyapatite crystals. Special attention should be given to an enzyme present in MVs: tissue-nonspecific alkaline phosphatase (TNAP). This enzyme regulates the phosphate and pyrophosphate levels, which are important compounds in the formation of calcium phosphate crystals, thus acting directly during the bone mineralization process.TNAP is a non-specific phosphomonohydrolase, able to hydrolyze phosphate monoesters, pyrophosphate, phosphate diesters, as well as catalyze transphosphorylation reactions. It is inserted to the plasma membrane of MVs by a glycosylphosphatidylinositol anchor and it's called "alkaline" due to its ability to perform these substrate hydrolysis reactions more efficiently at a pH above the neutral (pH 8-11). Recently, it has been observed in our laboratory that the catalytic properties of TNAP vary according to the microenvironment in which the enzyme is located, but there are still doubts about this effect on physiological substrates. So, we intend to study the kinetic characteristics of the enzyme, using ATP and PPi as substrates, due to the changes in the lipid microenvironment. For that, liposomes vesicular systems will be employed, with compositions similar to those found in MVs. Therefore, the main goal of this project is the construction of proteoliposomes systems containing TNAP to evaluate, according to the presence of different lipids, both the dependence of the enzyme's activity as well as the formation of hydroxyapatite crystals.