Kinetic analysis of substrate utilization by native and TNAP-, NPP1-, or PHOSPHO1-deficient matrix vesicles

Abstract

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.

Cellular function and molecular structure of ecto-nucleotidases

“…Of relevance in the present context, APs can hydrolyze extracellular ATP via ADP and AMP to adenosine [362,363,514] (Fig. 4).…”

“…Recently, it has been demonstrated that soluble heterologously expressed murine NPP1 [362,363] or NPP3 [7] hydrolyzes (at pH 7.4) PP i in addition to ATP. This result corroborates earlier observations that NPP2 cleaves PP i into P i [364].…”

“…As compared to that, a K m value of 1.5 mM was determined for a membrane-bound fraction of the enzyme [521]. Analyzing TNAP-, NPP1-, or POSPHO1-deficient mice, a recent study addressed the hydrolysis of ATP and ADP in isolated osteoblast-derived membranelimited matrix vesicles and compared it to recombinant enzymes (all at pH 7.4) [362]. It identified TNAP as the major ATP-and ADP-hydrolyzing enzyme of the mouse matrix vesicles.…”

Biophysical aspects of biomineralization

“…Biophysical and biochemical characterization of biomimetic models Biophysical tools applied to biomineralization studies A large number of studies have been carried out using proteoliposomes as chondrocyte-and osteoblast-derived MVs biomimetics (Andrade et al 2016;Bolean et al 2010Bolean et al , 2011Bolean et al , 2015Bolean et al , 2017Ciancaglini et al 2010;Favarin et al 2017;Simao et al 2010aSimao et al , b, 2013.…”

“…A relevant point to be observed is that the enzyme bound to liposomes retains the ability to hydrolyze different substrates, such as ATP, ADP, AMP, PP i , and p-nitrophenylphosphate Ciancaglini et al 2006Ciancaglini et al , 2010Simao et al 2010a, b).…”