Abstract
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. Therefore, we examined the roles of tissue nonspecific alkaline phosphatase within matrix vesicles isolated from femurs of 17-day-old chick embryos, under conditions where these both antagonistic and overlapping functions could be evidenced. Addition of 25 microM ATP significantly increased duration of mineralization process mediated by matrix vesicles, while supplementation of mineralization medium with levamisole, an alkaline phosphatase inhibitor, reduces the ATP-induced retardation of mineral formation. Phosphodiesterase activity of tissue nonspecific alkaline phosphatase for bis-p-nitrophenyl phosphate was confirmed, the rate of this phosphodiesterase activity is in the same range as that of phosphomonoesterase activity for p-nitrophenyl phosphate under physiological pH. In addition, tissue nonspecific alkaline phosphatase at pH 7.4 can hydrolyze ADPR. On the basis of these observations, it can be concluded that tissue nonspecific alkaline phosphatase, acting as a phosphomonoesterase, could hydrolyze free phosphate esters such as pyrophosphate and ATP, while as phosphodiesterase could contribute, together with plasma cell membrane glycoprotein-1, in the production of pyrophosphate from ATP.
Highlights
Matrix vesicles (MVs),2 released by budding from specialized areas of plasma membranes of osteoblasts, chondrocytes, or odonblasts, are involved in the initial step of mineralization in all calcifying tissues, by promoting the seeding of basic calcium phosphate crystals of hydroxy
The purpose of the present work was to examine the roles of tissue nonspecific alkaline phosphatase (TNAP) during the ATP-initiated deposition of calcium phosphate complexes by MVs isolated from femurs of 17-day-old chick embryos
We found that both sMV and pMV fractions were able to hydrolyze ADPR and that their ADPR hydrolysis activities were inhibited by levamisole, suggesting that the amount of PPi generated from ATP by MVs is reduced in the presence of levamisole
Summary
Matrix vesicles (MVs), released by budding from specialized areas of plasma membranes of osteoblasts, chondrocytes, or odonblasts, are involved in the initial step of mineralization in all calcifying tissues, by promoting the seeding of basic calcium phosphate crystals of hydroxy-. TNAP is a metalloenzyme with three divalent cations (two Zn2ϩ and one Mg2ϩ) and a serine residue in the active site [10]. It has been classified into a superfamily of phospho-/sulfo-coordinating enzymes catalyzing the hydrolysis of phosphate monoesters, diesters, triesters, and sulfate esters [11,12,13,14,15]. The PC-1 is a metalloenzyme with two divalent cations (Mn2ϩ, Zn2ϩ, Ca2ϩ, or Mg2ϩ) and a threonine residue in the active site (12, 15, 18 –20). Bovine intestinal alkaline phosphatase has been suggested to hydrolyze P3-[1-(2-nitrophenyl)]ethyl ester of ATP, an analogue of nucleotide without free phosphate monoester [24]. Chondrocytes in the growth plate export continually nucleotides (ATP/UTP) that may regulate cell maturation, energy metabolism and NOVEMBER 4, 2005 VOLUME 280 NUMBER 44
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