Isolation and Characterization of Calcifying Matrix Vesicles from Epiphyseal Cartilage

Abstract

Abstract. Matrix vesicles, associated with initial calcification in cartilage, have been isolated from bovine fetal epiphyseal cartilage. Cartilage was digested with collagenase, then partitioned into seven fractions by differential centrifugation. The cellular fractions contained over 80% of the DNA in the digest. The extracellular fraction that contained matrix vesicles, in which apatite crystals were often seen on electron microscopy, also displayed the highest specific activity for alkaline phosphatase, pyrophosphatase, ATPase, and 5'-AMPase (EC 3.1.3

Alkaline phosphatase activity associated to a calcium binding glycoprotein from calf scapula cartilage

“…These structures have been found also by other investigators [5,6]. They have been recently isolated and analyzed for their catalytic functions by Ali et al [7]. One of the enzymic activities bound to the globules, is alkaline phosphatase [7].…”

“…The function of splitting pyrophosphate has been also ascribed to alkaline phosphatases [22]. Biochemical analysis of isolated Bonucci vescicles has demostrated the presence of alkaline phosphatase, ATPase and pyrophosphatase activities [7]. These catalytic functions might be ascribed to the same enzyme.…”

“…They have been recently isolated and analyzed for their catalytic functions by Ali et al [7]. One of the enzymic activities bound to the globules, is alkaline phosphatase [7]. This enzyme was considered by Robison [8], to play an essential role in calcification.…”

Biophysical aspects of biomineralization

“…The deposited calcium phosphate is subsequently converted into hydroxyapatite (HA) [Ca 10 (PO 4 ) 6 (OH) 2 ] in specific areas of the extracellular matrix (ECM). Various experiments have revealed the presence of HA crystals both inside and outside collagen fibrils in the ECM (McNally et al 2012) and also within the lumen of matrix vesicles (MVs) (Ali et al 1970;Anderson 1969;Bonucci 1970). MVs are extracellular vesicles with a diameter ranging from approximately 100 to 300 nm (Anderson 2003;Anderson et al 2010;Cui et al 2016;Golub 2009;Wuthier and Lipscomb 2011) which harbor the biochemical machinery needed to establish the appropriate inorganic pyrophosphate (PP i ) to inorganic phosphate (P i ) ratio (Ciancaglini et al 2006Harmey et al 2004;Hessle et al 2002;Johnson et al 2000;Roberts et al 2007;Yadav et al 2011;Zhou et al 2012) required to initiate the synthesis of apatitic mineral (Bechkoff et al 2008;Thouverey et al 2009;Wu et al 1997), which will subsequently be propagated onto the collagenous ECM.…”

Ultrastructural localization of alkaline phosphatase in fish enamel organ (Oplegnathus fasciatus).

“…Even so, its exact role has not been determined and indeed, it may have several functions. Thus it has been associated with the hydrolysis of pyrophosphates to regulate crystal growth [4,5], the transport of calcium [6] and also the providing of phosphate ions for mineralization [3].…”