CHARACTERIZATION AND RECONSTITUTION OF THE NUCLEATIONAL COMPLEX RESPONSIBLE FOR MINERAL FORMATION BY GROWTH-PLATE CARTILAGE MATRIX VESICLES

Previous studies revealed that matrix vesicles (MV) have an acid-labil e nucleationally active core (ALNAC) essential for mineral formation; current studies were aimed at characterizing and reconstituting ALNAC. SDS-PAGE and FTIR analyses revealed the presence of lipids, proteins and amorphous calcium phosphate (ACP) in ALNAC. Extraction with chloro form-methanol reduced, but did not destroy MV calcification; treatment with chloroform-methanol-HCl destroyed all activity, This acidic solv ent extracted the annexins, (phosphatidylserine (PS)-dependent Ca2+-bi nding proteins), and dissociated PS-Ca2+-Pi complexes present in the M V. Attempts to reconstitute ALNAC, centered on the Ca2+-PS-Pi complex. Various pure lipids, electrolytes and proteins were combined to form a synthetic nucleationally active complex (SNAG), analyzing the rate o f Ca2+ uptake, Inclusion of phosphatidylethanolamine (PE) or sphingomy elin (SM) with PS, or Mg2+ or Zn2+ with Ca2+, strongly inhibited activ ity; incorporation of annexin V increased SNAG activity, Thus, approac hing from either deconstruction or reconstruction, it appears that ALN AC is composed of ACP complexed with PS and the annexins, Other lipids , proteins and electrolytes modulate its activity, These findings also indicate how ALNAC must be formed in vivo.