Localization of phosphatidylserine in isolated chick epiphyseal cartilage matrix vesicles with trinitrobenzenesulfonate

Summary

The localization of serine and ethanolamine phospholipids in isolated chick epiphyseal cartilage matrix vesicles was studied by time-course labeling with trinitrobenzenesulfonate (TNBS), a probe nonpermeant to most biological membranes. Significant amounts of all four of the primary amine-containing phospholipids in matrix vesicles reacted readily with TNBS, indicating that the vesicle membranes were unusually permeable to the label. We attribute this to the presence of large amounts of lysophospholipids present in the vesicle membranes. However, none of the lipids reacted with TNBS to completion. Plateau labeling values suggested that as much as 70 to 80% of the phosphatidylserine (PS) and 60 to 80% of the lysophosphatidylserine (LPS) are located inside the vesicles, and that 40 to 50% of the ethanolamine phospholipids are internal. The kinetic evidence further suggested that an additional 10 to 15% of these phospholipids are probably also internal. In the presence of chloroform-methanol, 1:1, which totally disrupts membrane structure, only 55 to 60% of the total PS and LPS reacted with TNBS, whereas all of the ethanolamine phospholipids did. We attribute this effect to the presence of PS-Ca-P_i complexes which inhibit the reaction of these lipids with TNBS under these conditions. Thus our findings add further evidence that binding of Ca by acidic phospholipids within the matrix vesicles may be involved in mineral deposition in epiphyseal cartilage.