Isolation of matrix vesicles by isoelectric focusing in Pevikon-Sephadex

We have investigated the use of an isoelectric focusing (IEF) technique for isolating and characterizing matrix vesicles. Focusing was performed on crude preparations of matrix vesicles isolated from collagenase digests of chick epiphyseal cartilage and purified by discontinuous sucrose gradient centrifugation. Crude and partially purified vesicle preparations were subjected to flat bed IEF in a slurry of Pevikon-Sephadex. Partially purified matrix vesicles focused as a narrow band (pI congruent to to 6.5). Alkaline phosphatase, solubilized from matrix vesicles, focused with a pl of 4.0-4.5. The IEF profile of matrix vesicles also differed from that of chondrocyte membranes. Thus, the membrane pls were congruent to to 5.4 and 6.6-7.8, respectively. The latter peak probably corresponded to the pl of the matrix vesicle preparation. This observation lends support to the view that vesicles originate from distinct regions of the chondrocyte membrane.