CHANGES IN PROTEOGLYCAN AND SULFATED PROTEIN SYNTHESIS DURING MEGAKARYOCYTE MATURATION IN VIVO

We investigated changes in sulfated proteoglycan (PG) and sulfated protein synthesis during megakaryocyte (MK) maturation in vivo by characterizing the (35S)-labeled molecules in MKs and platelets (PLTs) obtained daily from 3 hr to 5 days after injection of guinea pigs with (35S)sulfate. Radioactivity in macromolecules was maximal in MKs 3 hr and in PLTs 3 days after the injection. The cells were solubilized in 8M urea/50mM Tris/0.2% Triton X-100/0.1M NaCl, and PGs and sulfoproteins were separated by DEAE-Sephacel chromatography. PGs (65% of cell 35s) were eluted as two fractions, one (PG-1, 87%) with 4M Gdn HC1 and another (PG-2, 13%) with 4M Gdn HCl/2% TX-100. The Kav of PLT PG-1 on Sepharose CL-6B shifted gradually from 0.18 to 0.10 from 1-5 days after (35S) injection, and the smaller and larger PG-1 species were resolved on SDS-PAGE by fluorography. The size of PG-1 molecules was a function of glycosaminoglycan (GAG) chain length. The appearance of the different size PG-1 molecules in PLTs was accounted for by their disappearance from MKs over the same time period. Thus the size of the PG-1 synthesized by MKs decreased with MK maturation. The (35S)-PG-2 appeared in PLTs only 2-3 days after (35S) injection, had K_av 0.07 on CL-6B, but had GAGs of the same average size as those of PG-1. The hydrophobic character of PG-2 suggests that it might be the membrane PG. PG-1 and PG-2 were separated by SDS-PAGE and identified by fluorography. The core proteins of PG-1 and PG-2 were obtained by chondroitinase digestion and identified by SDS-PAGE and fluorography. The GAGs of PG-1 and PG-2 were almost entirely chondroitin-6-sulfate. The average size of PG-1 was 200,000 and its GAGs about 45,000.

The sulfated proteins (20-25% of total cell ³⁵S) eluted in the wash-through of the DEAE-Sephacel column and with 0.23M NaCl. Their isoelectric points were 4.0-6.5. They eluted as a small peak near the V0 and a major broad peak from Kav 0.3-0.6 on CL-6B columns, and could be identified as at least 8 distinct bands on SDS-PAGE by fluorography. Digestion with NaOH/NaBH₄, Pronase or papain released small (35S)-labeled fragments, and the (35S) appeared to be associated with oligosaccharides. The sulfoproteins appeared in PLTs primarily 2-4 days after (35S) injection, and different proteins were labeled at different time points.