The role of phosphorylation of proteoglycans in the intracellular sorting and control of biosynthesis of these molecules will be investigated using tissue culture, radioisotopic labeling and detailed chemical analysis. The detailed structure of the modified linkage region for the Swarm rat chondrosarcoma chondroitin sulfate proteoglycan and the EHS heparan sulfate proteoglycan will be determined using such methods as enzymatic digestions, methylation, GC-mass spectrophotometry and 31 P NMR. Enzymatic digestions will also be utilized to identify the sequence of phosphorylated peptides, and, to order the substitution sites with the proteoglycan. The phosphorylating enzymes, including the 2-phosphoxylose phosphorylating enzyme will be characterized as to donor and substrate specificity, size, location and chemical composition. The subcellular distribution of modifying enzyme(s) will be determined by standard subcellular fractionation and analysis. In addition, the regulation of the modification will be examined with cultured cells perturbed by reagents such as insulin, calmodulin inhibitors and cyclic AMP altering drugs, as well as by monesin and other blockers of intracellular transport.
