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Heparan sulfate and chondroitin sulfate glycosaminoglycans of BALB/c 3T3 fibroblasts, metabolically labeled with [3H]glucosamine and [35S]sulfate precursors, are resolved by preparative Sepharose CL-4B chromatography into distinct products, the proteoglycans and the glycosaminoglycan free chains, the latter resistant to appreciable molecular weight shift upon alkaline borohydride reduction. The in situ localization of these cell layer molecules was probed with glycosaminoglycan degrading enzymes (lyases) of bacterial origin, which were used to digest isotopically prelabeled monolayer cultures prior to extraction with a nonionic detergent in the presence of protease inhibitors. Most of the total cellular complement of glycosaminoglycan free chains, in addition to the proteoglycans, proved accessible to the lyases under conditions which did not appreciably affect cell viability or morphology. Because these results were also obtained under low temperature (4 degrees C) conditions and in the presence of phenylarsine oxide, a sulfhydryl reagent that irreversibly inhibits endocytosis, the effects of the lyases are not dependent upon internalization by the cells. The cellular production and cell surface expression of the glycosaminoglycan free chains were not materially altered when lysosomal function was pharmacologically inhibited, confirming that the free chains are not intracellular intermediates in the lysosomal degradation pathways of proteoglycans. Contrary to the prevailing model, our observations establish that, at least in the cell line under study, glycosaminoglycan free chains are located on the external leaflet of the plasma membrane, as such suggesting that these products are biologically active components of cell surfaces.

Crude enzyme obtained from heparin-induced flavobacteria has been fractionated into a heparitinase acting on heparitin sulfates and related compounds and a heparinase acting mainly on heparin. Purification achieved for each was from 50 to 100 times that of earlier preparations containing a mixture of the two enzymes. In agreement with previous data both enzymes act as eliminases rather than hydrolases yielding products containing Δ4,5-unsaturated uronic acid. Specificity of the heparitinase appears to require the absence of O-sulfate and the presence of N-acetyl or sulfamido groups. Specificity of the heparinase requires the presence of O-sulfate and sulfamido groups while derivatives containing free amino or N-acetyl are not substrates. The heparinase degrades heparitin sulfate to some extent acting apparently on the heparin-like portion.