Analysis of glycosaminoglycans of flow sorted cells: Incorporation of [ 35 S]sulfate and [ 3 H]glucosamine into glycosaminoglycans of B16‐F10 cells during the cell cycle

The incorporation of [ 35 S]sulfate and [ 3 H] glucosamine into cetyl pyridinium chloride (CPC) precipitable glycosaminoglycans was determined in B16‐F10 cultured cells sorted with respect to DNA content. Incorporation into surface material was measured indirectly as the difference between the radio‐activity of control and trypsin treated cells. Approximately 80% of the total cellular [ 35 S] sulfate labeled CPC precipitable material, but only 5% of that labeled by [ 3 H]glucosamine, was removed by this mild trypsin treatment. Incorporation of [ 35 S]sulfate into the trypsin removable surface material increased progressively from G 1 to S to G 2 + M in both long‐term (48 hours) and shortterm (1 hour) labeled cells, while the ratio of surface to total incorporated [ 35 S]sulfate remained relatively constant. Incorporation of [ 35 S]sulfate into total cellular glycosaminoglycans in long‐ and short‐term labeled cells increased as cells progressed from G 1 to S to G 2 + M; the incorporation of [ 3 H]glucosamine into CPC precipitable material also increased progressively from G 1 to S to G 2 + M in long‐term labeled cells but was greater during S phase relative to G 1 or G 2 + M in short‐term labeled cells. The degree of sulfation of glycosaminoglycans as represented by the ratio of [ 35 S]sulfate to [ 3 H]glucosamine of double labeled cells was relatively constant in long‐term labeled cells but was increased during the G 1 and G 2 + M phases of short‐term labeled cells. Comparison of the degree of sulfation of short‐term with long‐term labeled cells suggests that highly sulfated glycosaminoglycans may be turned over more rapidly during G 1 and G 2 + M phases of the cell cycle.