Studies on the Polysaccharide-Sulfating System in Sea Urchin Embryos. I. Characterization of the System in Sea Urchin Embryos*

Abstract

The sulfating system in sea urchin embryos was examined, using the labeled precursor inorganic [35S]sulfate in vivo and [35S]3 '-phosphoadenosine 5'-phosphosulfate ([35S]PAPS) in a cell-free system.In vivo incorporation of [3sS]sulfate into the trichloroacetic acid (TCA)-insoluble fraction increased gradually during sea urchin development, whereas radioactivity of [S5S]sulfate contained in the TCA-soluble fraction showed a conspicuous peak at the late gastrula stage.In a cell-free system, the particulate fraction showed marked incorporation of [a5S]PAPS. This sulfating activity was highest at pH 6.4 to 7.2 and at 27"C, and it was strongly inhibited by Hge+ and p-chloromercuribenzoic acid.The sulfating activity was quite low in fertilized eggs, but then increased rapidly up to the swimming blastula stage. The activity in the particulate fraction precipitated at 10,000 xg increased gradually and that in the particulate fraction precipitated at 100,000 xg was almost constant from the swimming blastula stage to the pluteus stage.GAG is thought to have important roles during embryonic development in morphogenetic movements and differentiation: Sulfated GAG synthesis may be involved in changes in cell surface properties and cell contact behavior during gastrulation of Rana pipiens (23), HA production may be associated with cell migration and expanding extracellular spaces during corneal development in the chick (39) and during formation of the primary mesenchyme and neural fold in rat embryos (39, and GAG may be involved in tissue interaction in mouse embryonic salivary glands (3, 4) and in somite induction and primary corneal differentiation in chick embryos (1 1, 25).The presence of GAG in sea urchin embryos has been demonstrated histochemically and biochemically (15-18, 20, 21, 26, 28-30, 37, 40), and one GAG was recently identified as DS (29, 40). In sea urchin embryos, possible roles of GAG in the cell movement involved in gastrulation (17, 18,28, 37), in protein synthesis (17) and in initiation of genomic RNA synthesis (20) have been proposed.

General Pattern, 35so4-Incorporation and Intracellular Localization of Glycans in Developing Sea Urchin (Anthocidaris) Embryos

“…Development of sea urchin embryos is arrested at the blastular stage in sulfate-free sea water (7,8) and this is consistent with reports of increased 35S04-incorporation (15,25,28,29) and related enzymic activities (4,19,24) in mesenchyme blastulae and later stage embryos. The 35S04-incorporation was reported to occur first in the vegetal region of embryos and then at the blastocoel in parallel with migration of mesenchyme cells (10,31).…”

Studies on the Polysaccharide-Sulfating System in Sea Urchin Embryos. Ii. Nature of Sulfated Materials Produced in a Cell-Free System*1

“…The TCA-insoluble fraction obtained was washed successively with 5 % TCA containing 50 mM Na,S04, ethanol, ethyl ether and acetone, and then digested with Pronase as described elsewhere (23) to obtain protein-free sulfated polysaccharide. After digestion, cold TCA (10% final concn.)…”

“…Solubilized [85S]sulfated polysaccharide was recovered in the TCA-soluble fraction. This fraction was dialyzed against distilled water, or after removal of TCA with ethyl ether, applied to a Sephadex G-25 column (23). The dialyzed solution or the polysaccharide eluted in the void volume from the column with 50 mM NaCl was used for analyses.…”