Graduate Thesis Or Dissertation
 

Synthesis and assembly of Fucus zygote cell walls with an emphasis on alignate and fucans

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  • Fucus distichus L. Powell zygotes were used to investigate cell wall synthesis and development and to construct a model of cell wall structure. Alginate synthesis was investigated. Zygotes were cultured in seawater containing ¹⁴C-bicarbonate. Alginate represented 12%, 37%, and 1.7% of the label recovered from whole zygotes, wall, and cytoplasm, respectively. Cytoplasmic alginate had 70% of the label in alternating blocks, but wall alginate had 58% of the label in polyguluronic acid blocks. The ratio of label in alginate monomers (M/G) was 0.8 (cytoplasm) and 0.5 (wall), suggesting that a C-5 alginate epimerase was active. Enzymes from marine bacteria were isolated, characterized, and used to disrupt Fucus zygote cell walls. Cell-bound lyase activity was specific for polymannuronic acid (M lyase), and released activity degraded polyguluronic acid (G lyase). A highly active M lyase from bacteria has not previously been reported. M lyase had a molecular weight of 100,000 daltons and acted in an exo-manner. The G lyase had a molecular weight of 35,000 daltons, was soluble in saturated ammonium sulfate, acted in an endo-manner, and was stimulated by calcium. Calcium reduced substrate specificity of G lyase but had no effect on the activity of M lyase. Cell walls from different stages were disrupted by G lyase and calcium chelation. The mature cell wall consisted of cellulose, alginate, and fucans (F₁ and F₂). F₂ was not present in the 6.5 hour walls, but all stages contained highly sulfated fucans covalently bound to the cellulosic portion of the wall. Calcium chelation released alginate, F₁ and F₂. Alginate and F₂ but not F₁ were degraded by G lyase treatment. F₂ was converted into a faster-moving component by mild acid hydrolysis and by G lyase treatment. The model of wall structure fits the events of development and allows a framework from which to make predictions about the behavior of wall components during subsequent experimental approaches.
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