Date of Award

6-1979

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Supervisor

Professor E.L. McCandless

Abstract

Biosynthesis of carrageenans, the sulphated polygalactans which consitute most of the cell wall and intercellular matrix in the marine red alga Chondrus crispus, was studied using in vivo labelling with ³⁵S - sulphate and ¹⁴C - bicarbonate. Pre-cultured wild-type gametophyte showed enhanced sulphate uptake and incorporation characteristic of the rapidly grown T₄ strain. It was found that sulphate uptake was related to growth status of the plants rather than to the life cycle stage (haploid gametophyte or diploid sporophyte) despite the difference in sulphation level of the major carrageenans in these different stages. An apparent Km for sulphate uptake was determined for the T₄ gametophyte.

The sporophyte produces mainly lambda carrageenan (soluble in 0.3M KCl) while the gametophyte produces kappa carrageenan (insoluble in 0.3M KCl) and a 0.3M KCl soluble fraction very unlike lambda carrageenan (47). Lambda carrageenan consists ideally of repeating disaccharide units of 4-linked galactose - 2,6-disulphate and 3-linked galactose (70% sulphated at C2) while kappa carrageenan consists ideally of 4-linked 3,6 anhydrogalactose and 3-linked galactose-4-sulphate (68,70).

The synthesis of lambda carrageenan was more rapid than that of kappa carrageenan. Neither molybdate nor tungstate (other Group VI anions) inhibited sulphate incororation into carrageenans in T₄ gametophyte nor did nitrate. NH₄Cl reduced incorporation into kappa carrageenan while urea inhibited incorporation into the 0.3M KCl soluble fraction in T₄. Plants kept in the dark showed very little incorporation of ¹⁴C - bicarbonate into carrageenan. Galactose and 2,4-D showed little effect on total incorporation of ¹⁴C-bicarbonate but tended to shift the labelling of 0.3M KCl soluble and insoluble fractions.

Pulse-chase experiments showed the precursor of kappa carrageenan to be present in the 0.3M KCl soluble fraction. Subfractionation of this soluble fraction with 3M KCl followed by infrared and immunological analyses of the fraction showed a progression from iota - (4-linked 3,6 anhydrogalactose-2-sulphate and 3-linked galactose-4-sulphate) and/or nu - (4-linked galactose-2,6-disulphate and 3-linked galactose-4-sulphate) like carrageenan to kappa type with the 3M KCl soluble carrageenan being least kappa-like. A pathway for the biosynthesis of kappa carrageeenan was proposed on the basis of radioisotope labelling, infrared, and immunological evidence.

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