CRYOTOXICITY OF ANTIFREEZE PROTEINS AND GLYCOPROTEINS TO SPINACH THYLAKOID MEMBRANES - COMPARISON WITH CRYOTOXIC SUGAR ACIDS

We have used thylakoids from spinach (Spinacia oleracea L.) chloroplasts to test the effects of antifreeze proteins (AFP) from the starry flounder (Platichthys stellatus; A.FP-SF) and from the antarctic eel pout (Austrolycichthys brachycephalus; AFP-AB), and antifreeze glycoproteins (AFGP) from the antarctic fish Dissostichus mawsoni on biological membranes during freezing. Freeze-thaw damage, measured as the release of the lumenal protein plastocyanin from the thylakoid vesicles, was strongly increased in the presence of all proteins tested. Measurements of the time dependence of plastocyanin release in a simplified artificial chloroplast stroma medium showed that all the fish proteins increased damage during the initial rapid phase while only AFGP increased plastocyanin release during the linearly time dependent slow phase. A slow plastocyanin release is also seen in the absence of freezing. It is increased by the presence of AFGP and AFP-AB, but not by AFP-SF. In order to distinguish between the contribution of the polypeptide and the carbohydrate part of AFGP on freeze-thaw damage we investigated the effects of galactose and N-acetylgalactosamine. While galactose was protective, N-acetylgalactosamine increased the rate of plastocyanin release in an artificial stroma medium at -20-degrees-C. It had no effect on the rapid phase of damage and was also ineffective at 0-degrees-C. The same was found for several other sugar derivatives (N-acetylglucosamine, gluconic acid, glucuronic acid, galacturonic acid). From these data we conclude that the increased plastocyanin release during the rapid phase of freeze-thaw damage is a function of the polypeptide part of AFGP. The increased rate of plastocyanin loss at longer incubation times both at 0-degrees-C and at -20-degrees-C may be mediated by the N-acetylgalactosamine moiety of the AFGP, but is strongly amplified by the polypeptide.