J. exp. Biol. 104, 149-162 (1983) 149 in Great Britain © The Company of Biologists Limited 1983 THE ROLE OF LOW MOLECULAR WEIGHT ANTIFREEZE GLYCOPEPTIDES IN THE BILE AND INTESTINAL FLUID OF ANTARCTIC FISH BY S. M. O'GRADY*, J. C. ELLORYf AND A. L. DEVRIES* • Department of Physiology and Biophysics, 524 Burrill Hall, University of Illinois, Urbana, Illinois 61801, U.SA. and f Physiological Laboratory, University of Cambridge, Cambridge, U.K. CB2 3EG {Received 20 August 1982—Accepted 19 January 1983) SUMMARY The role that low molecular weight antifreeze glycopeptides play in the physiology of polar fishes has been an open question. In this study, we demonstrate that antifreeze glycopeptides are present in the bile as well as the intestinal fluid of antarctic fishes. Isolation of antifreeze glycopeptides from these fluids by DEAE ion exchange chromatography followed by polyacrylamide gel electrophoresis revealed the presence of only low molecular weight glycopeptides (6, 7 and 8). Removal of the gall bladder with subsequent occlusion of the common bile duct eliminated the transport of antifreezes into the intestine. This suggests that antifreeze glycopeptides enter the intestinal lumen by biliary secretion. Measurements of reabsorp- tion, both in vivo and in vitro, indicate that antifreeze glycopeptides are not reabsorbed as intact molecules. Our results suggest that these glycopeptides are excreted. We conclude that low molecular weight antifreeze glycopep- tides are necessary to prevent the intestinal fluid from freezing and provide the first clear evidence that low molecular weight antifreeze glycopeptides have a specific biological function in polar fishes. INTRODUCTION Most antarctic fishes synthesize a group of eight glycopeptides that possess unique antifreeze properties (DeVries & Wohlschlag, 1969; DeVries & Lin, 1977; DeVries, 1980). The larger glycopeptides (1-5) have the greatest antifreeze activity and range in molecular weight between 10500 and 33700Da (DeVries & Lin, 1977). They contain two amino acids, alanine and threonine, which are arranged in a repeating sequence [ala-ala-thr] n . A disaccharide [galactose/3 (1-3) N-acetylgalactosamine] is attached to each threonine (Sheir, Lin & DeVries, 1975). The smaller glycopeptides, 6, 7 and 8, differ from the larger ones in that proline periodically replaces alanine at positions 7, 10, 13 and 16. The smaller glycopeptides range in molecular weight from 2600 to 8000 Da and exhibit only one-third of the antifreeze activity of glycopeptides (1-5). ^Bey words: Antifreeze, glycoprotein, teleost.