Characterization of receptors for sulphated polysaccharides on guinea-pig spermatozoa.

give 50"h inhibition of binding ( lC5,,) was derived from these graphs. The values for ram proacrosin were as follows: unlabelled fucoidan ( 19.1 nM), dextran sulphate SO0 000 M, (3 1.6 nM), dextran sulphate 5000 M, (251 nM), polyvinylsulphate (326 nM), xylan (299 p ~ ) and mannan ( 1000 p ~ ) . The following solutions were tested. but did not reach 50%) inhibition at the concentrations given: dextran 506 000 M, ( 160 p ~ ) , D( + )-fucose (0.8 M), D( + )-galactose (0.8 M), D( + )mannose (0.8 M), lactose (0.4 M), glucosamine (0.5 M), galactosamine (0.5 M), chondroitin sulphates A and C (0.4 mM). Comparable results were obtained using boar proacrosin. Fig. 1 shows the data for four blocking agents binding to ram proacrosin. The significance of molecular mass is demonstrated by the more potent effect of 500 000 M, over 5000 M , dextran sulphate. The importance of sulphation is shown by the lack of inhibition with dextran ( M , 506 000), versus dextran sulphate. However, absence of an inhibitory effect with chondroitin sulphates A and C (M, 40 000) demonstrates that the stereochemical arrangement of sulphate groups o n the polysaccharide structure must also be significant in order to block the fucoidan-binding sites on proacrosin. As proacrosin is a serine proteinase, other members o f this family (chymotrypsinogen. trypsinogen, thrombin. clastase. plasminogen, pepsin. Streptotnvces griserrs protease) have been screened for fucoidan binding activity. Only chymotrypsinogen and trypsinogen retained significant amounts o f the probe with K , values of 1.4 x lo-" M and 3.0 x l o ' M respectively [ S ] . The affinity o f fucoidan for proacrosin was K , 4.9 x 10Vx M. This unusual property of carbohydrate-binding activity suggests a second function for these three proteinases. This activity was not blocked by proteinase inhibitors, indicating that the fucoidan-binding site is different from the substrate-binding site. However, the tcrtiary structure o f the molecule is important, as reduction with 2-mercaptoethanol caused a SOYo decrease in binding o f the polysaccharide probe. These fucoidan-binding properties o f proacrosin arc comparable with those reported for bindin. the sca urchin sperm adhesion molecule [6]. Using group-specific chemical modification of basic amino acids, DeAngelis & Glabc 161 showed that arginine, lysine and histidine residues were important in fucoidan binding. To determine the location o f basic residues in chymotrypsinogen, trypsinogen and proacrosin, X-ray crystallographic data was used to construct three-dimensional molecular models using Evans and Suthcrland computer programs. As there is 40'% linear * 506000-M,