Kinetics of Vibrio cholerae sialidase action on gangliosidic substrates at different supramolecular-organizational levels

Gd1a, Gd1b and Gt1b gangliosides were dispersed in the following membrane-mimicking systems: (a) homogeneous micelles; (b) mixed micelles with Gm1 ganglioside (which is resistant to the enzyme action), Triton X-100 or bovine serum albumin; (c) small unilamellar vesicles of egg phosphatidylcholine. The effect of dispersion on sialic acid release by Vibrio cholerae sialidase was studied. As reference substrates freely interacting with the enzyme the lipid-free carbohydrates of Gd1a and 3′-sialosyl-lactose were employed. The apparent Vmax. of the enzyme was, with all the gangliosides, dependent on the type of ganglioside dispersion. It was lowest for homogeneous micelles and mixed micelles with ganglioside Gm1, and increased about 6-fold for ganglioside/bovine serum albumin lipoprotein micelles, 15-fold for mixed-ganglioside/Triton X-100 micelles (optimal molar ratio 1:7.5) and 30-fold for phosphatidylcholine vesicles containing 2.5 mol% ganglioside (this proportion was optimal for enzyme activity on the vesicles). For ganglioside Gd1a, the activity on Triton X-100 mixed micelles and on mixed vesicles was even greater (3- and 6-fold respectively) than that displayed on Gd1a lipid-free carbohydrate. With each of the used gangliosides the apparent Km values were very similar values for homogeneous micelles and vesicular dispersions, but showed marked increases for Triton X-100 mixed micelles, approaching the values exhibited by reference oligosaccharides. Triton X-100 micelles and phosphatidylcholine vesicles did not appreciably alter the kinetics of sialidase action on 3′-sialosyl-lactose and on Gd1a lipid-free carbohydrate, indicating that the above effects are dependent on the intrinsic characteristics of the membrane-like systems containing gangliosides.

Contribution of Sialic Acid to the Heterogeneity of Human Fibrinogen Polypeptide Chains

Human fibrinogen, isolated from single donor or from pooled plasma, shows a heterogeneity of the Bऔ- and the γ-polypeptide chains on CM-cellulose chromatography. In order to find out whether this heterogeneity is due to the observed differences in sialic acid content of the variants (2 and 1 residue per chain; Gati et al., J.Biol.Chem.253:1315,1978) pooled or single donor fibrinogen (clottability 93-95%) was incubated 24 hrs with either vibrio cholerae sialidase or buffer. The asialofibrinogen (10% of original sialic acid) was compared with intact fibrinogen. After dithiothreitol reduction and alkylation the chains were separated on CM Sepharose. The individual homogeneous Bऔ- and γ-chains were rechromatographed on CM-cellulose. The γ-chain heterogeneity of normal fibrinogen was absent in asialofibrinogen whereas the Bऔ-chain heterogeneity appeared unaffected. Although the variants were indistinguishable on SOS-PAGE, isoelectric focusing in presence of urea demonstrated heterogeneities of both Bऔ- and γ-chains even in asialofibrinogen. Thus, the differences in sialic acid content of the main polypeptide chain variants of pooled as wel l as single donor human fibrinogen can only explain a small part of the polypeptide chain heterogeneity.