Characterization of sialyltransferase-IV activity and its involvement in the c-pathway of brain ganglioside metabolism

To characterize the sialyltransferase-IV activity in brain tissues, the activities of GM1b-, GD1a-, GT1b-, and GQ1c-synthases in adult cichlid fish and rat brains were examined using GA1, GM1, GD1b, or a cod brain ganglioside mixture as the substrate. The GD1a-synthase activity in the total membrane fraction from cichlid fish brain required divalent cations such as Mg²⁺ or Mn²⁺ and Triton CF-54 for its full activity. The V(max) value was 1,340 pmol/mg of protein/h at an optimal pH of 6.5, whereas the apparent K(m) values for CMP-sialic acid and GM1 were 172 and 78 μM, respectively. Cichlid fish and rat brains also contained GM1b-, GT1b-, and GQ1c-synthase activities. The ratio of GM1b-, GD1a-, and GT1b- synthase activities in fish brain was 1.00:0.89:1.13, respectively, and in rat brain 1.00:0.60:0.63. Incubation of fish brain membranes with a cod brain ganglioside mixture, which contains GT1c, and [³H]CMP-sialic acid produced radiolabeled GQ1c. It is interesting that the adult rat brain also contains an appreciable level of GQ1c-synthase activity despite its very low concentrations of c-series gangliosides. The GD1a- or GQ1c- synthase activity in fish and rat brain was inhibited specifically by coincubation with the glycolipids that serve as the substrates for other sialyltranferase-IV reactions. Thus, the GD1a-synthase activity was inhibited by GA1 and GD1b, but not by LacCer, GM3, or GD3. In a simlar manner, the synthesis of GQ1c was suppressed by GA1, GM1, and GD1b, but not by LacCer, GM3, or GD3. The GD1a-synthase activity directed toward endogenous GM1 was inhibited by GA1 or GT1b, whereas the endogenous GT1b- synthase activity was suppressed by GA1 or GM1. GA1, GM1, and GD1b did not affect the endogenous GM3- and GD3-synthase activities. These results clearly demonstrate that sialyltransferase-IV in brain tissues catalyzes the reaction for GQ1c syntesis in the c-pathway as well as the corresponding steps in the asialo-, a-, and b-pathway in ganglioside biosynthesis.