Kinetics of endoglycoceramidase action toward cell‐surface glycosphingolipids of erythrocytes

As shown in the preceding paper [Ito, M., Ikegami, Y., Tai, T. & Yamagata, T. (1993) Eur. J. Biochem. 218, 637–643], endoglycoceramidase (EGCase; EC.3.2.1.123) was found to hydrolyze the cell‐surface glycosphingolipids (GSLs) of erythrocytes without any damage to other cell membrane components. This paper represents the kinetics of EGCase action toward cell‐surface GSLs of erythrocytes. Without activator or detergents, cell‐surface GSLs were found to be hydrolyzed by EGCase II very slowly at pH 7.0. The initial reaction velocity of EGCase II under this condition was 0.038 pmol · min⁻¹· mU⁻¹ for horse erythrocyte cell‐surface GM3 and 0.032 pmol · min⁻¹· mU⁻¹ for guinea pig erythrocyte cell‐surface Gg₃Cer. The addition of activator protein (60 μM), which stimulates EGCase II in the absence of detergents, increased the initial reaction velocity of the enzyme 616‐fold for cell‐surface GM3 and 468‐fold for Gg₃Cer, while no increased hemolysis was observed with the addition of activator. However, even in the presence of the activator, the cell‐surface GSLs were very resistant to hydrolysis by EGCase II compared to GSL vesicles (or micelles) under same conditions. In contrast to the activator, Triton X‐100 (0.4%, mass/vol.) not only stimulated the enzyme activity but also solubilized erythrocyte GSLs into detergent micelles, inducing further increment of the enzyme reaction velocity. The apparent K_m and V_max values of EGCase II were calculated from the Lineweaver‐Burk plot as 47 μM and 35 pmol min⁻¹ mU⁻¹ for horse erythrocyte cell‐surface GM3 and 44 μM and 27 pmol · min⁻¹· mU⁻¹ for guinea pig erythrocyte cell‐surface Gg₃Cer, at pH 7.0 in the presence of activator at a concentration of 60 μM.