New methods using polyvinylidene difluoride membranes to detect enzymes involved in glycosphingolipid metabolism

Two new methods are described using polyvinylidene difluoride (PVDF) membranes to detect enzymes involved in glycosphingolipid metabolism. One is the detection of enzymes on a PVDF membrane to which glycosphingolipids have been transferred from an HPTLC-plate by TLC blotting. The glycosphingolipids on the membrane were incubated with an enzyme preparation, and the resulting product was detected by immunostaining with a monoclonal antibody directed to the product. IV(3)NeuAc(alpha)Lc(4)Cer that had been transferred to a PVDF membrane was incubated with Clostridium perfringens sialidase. Lc(3)Cer then was transferred to the membrane, and the whole incubated with bovine milk beta1-4 galactosyltransferase, after which the product, nLc(4)Cer, was detected by immunostaining with the monoclonal antibody H11 that recognizes the GAl(beta)1-4GlcNAc(beta)1-3Gal structure of neolactoseries glycosphingolipids. This method detects glycosphingolipid-metabolizing enzymes that produce the same epitope. The second method is the detection of enzymes located on a polyacrylamide gel. A sialidase preparation from C. perfringens was subjected to native polyacrylamide electrophoresis then transferred to a PVDF membrane impregnated with IV(3)NeuAc(alpha)nLc(4)Cer as the enzyme substrate. The membrane then was incubated, and the resulting product, nLc(4)Cer, detected by immunostaining with the monoclonal antibody H11. The area stained shows the location of the sialidase on the polyacrylamide gel. The method is effective for determining the apparent molecular weight(s) of the enzyme(s) in the crude enzyme preparation. This was demonstrated by using crude sialidase preparation from C. perfringens.