Conversion of endoglycoceramidase-activator II by trypsin to the 27.9 kDa polypeptide possessing full activity: purification of activator for endoglycoceramidase by trypsin treatment followed by trypsin-inhibitor agarose column application.
Endoglycoceramidase (EGCase) cleaves the linkage between oligosaccharides and ceramides of various glycosphingolipids [Ito, M. & Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282]. A detergent was required for EGCase to express full activity, possibly due to its hydrophobic nature. Recently, activator proteins responsible for stimulating EGCase activity in the absence of detergents were isolated from the culture supernatant of Rhodococcus sp. [Ito, M., Ikegami, Y., & Yamagata, T. (1991) J. Biol. Chem. 266, 7919-7926]. The activity of activator II specific for EGCase II was heat-labile but insensitive to trypsin-treatment. This activator (69.2 kDa) was converted to the 27.9 kDa polypeptide via the 42 kDa intermediate by exhaustive trypsination, and the stimulatory activity of 27.9 kDa polypeptide on EGCase II was identical to that of the native form toward asialo GM1 and cell-surface GM3 of horse erythrocytes as substrates. This observation was successfully applied to obtain the purified activator without contamination with EGCase activity, which is abolished completely following treatment with trypsin.
Ito
M
M
Laboratory of Glycoconjugate Research, Mitsubishi Kasei Institute of Life Sciences, Tokyo.
Ikegami
Y
Y
Omori
A
A
Yamagata
T
T
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D006028:Glycosphingolipids / Q000097:blood; D006736:Horses; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012240:Rhodococcus / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism*
D006028:Glycosphingolipids; D010446:Peptide Fragments; D011506:Proteins; D006026:Glycoside Hydrolases; C050427:endoglycoceramidase; D014357:Trypsin