Human leucocyte aspartylglucosaminidase. Evidence for two different subunits in a more complex native structure.
Human leucocyte aspartylglucosaminidase (AGA: 1-aspartamido-beta-N-acetylglucosamine amidohydrolase, EC 3.5.1.26) was purified to homogeneity by using affinity chromatography, gel filtration, chromatofocusing and reverse-phase h.p.l.c. As shown by SDS/PAGE, the homogeneous purified enzyme preparation consists of four polypeptide chains with molecular masses of 25, 24, 18 and 17 kDa. In the native polyacrylamide gel these polypeptides migrate as one active enzyme complex, and by gel filtration the peak of enzyme activity can be detected in a position of about 65 kDa. Digestion with endoproteinase Lys-C or endoproteinase Asp-N, followed by peptide analysis with reverse-phase h.p.l.c., reveals an identical peptide pattern for the 24 and 25 kDa bands as well as for the 17 and 18 kDa bands. This treatment further demonstrated a totally different peptide pattern for the 24/25 kDa versus the 17/18 kDa subunit. The N-terminal sequences of the 17 kDa and the 18 kDa peptides were identical, as determined by Edman degradation. The N-termini of the 24 kDa and the 25 kDa peptides were blocked. The enzyme was partly resistant to endoglycosidases H and F, but N-glycosidase F transformed the 24/25 kDa band into one 23 kDa band and the 17/18 kDa band into one 16 kDa band. Also, immunological data obtained with antisera produced against these subunits showed that AGA consists of two non-identical polypeptides.
Halila
R
R
Laboratory of Molecular Genetics, National Public Health Institute, Helsinki, Finland.
Baumann
M
M
Ikonen
E
E
Enomaa
N
N
Peltonen
L
L
D000595:Amino Acid Sequence; D001227:Aspartylglucosylaminase / Q000097:blood* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007962:Leukocytes / Q000201:enzymology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification
D046911:Macromolecular Substances; D010446:Peptide Fragments; D001227:Aspartylglucosylaminase