Human leucocyte glycosylasparaginase is an alpha/beta-heterodimer of 19 kDa alpha-subunit and 17 and 18 kDa beta-subunit.
Human lysosomal glycosylasparaginase (AGA; EC 3.5.1.26) consists of two glycosylated subunits, alpha and beta. Treatment with 3% SDS at 45 degrees C as part of a new purification scheme did not affect enzyme activity, but the alpha-subunit migrated an apparent 19 kDa peptide on SDS/PAGE instead of as a 24 kDa peptide, as observed without this SDS treatment. The N-terminal sequence was similar to that of the 24 kDa form, and, after reversed-phase h.p.l.c., the 19 kDa form was transformed to an apparent 24 kDa peptide on SDS/PAGE, indicating that their primary structures were identical. As the molecular mass of the alpha-subunit deduced from its cDNA was 19.5 kDa, the variation might be due to incomplete SDS coating of the 24 kDa form. This was confirmed by the tendency of the 24 kDa variant to polymerize even in the presence of SDS. The molecular mass of the beta-subunit was 17 and 18 kDa in accordance with previous reports. Chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide resulted in the appearance of a 38 kDa peptide on SDS/PAGE which reacted with both the subunit-specific antisera on Western-blot analysis. On SDS/PAGE at pH 10.2 the active enzyme migrated as an apparent 43 kDa peptide. These results indicate that native human glycosylasparaginase is a heterodimer.
Tollersrud
O K
OK
Department of Human Molecular Genetics, National Public Health Institute, Helsinki, Finland.
Heiskanen
T
T
Peltonen
L
L
D000595:Amino Acid Sequence; D001227:Aspartylglucosylaminase / Q000737:chemistry* / Q000302:isolation & purification; 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 / Q000737:chemistry / Q000302:isolation & purification
D046911:Macromolecular Substances; D010446:Peptide Fragments; D001227:Aspartylglucosylaminase