HUMAN-LEUKOCYTE GLYCOSYLASPARAGINASE IS AN ALPHA BETA-HETERODIMER OF 19-KDA ALPHA-SUBUNIT AND 17-KDA AND 18-KDA BETA-SUBUNIT/

Human lysosomal glycosylasparaginase (AGA; EC 3.5.1.26) consists of tw o glycosylated subunits, alpha and beta. Treatment with 3% SDS at 45 d egrees C as part of a new purification scheme did not affect enzyme ac tivity, but the a-subunit migrated as an apparent 19 kDa peptide on SD S/PAGE instead of as a 24 kDa peptide, as observed without this SDS tr eatment. The N-terminal sequence was similar to that of the 24 kDa for m, 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 prim ary structures were identical. As the molecular mass of the cr.-subuni t deduced from its cDNA was 19.5 kDa, the variation might be due to in complete SDS coating of the 24 kDa form. This was confirmed by the ten dency 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 accordanc e with previous reports. Chemical crosslinking with 1-ethyl-3-(3-dimet hylaminopropyl)carbodi-imide resulted in the appearance of a 38 kDa pe ptide on SDS/PAGE which reacted with both the subunit-specific antiser a on Western-blot analysis. On SDS/PAGE at pH 10.2 the active enzyme m igrated as an apparent 43 kDa peptide. These results indicate that nat ive human glycosylasparaginase is a heterodimer.