Title : Purification and characterization of rat liver
glycosylasparaginase
Abstract :
- 1
- Rat liver glycosylasparaginase [ N4-(beta-N-acetylglucosaminyl)-L-asparaginase , EC 3.5.1.26] was purified to homogeneity by using salt fractionation, CM-cellulose and DEAE-cellulose chromatography, gel filtration on Ultrogel AcA-54, concanavalin A-Sepharose affinity chromatography, heat treatment at 70 degrees C and preparative SDS /polyacrylamide-gel electrophoresis
- The purified enzyme had a specific activity of 3.8 mumol of N-acetylglucosamine/min per mg with N4-(beta-N-acetylglucosaminyl)-L- asparagine as substrate
- 2
- The native enzyme had a molecular mass of 49 kDa and was composed of two non-identical subunits joined by strong non-covalent forces and having molecular masses of 24 and 20 kDa as determined by SDS /polyacrylamide-gel electrophoresis
- 3
- The 20 kDa subunit contained one high-mannose-type oligosaccharide chain, and the 24 kDa subunit had one high-mannose-type and one complex-type oligosaccharide chain.
- 4
- N-Terminal sequence analysis of each subunit revealed a frayed N-terminus of the 24 kDa subunit and an apparent N-glycosylation of Asn-15 in the same subunit
- 5
- The enzyme exhibited a broad pH maximum above 7
- Two major isoelectric forms were found at pH 6.4 and 6.6
- 6
- Glycosylasparaginase was stable at 75 degrees C and in 5% (w/v) SDS at pH 7.0