Purification, biochemistry and molecular cloning of an insect glycosylasparaginase from Spodoptera frugiperda.
Glycosylasparaginase (EC 3.5.1.26) from Sf9 cells (Spodoptera frugiperda) was purified to homogeneity with a specific activity of 2.1 unit/mg. The enzyme is composed of two non-identical alpha/beta subunits joined by strong non-covalent forces and has one glycosylation site located in the alpha subunit. Molecular masses of the subunits were determined to be 28 kDa and 17 kDa by SDS-PAGE. Native enzyme existed in quaternary structures of either heterodimer (alpha beta) or heterotetramer (alpha 2 beta 2). These forms exhibited different ionic characteristics during DE52 anion exchange chromatography, and their molecular masses were determined to be 47 kDa and 101 kDa by gel filtration. The enzyme was thermostable, requiring 65-70 degrees C to be denatured, and it had a broad pH optimum from 4-10.5 with a pKa around 6. SDS easily inactivated the enzyme. The K(m) of glycosylasparaginase for its normal substrate GlcNAc-Asn was 0.88 mM. The enzyme also exhibited asparaginase activity with a K(m) of 3.0 mM for asparagine. N-terminal amino acids of the denatured subunits were sequenced and degenerate primers were designed for cloning its cDNA using PCR and 5' and 3' RACE. Glycosylasparaginase cDNAs from bovine and rat were also cloned using similar strategies, and primary structures of glycosylasparaginases from six species (human, bovine, rat, mouse, Sf9 cells and Flavobacterium) have been compared and related to a recent crystal structure of the human enzyme.
Liu
Y
Y
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Alabama, Mobile 36688, USA.
Dunn
G S
GS
Aronson
N N
NN
D000595:Amino Acid Sequence; D000818:Animals; D001227:Aspartylglucosylaminase / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D051381:Rats; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013045:Species Specificity; D018411:Spodoptera / Q000201:enzymology / Q000235:genetics*; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature
D019476:Insect Proteins; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; D001227:Aspartylglucosylaminase