Structural and chemical characterization of a homogeneous peptide N-glycosidase from almond.
A peptide N-glycosidase that catalyzes the hydrolysis of N-linked oligosaccharide chains from glycopeptides and glycoproteins has been purified to homogeneity from almond emulsin and from almond meal. Purification from almond emulsin using ion-exchange chromatography, gel filtration chromatography, and preparative polyacrylamide gel electrophoresis gave an enzyme which was purified more than 700-fold and with a yield of 63%. An alternative procedure, more suitable for efficient large scale purification, used ion-exchange, affinity, and gel filtration chromatography. When purification began with almond emulsin, the enzyme was purified 1200-fold with a 37% yield, while when purification began with almond powder, the enzyme was purified 9000-fold with a yield of 45%. The homogeneous enzyme is stable at 4 degrees C for several months in 10 mM sodium acetate, pH 5.0, buffer. The peptide N-glycosidase is itself shown to be a glycoprotein consisting of a single polypeptide chain with a molecular weight of 66 800 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Circular dichroism spectra of the native molecule indicate the presence of a high (approximately 80%) alpha-helix content. The amino acid and carbohydrate contents of the enzyme are presented. When a convenient new assay with a turkey ovomucoid glycopeptide as a substrate is used, the enzyme preparation exhibits a broad pH optimum centered between pH 4 and pH 6. The enzyme is readily inactivated by SDS and guanidine hydrochloride, but it is stable in the presence of moderate concentrations of several other protein denaturants.(ABSTRACT TRUNCATED AT 250 WORDS)
D000581:Amidohydrolases / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002942:Circular Dichroism; D006020:Glycopeptides / Q000302:isolation & purification; D007700:Kinetics; D008970:Molecular Weight; D010047:Ovalbumin; D010059:Ovomucin / Q000302:isolation & purification; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011487:Protein Conformation; D012639:Seeds / Q000201:enzymology*; D014422:Turkeys
D000596:Amino Acids; D002241:Carbohydrates; D006020:Glycopeptides; D010059:Ovomucin; D010047:Ovalbumin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase