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A detailed study of the oligosaccharide specificity of the almond enzyme, peptide-N4-(N-acetyl-Pglucosaminy1)asparagine amidase A, was undertaken by comparing the rate of release of intact oligosaccharide chains from defined glycopeptides of all significant classes. The oligosaccharide of a trisialo-triantennary pentaglycopeptide from fetuin was released at the highest rate. A procedure was developed for the isolation of this glycopeptide in high yield from 5 g fetuin. Sequence analysis established the structure as Leu-Ala-Asn(CH0)-Cys-Ser. The Cys(Cm) and the Cys(Ae) derivatives of the glycopeptide were reacted with 4-(dimethylamino)-azobenzene-4-sulfonyl (dabsyl) chloride to yield a monosubstituted and a disubstituted glycopeptide respectively. This chromophore confers high sensitivity at 436 nm on a pentapeptide backbone having minimal bonds for protease cleavage. A procedure was developed wherein these dabsyl derivatives were used in a high-performance liquid chromatography assay. The dabsyl-pentapeptide was retarded significantly from the dabsyl-glycopeptide and provided a sensitive method (1 -2 nmol) of detection of peptide-N4-(N-acetylg1ucosaminyl)asparagine amidase activity. Enzyme was detected in crude extracts of all eight seed sources surveyed. The enzyme from Pisum sativum was partially purified and its properties were compared with the correponding enzyme from almonds.Takahashi and Nishibe [l, 21 first demonstrated the presence of a new amidase in almond emulsin that released intact oligosaccharides from defined glycopeptides of bromelain and ovalbumin. Unlike previously described endoglycosidases, such as endo H' [3], which hydrolyzed the di-N-acetylchitobiosyl core of N-linked glycans, this new type of enzyme hydrolyzed the p-aspartylglycosylamine linkage of asparagine-oligosaccharides in peptide linkage. Plummer and Tarentino [4] reported that this enzyme, peptide-N4-(N-acetyl-P-glucosaminyl)asparagine amidase (PNGase A), was much broader in specificity than all previously described endoglycosidases ; not only were high-mannose and hybrid-type oligosaccharide chains released, but also complex di-, tri-and tetraantennary chains [5, 61.Oligosaccharide-chain-cleaving enzymes with broad substrate specificity provide an attractive alternative to hydrazinolysis for isolating N-linked glycans from proteins, since yields are typically 90 -95% and there is no modification Abbreviations. endo, endo-P-N-acetylglucosaminidase; PNGase, peptide-N4-(N-acetyl-~-glucosaminyl)asparagine amidase (formerly known by the trivial name, peptide: N-glycosidase); dabsyl, 4-dimethylaminoazobenzene-4-sulfonyl; dns, 5-dimethylaminonaphthalene-1-sulfonyl; Ae, aminoethyl; Cm, carboxymethyl; SDS, sodium dodecylsulfate; CHO, intact oligosaccharide moiety.Enzymes. of the reducing end. Such enzymes continue to have great potential for investigating glycoprotein structure and function, and there is a need for specificities directed at other linkages on ohgosaccharide chains. Because of this we have developed a new method of detecti...

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