Asparaginyl endopeptidase of jack bean seeds. Purification, characterization, and high utility in protein sequence analysis.
Asparaginyl endopeptidase was highly purified from mature seeds of the jack bean (Canavalia ensiformis). The final enzyme preparation showed a single peak in high-performance liquid chromatography on a reversed-phase column, and the material in the peak gave the following NH2-terminal amino acid sequence on Edman degradation for 25 cycles: H-Glu-Val-Gly-Thr-Arg-Trp-Ala-Val-Leu-Val-Ala-Gly-Ser-Asn-Gly-Tyr-Gly-Asn-Tyr- Arg-His-Gln-Ala-Asp-Val-. Behavior of the enzyme toward various protease inhibitors suggested that it belongs to a family of cysteine proteases. Strict substrate specificity of this enzyme was verified by the use of 14 polypeptide substrates including those derived from proteins. Almost all the peptide bonds on the carboxyl side of Asn residues were susceptible to the enzyme. The exceptions were cases where the residue was at the NH2 terminus or the second position from the NH2 terminus of substrates and where it was N-glycosylated Asn. Peptide bonds on the carboxyl side of any other amino acid residues were not cleaved. These properties promise the high utility of this novel endopeptidase in protein sequence analysis. Identity of jack bean asparaginyl endopeptidase with a processing enzyme responsible for maturation of concanavalin A from its precursor is also discussed.
Abe
Y
Y
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Shirane
K
K
Yokosawa
H
H
Matsushita
H
H
Mitta
M
M
Kato
I
I
Ishii
S
S
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D006728:Hormones / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010940:Plant Proteins; D011480:Protease Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000235:genetics*; D012639:Seeds / Q000201:enzymology*; D013379:Substrate Specificity; D000099240:Asparaginyl Endopeptidase
D003546:Cysteine Endopeptidases; D006728:Hormones; D010940:Plant Proteins; D011480:Protease Inhibitors; D011506:Proteins; D000099240:Asparaginyl Endopeptidase