Abstract
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.
Highlights
From the $Departmentof Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060 and the §BiotechnologyResearch Laboratories, Takara Shuzo Co., Ltd., Otsu, Shiga 520-21,Japan
Purification of Asparaginyl Endopeptidase-Table I summarizes the steps pursued to purify the endopeptidase from an extract of 300 g of jack bean meal
The final enzyme preparation obtained after the second gel-permeation chromatography on G3000SW gave a single peak in HPLC on a reversed-phase column as shown in Fig. 1 and a single protein band with a mobility corresponding to a molecular mass of about 37,000 Da on SDS-polyacrylamide gel after electrophoresis under reductive conditions (Fig. 1, insert)
Summary
Yukichi AbeS, KatsunoriShiraneS, Hideyoshi YokosawaS, Hideyuki Matsushitag, Masanori Mittag, Ikunoshin Katot, and Shin-ichi IshiiST. Theenzyme purified fromjack beanmeal in this study high-performance liquid chromatography on a re- showed strict specificity toward asparaginyl bonds in various versed-phase column, andthe material in the peak gave polypeptide substrates andproved to beof much use in protein the following NHz-terminal amino acid sequence on sequence analysis. Posttranslational proteolysis and transpeptidation (ligation) All the other chromatographic media used for enzyme purification on the carboxyl side of Asn residues of its precursor Their were obtained from Tosoh Co. results suggest the presence of an asparaginyl endopeptidase Jack bean meal (300g) was defatted with acetone anhdomogenized in thebean. After 10-min incubation a t 35 "C, the amount of DNP-Pro-Glu-Ala-Asn-OH produced was analyzed by HPLC asdescribed above to determine the remaining enzyme activity. The composition analyses were carried out on a Hitachi L-8500 amino acid analyzer after the peptide was hydrolyzed with 6 M HCI vapor containing 0.05% phenol in an evacuated and sealed tube a t 110 "C for 24 h
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