Improved isolation, stability and substrate specificity of cucumisin, a plant serine endopeptidase.
Cucumisin (EC 3.4.21.25), a serine endopeptidase, was isolated by a simple purification procedure from the prince melon (Cucumis melo ssp. melo, cv. 'Prince Melon'). The enzyme is stable over a wide pH range (4-11) and to heat, 80% of its initial activity remaining even at pH 11.1 and at 60 degrees C for 20 min. The enzyme was inactive at 72 degrees C and pH 8.0, but 38% of the activity remained in the presence of 10% (w/v) glycerol. Caseinolysis by cucumisin indicated full activity in 8 M urea at pH 9.1 and 50 degrees C. Cucumisin was inactivated by treatment with trypsin at 37 degrees C for 24 h, but was not affected by alpha-chymotrypsin. The synthetic substrates benzyloxycarbonyltyrosine nitrophenyl ester (Z-Tyr-ONp) and benzoyltyrosine ethyl ester (Bz-Tyr-OEt) were cleaved, but Z-Lys-ONp and tosylarginine methyl ester (Tos-Arg-OMe) were not cleaved by cucumisin. Oxidized insulin B-chain was hydrolysed by cucumisin at 37 degrees C for 24 h, 21 cleavage sites being detected. Cucumisin could not cleave the C-termini of all the valine residues in the oxidized insulin B-chain molecule.
Kaneda
M
M
Department of Chemistry, Faculty of Science, Kagoshima University, Japan.
Yonezawa
H
H
Uchikoba
T
T
D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D019855:Ethylene Glycol; D005026:Ethylene Glycols / Q000494:pharmacology; D005638:Fruit / Q000201:enzymology*; D005990:Glycerol / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007328:Insulin / Q000378:metabolism; D008969:Molecular Sequence Data; D011092:Polyethylene Glycols / Q000494:pharmacology; D011489:Protein Denaturation; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D005026:Ethylene Glycols; D007328:Insulin; D011092:Polyethylene Glycols; D012697:Serine Endopeptidases; C041541:cucumisin; D019855:Ethylene Glycol; D005990:Glycerol