Isolation and characterization of a dual function protein from Allium sativum bulbs which exhibits proteolytic and hemagglutinating activities.
A dual function protein was isolated from Allium sativum bulbs and was characterized. The protein had a molecular mass of 25-26 kDa under non-reducing conditions, whereas two polypeptide chains of 12.5+/-0.5 kDa were observed under reducing conditions. E-64 and leupeptin inhibited the proteolytic activity of the protein, which exhibited characteristics similar to cysteine peptidase. The enzyme exhibited substrate specificity and hydrolyzed natural substrates such as alpha-casein (K(m): 23.0 microM), azocasein, haemoglobin and gelatin. It also showed a high affinity for synthetic peptides such as Cbz-Ala-Arg-Arg-OMe-beta-Nam (K(m): 55.24 microM, k(cat): 0.92 s(-1)). The cysteine peptidase activity showed a remarkable stability after incubation at moderate temperatures (40-50 degrees C) over a pH range of 5.5-6.5. The N-terminus of the protein displayed a 100% sequence similarity to the sequences of a mannose-binding lectin isolated from garlic bulbs. Moreover, the purified protein was retained in the chromatographic column when Con-A Sepharose affinity chromatography was performed and the protein was able to agglutinate trypsin-treated rabbit red cells. Therefore, our results indicate the presence of an additional cysteine peptidase activity on a lectin previously described.
Parisi
Mónica G
MG
Laboratorio de Química Biológica, Departamento de Ciencias Básicas, Universidad Nacional de Luján, Luján, Buenos Aires, Argentina. mgp@mail.unlu.edu.ar
Moreno
Silvia
S
Fernández
Graciela
G
D000818:Animals; D002364:Caseins / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004912:Erythrocytes / Q000737:chemistry; D005737:Garlic / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D005780:Gelatin / Q000737:chemistry; D006386:Hemagglutination Tests; D006388:Hemagglutinins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006454:Hemoglobins / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002364:Caseins; D006388:Hemagglutinins; D006454:Hemoglobins; D010940:Plant Proteins; D005780:Gelatin; D003546:Cysteine Endopeptidases