Isolation and characterization of a dual function protein from Allium sativum bulbs which exhibits proteolytic and hemagglutinating activities

Plant Physiol Biochem. 2008 Apr;46(4):403-13. doi: 10.1016/j.plaphy.2007.11.003. Epub 2007 Nov 29.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Caseins / chemistry
  • Cysteine Endopeptidases / chemistry*
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / isolation & purification
  • Erythrocytes / chemistry
  • Garlic / chemistry
  • Garlic / enzymology*
  • Garlic / genetics
  • Gelatin / chemistry
  • Hemagglutination Tests
  • Hemagglutinins / chemistry*
  • Hemagglutinins / genetics
  • Hemagglutinins / isolation & purification
  • Hemoglobins / chemistry
  • Plant Proteins / chemistry*
  • Plant Proteins / genetics
  • Plant Proteins / isolation & purification
  • Rabbits
  • Sequence Homology, Amino Acid
  • Substrate Specificity

Substances

  • Caseins
  • Hemagglutinins
  • Hemoglobins
  • Plant Proteins
  • Gelatin
  • Cysteine Endopeptidases