alpha-Galactoside binding lectin from Artocarpus hirsuta: characterization of the sugar specificity and binding site.
The hemagglutinin from the seeds of Artocarpus hirsuta was purified to homogeneity by ion-exchange chromatography on DEAE-cellulose and CM-sephadex. The native protein of molecular mass 60,000 (gel filtration) is made up of two pairs of unidentical subunits, alpha and beta with molecular masses of 15,800 and 14,130. The lectin is basic in nature (pI 8.5) and a glycoprotein with neutral sugar content of 6.25%. Rabbit as well as human erythrocytes (A, B and O) are agglutinated by the lectin. The lectin activity is neither affected by Ca2+, Mg2+ or Mn2+ nor by EDTA. Methyl alpha-D-galactopyranoside, pNP-alpha-D-galactopyranoside and pNP-alpha-D-N-acetylgalactosamine are the best inhibitors of the lectin. 4-Methylumbelliferyl-alpha-galactopyranoside fluorescence was quenched on binding to A. hirsuta lectin. The sugar has two binding sites per tetramer of the lectin with a Ka of 3.5x105 M-1 at 25 degrees C. Chemical modification studies indicate that the net positive charge associated with epsilon-NH2 of lysine residues and the phenyl ring of tyrosine are essential for the sugar binding activity of A. hirsuta lectin.
Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India.
M M
MM
Gurjar
M I
MI
Khan
S M
SM
Gaikwad
D000818:Animals; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D005638:Fruit / Q000737:chemistry*; D005697:Galactosides / Q000737:chemistry / Q000378:metabolism*; D006385:Hemagglutination Inhibition Tests; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D037102:Lectins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry; D008970:Molecular Weight; D037121:Plant Lectins; D011487:Protein Conformation; D011817:Rabbits; D012639:Seeds / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D005697:Galactosides; D037102:Lectins; D037121:Plant Lectins; D014443:Tyrosine; D008239:Lysine