Binding of hydrophobic ligands by Pseudomonas aeruginosa PA-I lectin.
The ability of Pseudomonas aeruginosa PA-I lectin to bind the fluorescent hydrophobic probe, 2-(p-toluidinyl) naphthalene sulfonic acid (TNS), and adenine was examined by spectrofluorametry and equilibrium dialysis. Interaction of TNS with PA-I caused significant enhancement of TNS fluorescence. The Hill coefficient (3.8+/-0.3) and the dissociation constant (8.7+/-0.16 microM) showed that TNS probably bound to four high affinity hydrophobic sites per PA-I tetramer. Interactions between PA-I and adenine were examined by equilibrium dialysis using [3H] adenine. The results indicated the presence of at least two classes of binding sites--one high and four lower affinity sites per tetramer with dissociation constants of 3.7+/-1.5 and 42.6+/-1.2 microM, respectively. These were distinct from the TNS sites as titration of TNS-equilibrated PA-I with adenine caused TNS fluorescence enhancement. The titration curve confirmed the existence of two classes of adenine-binding sites. Conversely, when PA-I was first equilibrated with adenine and then titrated with TNS, no TNS-binding was registered. This may indicate that conformational rearrangements of the lectin molecule caused by adenine prevent allosterically TNS binding.
Stoitsova
Stoyanka R
SR
Department of Microbiology and Immunology, University of Louisville, Louisville, KY 40202, USA. stoicova@hotmail.com
Boteva
Raina N
RN
Doyle
R J
RJ
D000225:Adenine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D003956:Dialysis; D005456:Fluorescent Dyes; D015801:Helminth Proteins; D037102:Lectins / Q000737:chemistry*; D008024:Ligands; D009282:Naphthalenesulfonates; D011485:Protein Binding; D011550:Pseudomonas aeruginosa; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D015801:Helminth Proteins; D037102:Lectins; D008024:Ligands; D009282:Naphthalenesulfonates; C419583:hydrophobic ligand binding protein, Hymenolepis diminuta; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D000225:Adenine