Use of monoclonal antibodies to demonstrate different sites with different functional characteristics in a bacterial lipase from Pseudomonas aeruginosa YS-7.
Structural and functional features of the extracellular lipase from the low-water-tolerant bacterium Pseudomonas aeruginosa YS-7 were studied immunochemically with the aid of monoclonal antibodies (MAbs) raised against the enzyme. Fourteen different MAbs were obtained, verified as immunoglobulin G types, and characterized by their interaction with the enzyme in relation to (i) inhibition of activity of free enzyme, (ii) inhibition of activity of adsorbed enzyme, (iii) interaction with the cell-bound enzyme, and (iv) inhibition of adherence to hexadecane droplets. Four of the MAbs exhibiting the highest binding constants (Kapp greater than 10(8) M-1) were selected for further study of the lipase. Their binding to the enzyme was assayed by means of adapted enzyme-linked immunosorbent assay techniques. Use of these MAbs in single or dual binding procedures made it possible to reveal several distinct sites on the lipase macromolecule. Two of these are functional sites, one for hydrophobic adhesion (binds MAb 5) and the other (binds MAb 1) for implementation of its hydrolytic activity. A third binding site (binds MAb 8) does not participate directly in either of the above functions. A fourth binding site (binds MAb 10) appears to be involved in the active expression of the enzyme. The cell-associated form of the lipase seems to be located on the external surface of the cells with its active site exposed. It appears to be anchored to the outer membrane of the cells by means of its hydrophobic region in a way that resembles its adherence to hydrophobic surfaces such as hexadecane droplets.
Daya-Mishne
N
N
Department of Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel.
Shabtai
Y
Y
D000473:Alkanes / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D005455:Fluorescent Antibody Technique; D008049:Lipase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D011489:Protein Denaturation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013696:Temperature
D000473:Alkanes; D000911:Antibodies, Monoclonal; D008049:Lipase; C007932:n-hexadecane