Calcium ion-dependent reactivation of a Pseudomonas lipase by its specific modulating protein, LipB.
LipB, the lipase activator protein of Pseudomonas aeruginosa TE3285, was overproduced in Escherichia coli, and purified 4.9-fold over the crude extract in the presence of SDS. The purified LipB reactivated the lipase from P. aeruginosa TE3285 denatured with guanidine hydrochloride, and its reactivation did not involve multiple turnover. In this reactivation, a 1:1 complex between the lipase and LipB was detected in a cross-linking experiment, suggesting that LipB still binds to the lipase after the reactivation. Calcium ion was essential for the complex formation and the reactivation, and addition of EDTA caused inactivation of the reactivated lipase bound to LipB more rapidly than the native lipase. These findings suggest that LipB could affect the calcium binding to the lipase in the reactivation process. LipB was unable to reactivate lipases from other sources except Pseudomonas sp. 109; this lipase has an amino acid sequence which is 98% identical to that of the lipase from P. aeruginosa TE3285. Thus, it may be concluded that LipB specifically recognizes a unique structural element of the lipase.
Shibata
H
H
Institute for Chemical Research, Kyoto University, Uji.
Kato
H
H
Oda
J
J
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D004926:Escherichia coli; D007700:Kinetics; D008049:Lipase / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D008049:Lipase; D002118:Calcium