Kinetic analysis of interactions between GroEL and reduced alpha-lactalbumin. Effect of GroES and nucleotides.
The real-time analysis of the association and dissociation of chaperonin with respect to its substrate protein was carried out using the BIAcore system. We immobilized alpha-lactalbumin (LA) as a substrate protein on the sensor chip and the GroEL solution was passed over it. Whereas GroEL did not bind to the immobilized native LA, it associated with the immobilized Ca(2+)-depleted, disulfide bond-reduced form of LA (rLA) rapidly (kon = 1.96 x 10(5) M-1 S-1) and dissociated extremely slowly (koff = 2.08 x 10(-4) S-1), giving a low dissociation constant (KD = 1.06 nM). MgATP greatly accelerated the dissociation (koff = 0.15 +/- 0.02 S-1). The KD value remained almost unchanged when GroES and/or 10 microM ADP was included in the GroEL solution. However, when 1 mM ADP was included, the KD value of GroEL increased by 2 orders of magnitude solely due to the change in koff. When GroES and 1 mM ADP were included, no interaction with rLA was detected due to changes in both kon and koff. These results indicate that GroEL/ES has high and low affinity ADP binding sites and that occupation of the low affinity sites by ADP was responsible for the loss of ability to interact with the substrate protein. The effect of excess GroES on the preformed GroEL.rLA and GroEL/ES.rLA complexes was also examined. With increasing GroES, the dissociation of GroEL and GroEL/ES from rLA was accelerated, and thus the possibility is suggested that the substrate protein and GroES compete for the same site on GroEL.
Murai
N
N
Tokyo Institute of Technology, Research Laboratory of Resources Utilization, Yokohama, Japan.
Taguchi
H
H
Yoshida
M
M
D000227:Adenine Nucleotides / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D018835:Chaperonin 10 / Q000494:pharmacology; D018834:Chaperonin 60 / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000187:drug effects
D000227:Adenine Nucleotides; D018835:Chaperonin 10; D018834:Chaperonin 60; D000244:Adenosine Diphosphate; D007768:Lactalbumin