Antibodies to Inactive Conformations of Glyceraldehyde-3-phosphate Dehydrogenase Inactivate the Apo- and Holoforms of the Enzyme

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E. I. Arutiunova¹, A. P. Pleten¹, N. K. Nagradova¹, and V. I. Muronetz^1,2*

¹Belozersky Institute of Physico-Chemical Biology and ²Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-3181; E-mail: vimuronets@belozersky.msu.ru

^* To whom correspondence should be addressed.

Received November 28, 2005; Revision received January 25, 2006

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Polyclonal antibodies produced after the immunization of a rabbit with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus were used to isolate two types of antibodies interacting with different non-native forms of the antigen. Type I antibodies were purified using Sepharose-bound apo-GAPDH that was treated with glutaraldehyde to stabilize the enzyme in the tetrameric form. Type II antibodies were isolated using immobilized denatured monomers of the enzyme. It was shown that the type I antibodies bound to the native holo- and apoforms of the enzyme with the ratio of one antibody molecule per GAPDH tetramer. While interacting with the native holoenzyme, the type I antibodies induce a time-dependent decrease in its activity by 80-90%. In the case of the apoenzyme, the decrease in the activity constitutes only 25%, this indicating that only one subunit of the tetramer is inactivated. Differential scanning calorimetry experiments showed that the formation of the complex between both forms of the enzyme and the type I antibodies resulted in a shift of the maximum of the thermal capacity curves (T_m value) to lower temperatures. The extremely stable holoenzyme was affected to the greatest extent, the shift of the T_m value constituting approximately 20°C. We assume that the formation of the complex between the holo- or apo-GAPDH and the type I antibody results in time-dependent conformational changes in the enzyme molecule. Thus, the antibodies induce the structural rearrangements yielding the conformation that is identical to the structure of the antigen used for the selection of the antibodies (i.e., inactive). The interaction of the antibodies with the apo-GAPDH results in the inactivation of the subunit directly bound to the antibody. Virtually complete inactivation of the holoenzyme by the antibodies is likely due to the transmission of the conformational changes through the intersubunit contacts. The type II antibodies, which were selected using the immunosorbent with unfolded enzyme form, do not affect the activity of native holo- and apo-GAPDH, but prevent the reactivation of the denatured GAPDH, binding the denatured forms of the enzyme.

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KEY WORDS: antibodies, glyceraldehyde-3-phosphate dehydrogenase, inactivation, renaturation, conformational states, differential scanning calorimetry

DOI: 10.1134/S0006297906060137

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