Conjugates of monoclonal antibodies with polyelectrolyte complexes-- an attempt to make an artificial chaperone.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rabbit muscle is a tetrameric enzyme. Inactivation of GAPDH at 50 degreesC or in the presence of 4 M urea proceeds via formation of inactive dimers followed by their aggregation. Antibodies (clone 6C5) were selected which bind specifically inactive dimers but not native tetramers. The simplified model of chaperone action when the inactive misfolded forms are removed from the reaction media preventing aggregation was developed using antibodies in combination with polyelectrolyte complexes. The antibodies were coupled covalently to polyanionic component of the complex, poly(methacrylic) acid. The treatment of inactivated GAPDH with this conjugate followed by its precipitation after equimolar addition of polycation, poly-(N-ethyl-4-vinylpyridinium) bromide, resulted in a significant increase in the specific activity of GAPDH. The restoration of specific activity was more complete in the experiments with lower GAPDH concentration and in the samples with lower inactivation degree, conditions where aggregation is less pronounced. Some aggregates are formed at high inactivation degree and high GAPDH concentration and observed as an increase in the solution turbidity. They could be removed by centrifugation. Antibody/polyelectrolyte complex treatment followed by centrifugation to remove insoluble aggregates resulted in nearly complete restoration of enzyme specific activity.
Dainiak
M B
MB
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-22100 Lund, Sweden.
Izumrudov
V A
VA
Muronetz
V I
VI
Galaev
I Y
IY
Mattiasson
B
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D019281:Dimerization; D004789:Enzyme Activation; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry* / Q000276:immunology; D018832:Molecular Chaperones; D000071228:Polyelectrolytes; D011108:Polymers; D011109:Polymethacrylic Acids / Q000737:chemistry; D017510:Protein Folding; D011817:Rabbits; D055672:Static Electricity; D014508:Urea / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D018832:Molecular Chaperones; D000071228:Polyelectrolytes; D011108:Polymers; D011109:Polymethacrylic Acids; D014508:Urea; C030613:polymethacrylic acid