Monomer-dimer equilibrium of uncomplemented M15 beta-galactosidase from Escherichia coli.
A series of gel filtration, native polyacrylamide gel electrophoresis (PAGE) and sucrose density experiments showed that uncomplemented M15 beta-galactosidase is in a monomer-dimer equilibrium and that only under some specific conditions does the equilibrium strongly favor dimerization. The ratio of dimer to monomer increased as a function of the protein concentration, and a very good fit to a theoretical plot of the effect of protein concentration on an associating system of this type was found. The Kdiss (equilibrium constant for dimer dissociation) was 2.5 x 10(-7) M. The addition of 20 mM Mg2+ lowered the Kdiss to 1.5 x 10(-7) M, and the addition of 150 mM NaCl lowered the value to 0.4 x 10(-7) M. Thiol reagents (2-mercaptoethanol and dithiothreitol) caused the equilibrium to shift totally to the dimeric form. The monomer-dimer equilibrium was also found to be dependent upon the pH. The dissociation increased as the pH was raised to 8.5, but there was a reversal of the equilibrium in favor of dimer formation at pH 9.0. This suggests that one (or more) residues with a pKa value of about 8.0 is involved. Tyr and Lys were eliminated as possible residues involved and it is, therefore, likely that one or more Cys are involved. Further evidence that uncomplemented M15 beta-galactosidase is in a monomer-dimer equilibrium was that the gel-filtration peaks were not totally resolved and that native PAGE bands were diffuse under all conditions except at high thiol concentration.
Gallagher
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Division of Biochemistry, Faculty of Science, University of Calgary, Alberta, Canada.
Huber
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D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D011487:Protein Conformation; D012965:Sodium Chloride / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism*
D012965:Sodium Chloride; D001616:beta-Galactosidase; D008274:Magnesium