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Research Article 2: Isoenzymes of Bacterial β-galactosidase: Renaturation of Heavier Isoenzymes from Dissociated E. coli β-galactosidase

Robert P. Erickson, Edward Steers, Jr.


The β-galactosidase of Escherichia coli has been shown to exist in multiple molecular forms. These forms consist of increasing mol wt aggregates (having sedimentation coefficients from 23 S to 45 S and designated “heavy” isoenzymes) of a basic monomer beginning with the predominant tetrameric form (16 S). These monomers were not detectably different from the monomers of the common tetrameric species by several criteria (Marchesi et al., 1969). Studies on the β-galactosidases of various species established that the ability to form the heavier isoenzymes (23 S-45 S) was determined by the structural gene for β-galactosidase and was correlated with the relative stability of the tetramer (Erickson and Steers, 1970a). This was corroborated by studies of the non-isoenzyme forming β-galactosidase from Aerobacter cloacae (Erickson and Steers, 1970b). The basis for the formation of heavier isoenzymes has been clarified as the process has now been carried out in vitro.

Preparation of β-Galactosidase
β-galactosidase was purified from a constitutive strain, 3300, of E. coli K12 as previously described (Marchesi et al., 1969). Briefly, a 20–30% ammonium sulfate fraction of the crude extract was gel filtered on Sepharose 4B followed by Sephadex G-200 and then chromatographed on DEAE-cellulose. Preparations containing heavier isoenzymes were obtained if the Sepharose 4B gel filtration step was eliminated. Purified heavier isoenzymes could be obtained from Sepharose 4B gel filtration of the 0–20% ammonium sulfate fraction (Marchesi et al., 1969).

Enzyme Assay
The activity of β-galactosidase was measured with the substrate O-nitrophenyl-β-D-galactopyranoside (ONPG) in buffer B...

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