Purification et propriétés de la β-galactosidase (lactase) d' escherichia coli

Abstract

1. 1. A method for the purification of the adaptive enzyme of E. coli, β-galactosidase (lactase) has been described. 2. 2. Lactose and o- nitrophenyl-β- d- galactopyranoside are quantitatively hydrolysed by this enzyme preparation which, however, is inactive on sugars not possessing the β-galactosidic configuration. 3. 3. The following results (4, 5, and 6) indicate that the hydrolysis of lactose and o- nitrophenyl-β- d- galactopyranoside i is catalyzed by the same enzyme. 4. 4. The speed of thermal inactivation is the same whether one determines the activity with lactose or o- nitrophenyl-β- d- galactopyranoside as substrate. 5. 5. The electrophoretic analysis of this purified preparation reveals the presence of at least two constituents. Each of these electrophoretically distinct constituents is active on lactose and on o- nitrophenyl-β- d- galactopyranoside , and the ratio of these activities is the same for both fractions. 6. 6. A precipitating anti-lactase serum has been obtained by immunization of rabbits with the purified enzyme preparation. A quantitative study of the precipitin reaction indicates that the enzymatic activity, whether it be determined on lactose or o- nitrophenyl-β- d- galactopyranoside , is associated with a single antigenic species. There has also been demonstrated the existence of a protein which possesses no enzymatic activity but which markedly crossreacts with the β-galactosidase. 7. 7. The action of various ions on the enzymatic hydrolysis of lactose has been investigated. The effects of monovalent cations are particularly marked and complex. Depending upon the conditions (the presence of certain other cations), a given ion (sodium) is able to behave as either an inhibitor or an activator. 8. 8. These effects are the result of the competetive relationship between the monovalent cations. In order to account for the above phenomena, a hypothesis is presented, bringing into play only two characteristic constants: the affinity (inverse of the apparent dissociation-constant of ion-enzyme complex) and the “activance” (activity of the enzyme when saturated by the ion). 9. 9. For the hydrolysis of lactose, potassium shows the maximum “activance”, while the sodium is only weakly active. On the other hand, with o- nitrophenol-β- d- galactopyranoside as substrate, the “activance” of sodium is higher than that of the potassium. This remarkable inversion of the effects of ions demonstrates that the activance is linked to the chemical structure of the substrate.