Regulation of enzymatic activity in the intact cell: the beta-D-galactosidase of Escherichia coli.

Large differences in enzymatic activity are often found between intact cells and extracts. Lacking a better explanation, it has frequently been assumed that the penetration of the substrate is the rate limiting step in the intact cells. The experiments reported here were designed to test several hypotheses which might account for the discrepancy. The 3-galactosidase (lactase) in Escherichia coli was selected as a model system because of the sensitivity and ease of assay, together with the extensive knowledge of the induction, isolation, and general properties of the system (Lederberg, 1950; Cohn and Monod, 1951; Kuby and Lardy, 1953; Rotman and Spiegelman, 1954). Deere (1939) reported that drying E. coli-mutabile appeared to "activate" the lactase. Lederberg (1950) studied the ,B-galactosidase of E. coli strain K-12 with a chromogenic substrate and found discrepancies of 10to 47-fold between the activity of the intact cells and of disrupted cells or extracts. In addition, Lederberg found that the activity of the intact cell was protected from pH changes and inhibitory cations. The hypotheses tested in this investigation were: passive diffusion, inhibitors complexed with the enzyme, and product inhibition of the enzyme. All three were ruled out by the data presented below. Consequently, a more compatible proposal is given which assumes the existence of a specifically induced penetration mechanism. During the course of this work, Monod and his associates postulated an inducible enzymelike mechanism for specific control over galactoside penetration and termed the agent a "permease" (see Cohen and Monod, 1957). The