Adam Kepes

The membrane ATPase of Escherichia coli. II. Release into solution, allotopic properties and reconstitution of membrane-bound ATPase

Abstract Membrane-bound ATPase of Escherichia coli was released in a soluble form by decreasing the Mg 2+ concentration to 0.05 mM. The particulate fraction left behind was depleted by more than 90% from its initial ATPase activity. Soluble ATPase exhibits a number of different properties as compared with membrane-bound ATPase. These are a 2-fold increased K m toward ATP, a shift of 1–1.5 pH units in the pH-dependence curve, a greatly increased resistance to inhibition by N , N ′-dicyclohexylcarbodiimide (DCCD) and a stimulation by Dio 9 instead of an inhibition. Upon mixing the soluble fraction and the depleted membrane fraction, the initial properties of native membrane-bound ATPase reappear. This reconstitution requires Mg 2+ and results in the physical binding of the activity to sedimentable material. Soluble ATPase and depleted membrane can be titrated against each other until an equivalence point is reached, beyond which the component in excess keeps its previous characteristics. During the release procedure, DCCD remains associated with the particulate fraction with conservation of the ATPase-binding sites. Such DCCD-treated depleted membranes behave as a specific inhibitor of soluble ATPase.

The membrane ATPase of Escherichia coli: I. Ion dependence and ATP-ADP exchange reaction

The properties of the membrane-bound ATPase (EC 3.6.1.3) of Escherichia coli have been reexamined using membranes obtained by mechanical disruption of exponentially growing cells. The activity exhibited an absolute requirement for Mg2+ in the neutral pH range, while Ca2+ was found able to activate ATPase at more alkaline pH. Optimal activity was observed at pH 7.5, with a Mg/ATP ratio of 0.5. ADP was found to inhibit ATP hydrolysis and to transform the Michaelian ATP concentration dependence with a Km of 0.5 mM into a sigmoid curve with increasing Km and decreasing V. In contrast ADP activated an ATP-ADP exchange process and this shift from hydrolysis to exchange was stimulated by high Mg2+ and by orthophosphate. All nucleoside triphosphates tested interfered with ATP hydrolysis, all could be hydrolyzed and could donate their terminal phosphate group to ADP. The relative efficiencies of nucleoside triphosphates in these three processes varied in parallel with minor discrepancies. ATP hydrolysis was inhibited by N,N′-dicyclohexylcarbodiimide (DCCD) Dio 9, NaN3 and pyrophosphate, the first two being ineffective against ATP-ADP exchange, the third being stimulatory and the last inhibitory. ATP hydrolysis and ATP-ADP exchange are tentatively attributed to the terminal enzyme of oxidative phosphorylation.

The mechanism of maintenance of electroneutrality during the transport of gluconate by E. coli

A gluconate kinase deficient mutant AR13 has been isolated from E. coli strain DF1070 [l]. This mutant has an inducible gluconate permease and allows the study of this transport system in the absence of metabolic change of the transport substrate. Gluconate transport was found to achieve concentration gradients of 200-fold and above. When a plateau of accumulation was reached, the rate of uptake was found unchanged but balanced by an equal rate of exit. A more detailed report on the isolation of the mutant and on full kinetic description of the gluconate transport is the subject of another article [2], the present paper is devoted to the study of the electrical balance during the uptake of a negatively charged substrate.

Segregation of membrane markers during cell division in Escherichia coli. II. Segregation of Lac-permease and Mel-permease studied with a penicillin technique

Abstract A rapid method is described for the detection of heterogeneity in a bacterial population with respect to a membrane marker. The technique involves treatment with penicillin in the presence of a carbon source, the utilization of which is limited by the inducible membrane marker, usually a permease. The part of the population containing the marker is lysed rapidly, and the cells devoid of the marker can be separated. The distribution of Lac-permease and Mel-permease in the progeny of fully induced populations during growth and cell division in the absence of inducer was examined using this technique. A non-synchronous population gives rise to approximately 50% of permease-less segregants after three generation times in mineral medium with glycerol or glucose as sole carbon source. These results can be interpreted in terms of a simple model of membrane growth.

Relationship between intracellular K+ concentrations and K+ fluxes in growing and contact-inhibited cells.

The K+ content and the K+ flux were measured in the cell lines ME2 and MF2 isolated from plasmocytoma MOPC 173. Both cell lines were shown to have the seem K+ content and the same K+ steady state flux per unit of surface area. In ME2 cells, no modification of the exchange movement was observed during contact inhibition. However, contact-inhibited cells exhibited an increased resistance to depletion, characterized by a lower K+ net movement. The (Na+ plus K+)-ATPase measured in homogenates is poorly correlated to in vivo cation fluxes both because of the enhancement due, presumably, to the drop of K+ concentration on the cytoplasmic face of the membrane and because of losses during preparation which can be conspicuous, especially in contact-inhibited cells. The K+ net flux is considerable increased when the intracellular K+ level is reduced after preincubation of the cells in a K+ -free medium. Thus, internal K+ seems to regulate the K+ influx.

Segregation of galactoside permease, a membrane marker during growth and cell division in Escherichia coli.

SummaryIncubation of Escherichia coli with chloramphenicol causes metabolic and biosynthetic disturbances, the best known of which is the synthesis of RNA and formation of incomplete ribosomes (chloramphenicol particles). As a result of the unbalanced biosynthesis the bacteria transferred in a growth medium exhibit a prolonged lag of recovery and also a lag before development of and lysis by phage λ857 occurs. If lactose is the sole carbon source during incubation with chloramphenicol, the extent of these disturbaces is strongly dependent on the relative amount of β-galactoside permease.This effect can serve to demonstrate heterogeneity of permease content in a population and permits to physically separate the fraction rich in permease.If bacteria fully induced for the lactose operon are grown without inducer, the permease is distributed among the progeny and unequal distribution will result in a heterogeneous population. It is shown, that using chloramphenicol treatment in the presence of lactose, followed by thermal induction of phage λ857, bacteria previously deinduced during two doubling periods appear heterogeneous, about half the population being poor in permease.The significance of these results in terms of the pattern of growth of membrane is discussed.

Bacterial Membrane Transport Proteins

Bacterial transport systems are historically associated with the acceptance of the idea that the crossing of the cell membrane by a physiologically significant solute was mediated by the specialized operation of a protein or an array of molecules including specific proteins. This idea was in opposition to the predominant-view of permeability, a membrane property, as the principal factor governing the passage of solutes.