Jeffrey B. Smith

Inhibitory properties of endogenous subunit ϵ in the Escherichia coli F1 ATPase

Abstract Homogeneous ϵ bound tightly to the purified Escherichia coli ATPase (ECF 1 from which ϵ had been removed and strongly inhibited its ATPase activity. ECF 1 containing ϵ had a lower specific activity than ECF 1 missing ϵ, provided that the ATPase assay was carried out at relatively high concentrations of enzyme. Antiserum specific for the ϵ subunit stimulated the ATPase, as did diluting the enzyme, apparently by dissociating ϵ. When the ATPase reaction was started by the addition of enzyme, the rate of ATP hydrolysis increased progressively during the first 3 min until a linear steady-state rate was reached. A prior incubation with ATP abolished the lag period and ADP prevented the ATP effect. ECF 1 missing ϵ gave a linear rate of ATP hydrolysis without a lag, unless ϵ was rebound to it before the assay. These results suggest that ECF 1 as purified is in an inhibited state due to the presence of the ϵ subunit, whose interaction with ECF 1 is governed by an equilibrium binding. ATP appears to convert ECF 1 to a form which more readily binds and releases ϵ.

Restoration of coupling factor activity to Escherichia coli ATPase missing the delta subunit

We separated the two minor subunits (δ and e) of the E. coli ATPase from the major subunits (α, β, and γ). The minor subunit fraction was obtained by treating purified ATPase with pyridine following the procedure that Nelson et al. (J. Biol. Chem. 348, 2049 [1973]) used to separate the subunits of chloroplast ATPase. The minor subunit fraction restored the capacity of ATPase lacking the delta subunit to recombine with ATPase-depleted membrane vesicles and to reconstitute energy coupling to the transhydrogenase and oxidative phosphorylation in the vesicles. These results clearly implicate the delta subunit in the attachment of the ATPase to the membrane.

Subunit specific antisera to the Escherichia coli ATP synthase: Effects on ATPase activity, energy transduction, and enzyme assembly

Abstract We obtained antisera to each of the five subunits (α, β, γ, δ, and ϵ) of the F 1 portion of the proton-translocating ATPase from Escherichia coli (ECF 1 ). No cross-reaction between the antiserum to a given subunit and any of the other four subunits was observed by Ouchterlony immunodiffusion. The α antiserum reacted only with the denatured α chain. Antibodies to either subunit β or subunit γ inhibited the ATPase activity of the enzyme. The ATPase activity of the holoenzyme in the everted membrane vesicles was just as sensitive as purified ECF 1 to inhibition by the anti-β or anti-γ serum. A prolonged digestion of ECF 1 with trypsin removed intact γ from ECF 1 , but did not alter the sensitivity of the ATPase to inhibition by the anti-γ serum. Proteolytic fragments were isolated from the trypsinized enzyme. They gave an immunoprecipitation band with the anti-γ serum, but none of the other subunit antisera. The antiδ serum detached ECF 1 from everted membrane vesicles and completely blocked both the ATP- and respiration-dependent pyridine nucleotide transhydrogenase, an energylinked membrane function. The δ antiserum had no effect on the ATPase activity of the ECF 1 . The e antiserum stimulated the ATPase activity of purified ECF 1 as shown previously (P. P. Laget and J. B. Smith, Arch. Biochem. Biophys. 197 , 83, 1979), but strongly inhibited the holoenzyme in membrane vesicles. The α antiserum completely blocked the ATP-driven transhydrogenase. The same antiserum maximally inhibited the respiratory chain-driven reaction by only 35%. These observations indicate that the antiserum selectively affected energy transduction mediated by the ATPase. The protonmotive force generated by substrate oxidation was probably not dissipated by the ϵ antiserum. Adsorbing the δ or ϵ antiserum with everted membrane vesicles selectively removed those antibodies that reacted with membrane-bound ATPase. The adsorbed sera still reacted strongly with purified ECF 1 , and prevented it from restoring ATP-dependent proton translocation in ECF 1 -depleted vesicles. Therefore, it appears that more of the δ and the ϵ subunit is exposed in the purified ECF 1 molecule than in the membrane-bound enzyme.

Activating the NaK pump with monensin increases aminoisobutyric acid uptake by mouse fibroblasts

Abstract Monensin rapidly tripled the initial rate and extent of α-aminoisobutyric acid accumulation by Swiss 3T3 cells. This ionophore catalyzes the electroneutral exchange of external Na for cellular protons and stimulates the Naue5f8K pump by suppling it with more Na. The stimulation of the Naue5f8K pump and α-aminoisobutyric acid uptake exhibited a similar dependence on monensin concentration. Ouabain prevented monensin from increasing α-aminoisobutyric acid transport. Aminoisobutryic acid transport was more than doubled at low doses of monensin that activated the Naue5f8K pump by elevating cell Na without significantly changing cell K. The rapid activation of α-aminoisobutyric acid transport is probably due to the hyperpolarizing effect of stimulating the electrogenic Naue5f8K pump. The stimulation of the Naue5f8K pump is quiescent fibroblasts by serum or growth factors may be sufficient to activate the Na-dependent amino acid transport systems.