Mechanisms of active transport in isolated bacterial membrane vesicles

Kaback, H. Ronald; Reeves, John P.; Short, Steven A.; Lombardi, Frank J.

doi:10.1016/s0003-9861(74)80028-7

Cited by 116 publications

(13 citation statements)

References 34 publications

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“…Carbonylcyanide m-chlorophenylhydrazone as a protonophore was added to the cell suspension of the Rhodococcus transformant harboring pLJK50, 10 min prior to the addition of 57 10 M in the reaction mixture inhibited the uptake by 25% and 85%, respectively, after 25 min of the reaction time. Since carbonylcyanide m-chlorophenylhydrazone is known to exhibit side effects-i.e., blockage for sulfhydryl groups in membrane proteins (24,25)-the effect of an uncoupler 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile (SF6847) on the cobalt uptake was investigated. 3,5-Di-tert-butyl-4-hydroxybenzilidenemalononitrile at the final concentration of 0.1 M, 1 M, and 10 M inhibited the uptake by 55%, 85%, and 85%, respectively, after the reaction time of 25 min.…”

Section: Resultsmentioning

confidence: 99%

A novel transporter involved in cobalt uptake

Komeda

Kobayashi

Shimizu

1997

Proc. Natl. Acad. Sci. U.S.A.

114

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Cobalt is an essential component of a low molecular-mass nitrile hydratase (L-NHase) from Rhodococcus rhodochrous J1. We have found a new gene, nhlF, in the DNA region sandwiched between nhlBA encoding L-NHase and amdA encoding amidase, which are involved in the degradation of nitriles. The product of nhlF, NhlF, shows a significant sequence similarity with those of hoxN from Alcaligenes eutrophus, hupN from Bradyrhizobium japonicum, nixA from Helicobacter pylori, and ureH from Bacillus sp., which are considered to be involved in nickel uptake into these cells. Sequence and hydropathy plot analyses have shown that NhlF encodes a 352-amino acid (aa) protein with eight hydrophobic putative membrane-spanning domains. nhlF expression in R. rhodochrous ATCC 12674 and Escherichia coli JM109 confers uptake of 57 Co in their cells, but not of 63 Ni. The expression of both nhlF and nhlBA in R. rhodochrous ATCC 12674 exhibited higher NHase activity than nhlBA expression. These findings together with the inhibitory effect by uncouplers (CCCP and SF6847) for the cobalt uptake suggest that NhlF mediates the cobalt transport into the cell energy-dependently finally to provide L-NHase.

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Section: Resultsmentioning

confidence: 99%

A novel transporter involved in cobalt uptake

Komeda

Kobayashi

Shimizu

1997

Proc. Natl. Acad. Sci. U.S.A.

114

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“…Upon addition of CCCP to EDTA-treated cells preloaded with TMG, the cells rapidly lose all of their accumulated TMG (Fig. 4) (28), might simply allow CCCP to act as a sulfhydryl reagent on the TMG carrier protein (29,30). Yet Two recently received manuscripts bear on our findings and conclusions.…”

Section: Resultsmentioning

confidence: 99%

Reduction of membrane potential, an immediate effect of colicin K.

Weiss¹,

Luria²

1978

Proc. Natl. Acad. Sci. U.S.A.

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(2), and inhibition of several active transport systems (3-6). Uptake and respiration of glucose remain normal, as do retention of a-methyl glucoside and facilitated diffusion of flD-galactosides (2).Previous experiments have shown that some of the observed changes are secondary effects (7). The processes that seem to be directly affected are those that are presumed to be dependent on an "energized membrane state." According to Mitchell's chemiosmotic hypothesis (8,9), the energized membrane state is identified as an electrochemical gradient of protons or protonmotive force (Ap), composed of a concentration gradient (ApH) and an electrical gradient (A41). The value of Ap is given in mV by the formula Ap = A4' -6OApH at 27°. Ap is postulated to be the driving force that energizes, either directly or indirectly, a wide variety of metabolic processes, including motility and various active transport systems. The primary effect of colicin K is apparently to uncouple the energy produced by respiration or by ATP hydrolysis from energy-utilizing processes such as active transport and motility. Colicin K might act either by lowering the energized membrane state or by preventing the utilization of the energized state. This paper presents evidence that colicin K acting on E. coil cells causes rapid membrane depolarization as measured by the accumulation of the lipophilic cation triphenylmethylphosphonium (TPMP+) (10). This conclusion is consistent with previous findings of Brewer (11) and Gould and Cramer (12). From measurements of the uptake of a weak acid analyzed by the flow dialysis method of Ramos et al. (13), we conclude that colicin K has only a slight effect on the pH gradient.In the course of these experiments we noticed that both the commonly used pretreatment of bacterial cells with EDTA and the presence of the lipophilic ions tetraphenylboron and TPMP+ have unexpected effects on certain bacterial membrane functions, effects that must be considered in the interpretation of experimental results. Ramos et al. (13). A pH electrode and a reference electrode (Radiometer G222C and K4112) were inserted in the cell suspension and connected to a recording pH meter. The flow rate of the dialysis buffer was about 5.1 ml/min. Fractions were collected directly into scintillation vials and sampled for radioactivity. The fraction collector and recorder were wired so that a small blip was recorded on the pH trace at the beginning of every fifth fraction.The method used by Ramos et al. (13) to calculate the amount of weak acid taken up and hence the internal pH had to be modified because our experiments, involving lower pH gradients and using dilute cell suspensions to maintain aerobiosis, required more precise measurements of relatively small changes in substrate concentration. A computer program was therefore devised § that calculates and plots the internal and external concentrations and the pH gradient. The same program was also used for proline uptake and can be used for any substrate that is actively concentrate...

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“…First, energy-dependent glutamine uptake is inhibited by 60-80 % by carbonyl cyanide m-chlorophenylhydrazone (under similar conditions energy-dependent proline uptake is inhibited by 85-100%) ( Table 2). Since the uncoupler inhibits anaerobic glutamine uptake in the presence of glucose in strain AN480 (uncB401, frd-1) (Table 2), it is likely that the uncoupler does not merely dissipate the 'high-energy membrane state', but also has secondary inhibitory effects, which may possibly be related to the ability of carbonyl cyanide m-chlorophenylhydrazone to form complexes with thiol groups (Kaback et al, 1974). Secondly, nitrate stimulates anaerobic glutamine uptake in strain AN480 (uncB401, frd-1).…”

Section: Resultsmentioning

confidence: 99%

Energy coupling to active transport in anaerobically grown mutants of Escherichia Coli K12

Gutowski

Rosenberg

1976

Biochemical Journal

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1. Anaerobic uptake of proline requires either the presence of a coupled Mg2+-stimulated adenosine triphosphatase or anaerobic electron transport. 2. Anaerobic uptake of glutamine does not require anaerobic electron transport even in the absence of a coupled Mg+2-stimulated adenosine triphosphatase. 3. These results support previous suggestions [Berger (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 1514––1518; Berger & Heppel (1974) J. Biol. Chem. 249, 7747-7755; Kobayashi, Kin & Anraku (1974) J. Biochem. (Tokyo) 76, 251-261] that two distinct mechanisms of energy coupling to active transport exist in Escherichia coli in that energization of anaerobic proline uptake requires the ‘high-energy membrane state’, whereas the energization of anaerobic glutamine uptake does not.

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Mechanisms of active transport in isolated bacterial membrane vesicles

Cited by 116 publications

References 34 publications

A novel transporter involved in cobalt uptake

A novel transporter involved in cobalt uptake

Reduction of membrane potential, an immediate effect of colicin K.

Energy coupling to active transport in anaerobically grown mutants of Escherichia Coli K12

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