Respiration dependent transport of proline by electron transport particles from Mycobacterium phlei

Hirata, Hajime; Asano, Atsushi; Brodie, Arnold F.

doi:10.1016/0006-291x(71)90609-7

Cited by 48 publications

(21 citation statements)

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“…probably occurs quite directly at the level of the membrane and its constituent proteins (5)(6)(7). Kaback and Barnes (8) proposed a tentative mechanism by which the coupling might be effected: the transport carriers are thought to monitor the redox state of the electron-transport chain and themselves undergo cyclic oxidation and reduction of critical sulfhydryl groups; each cycle is accompanied by concurrent changes in the orientation of the carrier center and in its affinity for the substrate, leading to accumulation of the substrate in the lumen of the vesicle.…”

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Role of an Electrical Potential in the Coupling of Metabolic Energy to Active Transport by Membrane Vesicles of Escherichia coli

Hirata

Altendorf

Harold

1973

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

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172

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Membrane vesicles from E. coli can oxidize D-lactate and other substrates and couple respiration to the active transport of sugars and amino acidl8. The present experiments bear on the nature of the link between respiration and transport.Respiring vesicles were found to accumulate dibenzyldimethylammonium ion, a synthetic lipid-soluble cation that serves as an indicator of an electrical potential. Escherichia coti, like other bacteria, accumulates nutrients from the medium by virtue of specific transport systems ("permeases"). Many nutrients, including amino acids, ,-galactosides, and certain inorganic ions, are transported in the face of large concentration gradients without any apparent chemical modification. Such "active transport" requires the performance of work and implies the coupling of transport systems to the major metabolic pathways of the cell (1-4).A partially resolved system, in which the mechanism of energy coupling may be more amenable to analysis than it is in the intact cell, became available through the work of H. R. Kaback and his associates. In a remarkable research program (5), these investigators demonstrated that isolated membrane vesicles of E. coti and other bacteria can couple active transport of many sugars, amino acids, and certain ions to the oxidation of particular respiratory substrates such as Dlactate. Since these vesicles neither make ATP by oxidative phosphorylation nor utilize exogenous ATP as an energy donor for transport, the coupling of oxidation to transport Abbreviations: DDA+, dibenzyldimethylammonium ion; TPB-, tetraphenylboron; TCS, tetrachlorosalicylanilide; CCCP, carbonylcyanide m-chlorophenylhydrazone; HOQNO, 2-heptyl-4 hydroxyquinoline-N-oxide. * To whom requests for reprints should be addressed, at National Jewish Hospital and Research Center. 1804probably occurs quite directly at the level of the membrane and its constituent proteins (5-7). Kaback and Barnes (8) proposed a tentative mechanism by which the coupling might be effected: the transport carriers are thought to monitor the redox state of the electron-transport chain and themselves undergo cyclic oxidation and reduction of critical sulfhydryl groups; each cycle is accompanied by concurrent changes in the orientation of the carrier center and in its affinity for the substrate, leading to accumulation of the substrate in the lumen of the vesicle. Serious shortcomings of this hypothesis have been pointed out by several investigators (1, 9-11).An alternative mechanism for the coupling of respiration to transport can be envisaged in terms of Mitchell's chemiosmotic hypothesis (12, 13). Briefly, he postulates that the respiratory chain translocates protons outward, thereby generating across the membrane an electrical potential (interior negative) and, under certain conditions, a pH gradient as well (interior alkaline). These gradients, which together constitute a force tending to pull protons back into the vesicle, are held to effect active transport by virtue of carriers that translocate simultaneousl...

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Role of an Electrical Potential in the Coupling of Metabolic Energy to Active Transport by Membrane Vesicles of Escherichia coli

Hirata

Altendorf

Harold

1973

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

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172

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“…7,1975 on May 9, 2018 by guest http://aac.asm.org/ has little apparent effect on the rate of this efflux (Fig. 7).…”

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confidence: 95%

“…Although the mechanism of energy coupling to active transport in bacteria is not completely understood, work with isolated membrane vesicles from a number of bacterial species provides evidence that ATP is not required for coupling energy to the transport of various amino acids and sugars by these vesicles (1,7,22; W. L. Klein, A. S. Dahms, and P. D. Boyer, Fed. Proc.…”

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Inhibition by Levorphanol and Related Drugs of Amino Acid Transport by Isolated Membrane Vesicles from Escherichia coli

Holland

Simon

1975

Antimicrob Agents Chemother

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Levorphanol inhibits the transport of the amino acids proline and lysine by cytoplasmic membrane vesicles derived from Escherichia coli. The degree of inhibition increases with increasing levorphanol concentration and ranges from 26% at 10-6 M levorphanol to 92% at 10-3 M levorphanol. The effect is independent of the energy source, since levorphanol inhibits proline uptake to the same extent in the presence of 20 mM D-lactate or 20 mM succinate and in the absence of an exogenous energy source. Levorphanol does not irreversibly alter the ability of membrane vesicles to transport proline, since incubation of membrane vesicles for 15 min in the presence of 0.25 mM levorphanol, a concentration which inhibits proline transport by more than 75%, has no effect on the rate of proline transport by these vesicles once the drug is removed. Both the maximum velocity and the Km of proline transport are modified by levorphanol, hence, the type of inhibition produced by levorphanol is mixed. The inhibitor constant (K) for levorphanol inhibition of proline transport is approximately 3 x 10-4 M. Membrane vesicles incubated in the presence of levorphanol accumulate much less proline at the steady state than do control vesicles. Furthermore, the addition of levorphanol to membrane vesicles preloaded to the steady state with proline produces a marked net efflux of proline. Levorphanol does not block either temperature-induced efflux or exchange of external proline with ["4C ]proline present in the intravesicular pool. Dextrorphan, the enantiomorph of levorphanol, and levallorphan, the N-allyl analogue of levorphanol, inhibit proline and lysine transport in a similar manner. Possible mechanisms of the effects of these drugs on cell membranes are discussed.

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“…With membrane vesicles from Mycobacterium phlei (ETP), there is no apparent correlation between oxidation rates of substrates (4), the ability to couple phosphorylation, or the ability to mediate active transport of proline (5,6). However, others have provided evidence for transport not supported by the respiratory chain (7)(8)(9)(10).…”

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Relationship of a Proton Gradient to the Active Transport of Proline with Membrane Vesicles from Mycobacterium phlei

Hinds

Brodie

1974

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

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Electron transport particles prepared from Mycobacterium phlei were depleted of bound coupling factors by washing with water in the absence of inorganic ions. The depleted electron transport particles were void of latent ATPase activity and were capable of oxidation, but were unable to support coupled phosphorylation. Nevertheless, the depleted electron transport particles were capable of suxbstratie-induced active transport of proline. It has been shown in Escherichia coli that oxidation of Dlactate by the respiratory linked lactate dehydrogenase can be coupled to the active transport of amino acids (1-3). With membrane vesicles from Mycobacterium phlei (ETP), there is no apparent correlation between oxidation rates of substrates (4), the ability to couple phosphorylation, or the ability to mediate active transport of proline (5, 6). However, others have provided evidence for transport not supported by the respiratory chain (7-10). The energy for transport can be derived from oxidation of substrate or from the hydrolysis of ATP. The mechanism(s) responsible is not known, although several proposals have been made (7,(11)(12)(13)(14). MATERIALS AND METHODSThe growth conditions for M. phlei (ATCC 354) External pH measurements were made with an external electrode. When simultaneous pH and 02 measurements were made, the entire system (pH meter, recorder, and oxygen amplifier) was allowed to float above ground. A special Lucite holder, which sealed the reaction vessel from the atmosphere, was made to hold both the pH and oxygen electrodes. RESULTS AND DISCUSSIONTo determine the role of oxidative phosphorylation in hctive transport, we compared membrane vesicles that were capable of oxidative phosphorylation (ETP) to those that were unable to couple phosphorylation to oxidation (DETP), with either succinate or NADH as substrate. As seen in Table 1, washing ETP with water was sufficient to inhibit coupled phosphorylation. The P/O value for succinate was higher thaf the value for exogenous NADH, as exogenous NADH can be oxidized by a bypass of the major respiratory chain (24).Washing ETP in a sucrose gradient in the absence of Mg++ ion has been used as a means to remove membrane-bound coupling factor-latent ATPase (6, 25). However, washing ETP with water was better than the sucrose wash, as there was only a 41% loss of the total protein after the water wash, as compared to a 71% loss with the sucrose wash. This drastic reduction has been observed previously (Higashi, Bogin, and Brodie, unpublished observations) and appeared 1202 Abbreviations: ETP, electron transport particle; DETP, depleted electron transport particle; BTB, bromthymol blue.

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Respiration dependent transport of proline by electron transport particles from Mycobacterium phlei

Cited by 48 publications

References 10 publications

Role of an Electrical Potential in the Coupling of Metabolic Energy to Active Transport by Membrane Vesicles of Escherichia coli

Role of an Electrical Potential in the Coupling of Metabolic Energy to Active Transport by Membrane Vesicles of Escherichia coli

Inhibition by Levorphanol and Related Drugs of Amino Acid Transport by Isolated Membrane Vesicles from Escherichia coli

Relationship of a Proton Gradient to the Active Transport of Proline with Membrane Vesicles from Mycobacterium phlei

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