A fluorescent label for the outer components of the plasma membrane

Strains of Escherichia coli carrying R-factor R71(a), which codes for a streptomycin-spectinomycin adenylyltransferase, have elevated levels of resistance to dihydrostreptomycin (DHS) compared with isogenic R-bacteria. DHS accumulated by whole cells and spheroplasts of R+ bacteria is lower than that observed for R-strains, a result of the absence of the second and more rapid of the two energy-dependent phases of DHS uptake seen in susceptible E. coli. A mutant of R+ E. coli with reduced DHS resistance has been shown to have reduced levels of streptomycin-spectinomycin adenylyltransferase activity as well as enhanced drug accumulation. Actively accumulated DHS was recovered from R+ cells as the adenylylated derivative. Neither was inactivated antibiotic detected in culture filtrates, nor was actively accumulated drug lost from R+ cells under normal conditions. The cellular distribution of actively accumulated DHS in R+ and Rcells was found to be the same. Membranes isolated from these cells retained only a small fraction (=1%) of the total cell-associated drug. The R+ derivative of a mutant with defective energy transduction (E. coli NR-70) and reduced ability to transport aminoglycosides has a significantly higher minimal inhibitory concentration of DHS than its R+ parent (strain 7). Streptomycin-spectinomycin adenylyltransferase activity, from comparisons of Km values and total activities of enzyme, was the same in both strains. The enzyme has been localized to the exterior surface of the bacterial inner membrane, although isolated membranes lacked detectable enzyme activity. The preceding observations are consistent with the proposal that the level of R71(a)-mediated DHS resistance is the outcome of competition between the rate of adenylylation and the rate of the first energy-dependent phase of DHS transport. When the rate of adenylylation exceeds the first energy-dependent phase, adenylylated DHS is accumulated, apparently in a manner identical to the accumulation of DHS. Unlike DHS, adenylylated DHS does not interact with ribosomes, and, consequently, there is a failure to initiate ribosomally dependent sequelae such as the second energydependent phase of accumulation, inhibition of protein synthesis, and/or misreading of mRNA. R-factor-specified enzymatic modification of aminoglycoside antibiotics has been shown repeatedly to be a clinically important mechanism of resistance to aminoglycosides in bacteria. However, the precise mechanism by which enzymatic modification causes resistance is unknown. It has been shown in certain instances that modified aminoglycosides are no longer as effective at inhibiting protein synthesis or producing misreading of mRNA (23). These observations fail to explain aminoglycoside resistance because it has been demonstrated that only a very small fraction (less than 1%) of the total available, active aminoglycoside in the bacterial growth medium is detoxified. Thus, bacteria possessing aminoglycoside-inactivating enzymes are able to multiply in a milieu of almost entirely activ...

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“…The proteolytic enzyme trypsin and the reagent dye SITS were used. SITS reacts with amino, histidyl, and guanidyl groups of peptides (12).…”

Section: Resultsmentioning

confidence: 99%

Effect of Enzymatic Adenylylation on Dihydrostreptomycin Accumulation in Escherichia coli Carrying an R-Factor: Model Explaining Aminoglycoside Resistance by Inactivating Mechanisms

Dickie

Bryan

Pickard

1978

Antimicrob Agents Chemother

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“…Studies by Rothstein, Cabantchik, and Knauf (15)(16)(17) in erythrocytes, indicate that the disulfonic stilbenes inhibit anion transport by binding to a membrane protein, and that they appear to react with a site near the outside of the cell membrane (15)(16)(17)(18). Inhibition of anion transport by SITS has been demonstrated in a variety of other tissues as well (19)(20)(21).…”

Section: Discussionmentioning

confidence: 99%

Inhibition of the Bicarbonate Exit Step in Urinary Acidification by a Disulfonic Stilbene

Cohen¹,

Mueller²,

Steinmetz³

1978

J. Clin. Invest.

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A B S T R A C T Acidification of the luminal solution by the isolated turtle bladder involves H+ secretion by a pump at the luminal membrane. The OH-dissociated in this process reacts with CO2 and forms HCO-which moves passively out of the cell across the serosal cell membrane. In the present study, this exit step for HCO3 was inhibited by serosal addition of the disulfonic stilbene, SITS, an agent which is thought to bind to a transport protein at the serosal cell membrane. 90 min after serosal addition of 0.5 mM SITS, H+ secretion decreased by > 80%. In contrast, luminal addition of SITS had no effect. During inhibition of H+ secretion by serosal SITS, overall cell pH, measured by the 5,5-dimethyl-2,3-oxazolidinedione method, increased from 7.48+0.03 to 7.61+0.02. This increase of0. 13+0.02 pH U was associated with a much larger regional pH increase as judged from the decrement in the attainable pH gradient across the epithelium. After serosal SITS, this gradient was reduced from 2.88+0.06 to 2.09±0.11 pH U. In the absence of evidence for increased H+ permeability or a change in the force of the H+ pump, the gradient decrement of 0.79±0.08 U reflects a similar pH increment on the cytoplasmic side of the pump.SITS inhibits the exit of bicarbonate across the serosal cell membrane and, thereby, creates a compartment of high alkalinity in series with the pump.

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“…PCMBS is a reagent which binds specifically to sulfhydryl groups (Sutherland et al, 1967), while TNBS, SITS, and 2-methoxy-5-nitrotropone (MNT) are used as amino-specific reagents (Satake et al, 1960;Maddy, 1964;and Tamaoki et al, 1967). It should be noted that TNBS and SITS reactions with other groups (e.g., sulfhydryl groups) may be possible.…”

Section: Modification Of the Sodium Channel With Amino And Sulfhydrylmentioning

confidence: 99%

Sodium permeability of dog red blood cell membranes. I. Identification of regulatory sites.

Castranova¹,

Miles²

1976

The Journal of General Physiology

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A B S T R A C T Divalent cations and group-specific chemical modifiers were used to modify sodium efflux in order to probe the molecular structure of sodium channels in dog red blood cells. Hg ++, Ni ÷+, Co ++, and PCMBS (parachloromercuribenzene sulfonic acid), a sulfhydryl reactive reagent, induce large increases in Na + permeability and their effects can be described by a curve which assumes 2:1 binding with the sodium channel. The sequence of affinities, as measured by the dissociation constants, reflects the reactivity of these divalent cations with sulfhydryl groups. In addition, the effects of Hg ++ and PCMBS can be reversed by the addition of dithiothreitol, an SH-containing compound, to the medium. Much smaller increases in Na + permeability are produced by Zn +÷ and the amino-specific reagents, TNBS (2,4,6-trinitrobenzene sulfonic acid) and SITS (4-acetamido-4'-isothiocyano-stilbene-2-2'-disulfonic acid). The Zn ++ effect can be described by a curve which assumes bimolecular binding with the channel, and its effect on Na + permeability can be reversed by the addition of glycine to the medium. The effects of Ni ++ and SITS can be completely reversed by washing the cells in 0.16 M NaC1 while TNBS binding is partially irreversible. Measurements of mean cell volumes (MCV) indicate that the modifier-induced increases in Na + permeability are not caused by shrinkage of the cells. It is concluded that the movement of sodium ions through ionic channels in dog red blood cells can be enhanced by modification of amino and sulfhydryl groups. Zn ++, TNBS, and SITS increase Na + permeability by modifying amino groups in the channel while Hg ++ , Ni ++ , Co ++, and PCMBS act on sulfhydryl groups.

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A fluorescent label for the outer components of the plasma membrane

Cited by 94 publications

References 31 publications

Effect of Enzymatic Adenylylation on Dihydrostreptomycin Accumulation in Escherichia coli Carrying an R-Factor: Model Explaining Aminoglycoside Resistance by Inactivating Mechanisms

Effect of Enzymatic Adenylylation on Dihydrostreptomycin Accumulation in Escherichia coli Carrying an R-Factor: Model Explaining Aminoglycoside Resistance by Inactivating Mechanisms

Inhibition of the Bicarbonate Exit Step in Urinary Acidification by a Disulfonic Stilbene

Sodium permeability of dog red blood cell membranes. I. Identification of regulatory sites.

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