Separation of phenylalanine transport events by using selective inhibitors, and identification of a specific uncoupler activity in Yersinia pestis

Abstract

Phenylalanine transport in Yersinia pestis TJW was differentially inhibited by sulfhydryl blocking reagents, uncoupling agents, and respiratory inhibitors. Kinetic studies with potassium cyanide and sodium azide showed that these compounds have no immediate effect on the initial rate of phenylalanine transport, but have an immediate and severe inhibitory effect on the rate of oxygen uptake. Identical studies with p-chloromercuribenzoate (pCMB) and 2,4-dinitrophenol (DNP) showed that these compounds have an instantaneous

Methionine transport in Yersinia pestis

“…Electron transport inhibitors such as KCN and amytal were the most effective inhibitors. KCN and azide have been shown to be effective inhibitors of respiration under similar conditions to those used for these transport studies (21). Similar results with respiratory inhibitors were obtained with DNPstarved cells using either D-glucose or D-lactate as substrates (Table 3).…”

“…Thiosalicylate, a weak uncoupler, was completely ineffective. Unlike the phenylalanine transport system of Y. pestis (21), sulfhydryl reagents and uncouplers did not cause efflux of accumulated methionine or its products. Also, in counterflow experiments, cells preloaded with uniformly labeled methionine for 2 min, or 2 min and 45 s, did not exhibit efflux when excess cold methionine was added immediately at the latter two time points (data not shown).…”

“…8), suggesting that an accumulated form of energy was dissipated. These compounds, whether preincubated or added sometime after labeled amino acid, immediately and completely blocked energy couple to phenylalanine transport, causing rapid efflux of phenylalanine (21). Thiosalicylate, a weak uncoupler, gave partial inhibition of phenylalanine transport but had no effect on methionine transport.…”

“…Thiosalicylate, a weak uncoupler, gave partial inhibition of phenylalanine transport but had no effect on methionine transport. It is also interesting that at 5 C, DNP is not effective in blocking transport of phenylalanine (21), whereas methionine uptake shows the same amount of inhibition at 28 or 5 C. We have proposed that DNP is altering membrane conformation which in turn affects phenylalanine transport selectively (21). Another contrast is seen in the 5-to 20-fold stimulation of methionine transport by D-lactate or glucose, whereas phenylalanine transport is not stimulated under the same conditions by exogenous energy sources.…”