Properties and Characterization of Binding Protein Dependent Active Transport of Glutamine in Isolated Membrane Vesicles of Escherichia coli

Arthur G. Hunt, Jen shiang Hong

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17 Scopus citations

Abstract

The reconstituted binding protein dependent active transport of glutamine in isolated membrane vesicles of Escherichia coli [Hunt, A. G., & Hong, J. (1981) J. Biol. Chem. 256, 11988–11991] is characterized in some detail. Transport activity exhibits a rather narrow pH optimum at about 5.8 with apparent pKas of 5.3 and 6.6, is inhibited by increasing ionic strength, and requires potassium and phosphate ions. However, the binding of glutamine to the glutamine binding protein is unaffected by pH over a range of 5–8, is relatively insensitive to variation in ionic strength up to 1.0 M KCl, and does not require potassium and phosphate ions. Since the internal pH of vesicles does not change over the range of 5–8, the pH-dependent transport profile most probably reflects the interaction of liganded glutamine binding protein with the membrane-bound components of the glutamine transport system. Two classes of compounds can serve as exogenous sources of energy for glutamine transport. One class consists of those compounds that can be metabolized to pyruvate. This class of compounds is effective only if NAD is incorporated into vesicles, and only if vesicles are prepared from strains containing active phosphotransacetylase and acetate kinase. The second class of compounds, of which succinate is the sole member, is effective in vesicles containing only those small molecules present in the lysis buffer, and in vesicles prepared from phosphotransacetylase and acetate kinase mutant strains as well as from the parent PSM116. It appears that succinate or a yet to be determined metabolite derived from succinate or a common product of pyruvate and succinate metabolism is the energy donor for glutamine transport. ATP and/or acetyl phosphate are found to be inactive as a source of energy in vesicles. Substances that abolish the electrochemical proton gradient (Δμ̄H), either by conducting protons across the vesicular membrane or by halting respiration, inhibit glutamine transport in vesicles. However, it appears that [formula omitted] is required for glutamine transport for a role other than serving as an energy donor as in the [formula omitted]-driven shock-resistant transport. The membrane vesicle preparations described here possess considerable metabolic capabilities. Vesicles are capable of incorporating 32P from Pi into ATP, ADP, AMP, GTP, acetyl phosphate, and several unidentified phosphate-containing compounds, indicating the presence of pyruvate dehydrogenase, phosphotransacetylase, acetate kinase, Mg2+-ATPase, adenylate kinase, nucleoside (AMP) phosphatase, nucleotide (adenosine) kinase, and nucleoside diphosphate kinase in vesicles.

Original languageEnglish
Pages (from-to)844-850
Number of pages7
JournalBiochemistry
Volume22
Issue number4
DOIs
StatePublished - 1983

ASJC Scopus subject areas

  • Biochemistry

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