Photoaffinity inhibition of dipeptide transport in Escherichia coli

Abstract

A dipeptide containing a nitrene precursor, glycyl-4-azido-2-nitro-L-phenylalanine, has been synthesized. This compound is a photoaffinity inhibitor of dipeptide transport in E. coli. In the dark, the dipeptide is a reversible inhibitor of glycylglycine uptake by live E. coli W cells. The 14C-labeled compound is a substrate for the transport system, with a Km of 7 micrometer and V max of 5 x 10(3) molecules cell-1 s-1 (compare 9 micrometer and 1 x 10(4) molecules cell-1 s-1, respectively, for the transport of glycylglycine under the same conditions). When intact E. coli cells are photolyzed at approximately 350 nm in the presence of the photolabile dipeptide, their ability to transport either glycylglycine or unphotolyzed glycyl-4-azido-2-nitro-L-phenylalanine is irreversibly inhibited, but their ability to transport arginine is unaffected. The presence of glycylglycine in the medium during photolysis protects the cells against the light-dependent inactivation of dipeptide transport.

Cloning and functional characterisation of a peptide transporter expressed in the scutellum of barley grain during the early stages of germination

“…Alanyl-[U 14 C]phenylalanine (Ala-[U 14 C]Phe) was synthesised as described by Staros and Knowles (1978) Isolation of total RNA and Northern analysis RNA was isolated using a modified version of the method of Knight and Gray (1994). Fresh tissue (500 mg) was ground in liquid nitrogen and added to 4 ml extraction buffer (100 mM TrisHCl pH 8.5, 100 mM NaCl, 20 mM EDTA, 1% (w/v) sodium lauryl sarcosinate, 1.7% (w/v) diethyldithiocarbamate) and vortexed.…”

Analysis of the peptide carrier in the scutellum of barley embryos by photoaffinity labelling

“…All other laboratory chemicals were from either Sigma Chemical Co. or BDH, both of Poole, Dorset, UK, and were of the highest purity available. Ala[UJgC]Phe (2.0 GBq-mmol-1) was prepared in our laboratory by coupling [U-a4C]Phe (15.7 GBq' mmo1-1, Amersham International), with N-t-butoxycarbonyl (Boc)-Ala-N-hydroxysuccinimide ester as described by Staros and Knowles (1978). Aluminium-backed silica gel TLC plates (20 cm" 20 cm) were from BDH.…”

“…4-Azido-2-nitrophenylalanine hydrochloride (Phe(N3NO2) -HCI) was synthesized with minor modifications by the method of Staros and Knowles (1978). The product gave a single spot by TLC as described below (Rf of Phe(N3NO2)" HCI in System A 0.31-0.34, System B 0.5%0.64) and gave a single peak on reverse-phase chromatography [Pharmacia PEP-RPC (C2C18) column equilibrated with aqueous trifluoroacetic acid (TFA) (0.1%) and eluted with a gradient of CH3CN containing TFA (0.1%)].…”

“…Glyand Ala-Phe(N3NOz) were synthesised by the method of Staros and Knowles (1978) with minor modifications. The Boc-dipeptides, after partitioning into ethyl acetate as described, were dried down and deblocked by treatment with ice-cold anhydrous TFA for 60 min.…”

“…One of the most commonly used active moieties in photolabetling studies is the nitrene radical, which is most commonly derived from photolysis of aryl-or nitro-aryl azides (Bayley and Knowles 1977). Azido derivatives of phenylalanine (Phe) have been used as photoaffinity probes for amino-acid carriers (Noren et al 1983;Schlichter and Bajorat 1990) and also for dipeptide uptake in Escheriehia coli when combined into the dipeptide, Gly-Phe (Staros and Knowles 1978). In the rat intestine, a membrane glycoprotein of Mr = 127 000 has been labelled with photoactivatable derivatives of Gly-Pro and of the 13-1actam antibiotic cephalexin, which are purported to share a common transport system (Kramer 1987;Kramer et al 1990a).…”