BBA - Reviews on Biomembranes 737 (1983) 267-284. doi:10.1016/0304-4157(83)90003-5Received by publisher: 1982-10-06Harvest Date: 2016-01-04 12:22:08DOI: 10.1016/0304-4157(83)90003-5Page Range: 267-28

The system L transporter is generally considered to be one of the major Na+‐independent carriers for large neutral α‐amino acids in mammalian cells. However, we found that cultured astrocytes from rat brain cortex accumulate gabapentin, a γ‐amino acid, predominantly by this α‐amino acid transport system. Uptake of gabapentin by system L transporter was also examined in synaptosomes and Chinese hamster ovary (CHO) cells. The inhibition pattern displayed by various amino acids on gabapentin uptake in astrocytes and synaptosomes corresponds closely to that observed for the system L transport activity in CHO cells. Gabapentin and leucine have Km values that equal their Ki values for inhibition of each other, suggesting that leucine and gabapentin compete for the same system L transporter. By contrast, gabapentin exhibited no effect on uptake of GABA, glutamate, and arginine, indicating that these latter three types of brain transporters do not serve for uptake of gabapentin. A comparison of computer modeling analysis of gabapentin and l‐leucine structures shows that although the former is a γ‐amino acid, it can assume a conformation that can resemble the L‐form of a large neutral α‐amino acid such as l‐leucine. The steady‐state kinetic study in astrocytes and CHO cells indicates that the intracellular concentrations of gabapentin are about two to four times higher than that of leucine. The uptake levels of these two substrates are inversely related to their relative exodus rates. The concentrating ability by system L observed in astrocytes is consistent with the substantially high accumulation gradient of gabapentin in the brain tissue as determined by microdialysis.

The major component of leucine uptake in Escherichia coli K-12 is a common system for l -leucine, l -isoleucine, and l -valine (LIV-I) with a Michaelis constant ( K m ) value of 0.2 μM (LIV-I system). The LIV-binding protein appears to be associated with this system. It now appears that the LIV-I transport system and LIV-binding protein also serve for the entry of l -alanine, l -threonine, and possibly l -serine. A minor component of l -leucine entry occurs by a leucine-specific system (L-system) for which a specific leucine-binding protein has been isolated. A mutant has been obtained that shows increased levels of the LIV-I transport activity and increased levels of both of the binding proteins. Another mutant has been isolated that shows only a major increase in the levels of the leucine-specific transport system and the leucine-specific binding protein. A third binding protein that binds all three branched-chain amino acids but binds isoleucine preferentially has been identified. The relationship of the binding proteins to each other and to transport activity is discussed. A second general transport system (LIV-II system) with a K m value of 2 μM and a relatively low V max can be observed in E. coli . The LIV-II system is not sensitive to osmotic shock treatment nor to growth of cells in the presence of leucine. This high K m system, which is specific for the branched-chain amino acids, can be observed in membrane vesicle preparations.

The secondary structure of the terminated tip leader transcript from Escherichia coli was analyzed by RNase Ti partial digestion. Base-paired regions were recovered by nondenaturing gel electrophoresis and identified by denaturing gel electrophoresis and fingerprinting. The tandem tryptophan codons in the leader peptide coding region were found to be base paired with a more distal region of the transcript. This and other secondary structures that the tip leader RNA can form help explain the physiological response of the operon as well as the behavior of regulatory mutants. Transcription of the tryptophan (trp) operon of Escherichia coli is regulated at two sites, a promoter-operator and an attenuator.At the promoter-operator the rate of transcription initiation is regulated in response to chaqges in the intracellular level of free tryptophan (1, 2). At the attenuator, a site located within the transcribed 162-base-pair leader region that precedes the structural genes of the operon, transcription is either terminated to give a 140-residue leader transcript or allowed to proceed into the structural genes (3). Termination at the attenuator is regulated by the levels of charged and uncharged tRNATrP (4,5). Charged tRNATrP presumably is required for translation (6) of the segment of the leader transcript that codes for a 14-residue peptide containing adjacent Trp residues (7). The 3' half of the terminated tip leader transcript exhibits extensive secondary structure in vitro (8). Studies of tip leader mutants indicate that the capacity to form this secondary structure is essential for normalregulation of transcription termination at the attenuator (6, 9, 1(0). In the present study we demonstrate that the RNA region containing the adjacent Trp codons also participates in secondary structure. The various secondary structures that tip leader RNA can form help explain the physiological response ,of the operon as well as the behavior of the regulatory mutants we have studied. MATERIALS AND METHODSPreparation of Fingerprint Analysis of RNase Ti Oligonucleotides. Two-dimensional fingerprints of RNase Ti digests were prepared as described (11). RESULTSThe existence of extensive secondary structure in the 3' half of the E. coli terminated tip leader transcript is inferred from the resistance of this region to RNase T1 digestion (8). Analysis of the oligonucleotide products of RNase T1 partial digestion of tip leader RNA on denaturing gels suggested that the secondary structure consisted of two alternative stem and loop structures (8). To analyze further the extent and nature of the secondary structure of the terminated top leader transcript we used native gel electrophoresis to recover base-paired RNA fragments. We were particularly interested in determining the portion of the transcript that pairs with the Trp codon region, a segment known to be protected from RNase T1 attack (8). Fig. 1 shows an autoradiograph of RNase T1 partial digests of terminated Abbreviations: TBE, Tris/borate/EDTA; TME, Tris/MgCI2/ EDTA.

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