DL-threo-beta-Benzyloxyaspartate (DL-TBOA), a novel derivative of DL-threo-beta-hydroxyaspartate, was synthesized and examined as an inhibitor of sodium-dependent glutamate/aspartate (excitatory amino acid) transporters. DL-TBOA inhibited the uptake of [14C]glutamate in COS-1 cells expressing the human excitatory amino acid transporter-1 (EAAT1) (Ki = 42 microM) with almost the same potency as DL-threo-beta-hydroxyaspartate (Ki = 58 microM). With regard to the human excitatory amino acid transporter-2 (EAAT2), the inhibitory effect of DL-TBOA (Ki = 5.7 microM) was much more potent than that of dihydrokainate (Ki = 79 microM), which is well known as a selective blocker of this subtype. Electrophysiologically, DL-TBOA induced no detectable inward currents in Xenopus laevis oocytes expressing human EAAT1 or EAAT2. However, it significantly reduced the glutamate-induced currents, indicating the prevention of transport. The dose-response curve of glutamate was shifted by adding DL-TBOA without a significant change in the maximum current. The Kb values for human EAAT1 and EAAT2 expressed in X. laevis oocytes were 9.0 microM and 116 nM, respectively. These results demonstrated that DL-TBOA is, so far, the most potent competitive blocker of glutamate transporters. DL-TBOA did not show any significant effects on either the ionotropic or metabotropic glutamate receptors. Moreover, DL-TBOA is chemically much more stable than its benzoyl analog, a previously reported blocker of excitatory amino acid transporters; therefore, DL-TBOA should be a useful tool for investigating the physiological roles of transporters.

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The sequence of the entF gene which codes for the serine activating enzyme in enterobactin biosynthesis is reported. The gene encodes a protein with a calculated molecular weight of 142,006 and shares homologies with the small subunits of gramicidin S synthetase and tyrocidine synthetase. We have subcloned and overexpressed entF in a multicopy plasmid and attempted to demonstrate L-serine-dependent ATP-[32P]PPi exchange activity and its participation in enterobactin biosynthesis, but the overexpressed enzyme appears to be essentially inactive in crude extract. A partial purification of active EntF from wild-type Escherichia coli, however, has confirmed the expected activities of EntF. In a search for possible causes for the low level of activity of the overexpressed enzyme, we have discovered that EntF contains a covalently bound phosphopantetheine cofactor.

Four subtypes of excitatory amino acid transporters (EAAT1-4) have been identified in the mammalian brain. A number of pharmacological agents have been developed to study their intrinsic properties and function. Up to now, blockers were available only for EAAT2, whereas all the inhibitors of glutamate uptake active on the other subtypes were proved to be substrates of the transporters. We synthesized five new derivatives of DLthreo-␤-hydroxyaspartic acid, a well known general substrate of EAATs, and investigated their potential blocking activity on the cloned bovine EAAT1 expressed in the Xenopus oocyte system, by using radiotracer and voltage-clamp techniques. Two of our derivatives proved to be substrates for bovine EAAT1, with reduced electrogenicity compared with their parent compound, and an affinity of 40 and 64 M. The last three derivatives displayed a blocking activity on bovine EAAT1. The affinity of DL-threo-␤-benzoyloxyaspartate and DLthreo-␤-(1-naphthoyl)oxyaspartate was determined by Schild analysis as 17.2 and 52.1 M, respectively. These blockers should help in the better understanding of the key intrinsic properties of EAAT1. Moreover, they appear as good candidates for a general blocking activity on EAATs.