Kinetics of 4-aminobutyrate:2-oxoglutarate aminotransferase from Nippostrongylus brasiliensis.

A gamma-aminobutyric acid transferase (4-aminobutyrate:2-oxoglutarate aminotransferase; EC 2.6.1.19) preparation from Nippostrongylus brasiliensis was found to contain only one peak of enzyme activity with a highly basic pI of 10.5 when analysed by isoelectric focusing and chromatofocusing. This material was used in kinetic studies to demonstrate that the parasite enzyme reaction mechanism conforms to the usual binary, non-sequential ('Bi Bi Ping Pong') type found with aminotransferases. The Km for 4-aminobutyrate was 0.33 mM, the Km for 2-oxoglutarate was 0.57 mM and Ki for glutamate was 0.35 mM. In holoenzyme reconstitution experiments with the cofactor, pyridoxal 5-phosphate, the KD was 1.54 microM. The values are comparable to those reported for other tissues. Only 2-oxoglutarate could function as the keto acid substrate whereas several amino acids besides 4-aminobutyrate (beta-alanine, alpha-L-alanine, L-aspartate and L-arginine) could apparently act as substrate although the possible presence of other amino acid:2-oxoglutarate aminotransferases was not excluded. In preliminary studies on the usefulness of conventional substrate analogues as parasite gamma-aminobutyric acid transferase inhibitors only canaline was effective.

Application of a quick, simple and direct radiometric assay for 4-aminobutyrate:2-oxoglutarate aminotransferase to studies of the parasitic nematode Nippostrongylus brasiliensis.

A preparation of rat brain was used to develop a quick and simple radiometric assay for 4-aminobutyrate: 2-oxoglutarate aminotransferase (GABA-T), an important enzyme in neural tissue of vertebrates and invertebrates. Application of the methodology to the parasitic nematode Nippostrongylus brasiliensis revealed that a soluble preparation of partially purified GABA-T could be recovered in high yield. This enzyme had a specific activity comparable to that observed in rat brain after similar treatment, was very stable when frozen, depended for activity upon tightly-bound pyridoxal 5-phosphate, had an apparent molecular weight of 72,000 and was strongly inhibited by NaCl. The inhibition was competitive with 4-aminobutyrate (Ki = 20 mM) but uncompetitive with 2-oxoglutarate (Ki = 160 mM).

Inhibition of acetylcholinesterase secretion from Nippostrongylus brasiliensis by benzimidazole anthelmintics.

Treatment of rats infected with Nippostrongylus brasiliensis with a single, oral therapeutic dose of the anthelmintic benzimidazole carbamates oxfendazole or mebendazole resulted, 24 hr later, in a marked reduction (60-90%) in the secretion of a low molecular weight acetylcholinesterase from the parasites when they were incubated in vitro. This effect coincided with the expulsion of parasites from the host as a result of the therapy. When parasites were incubated in vitro with 0.1 mM oxfendazole, mebendazole, flubendazole, parbendazole, cambendazole or thiabendazole a similar effect was observed; with oxfendazole and mebendazole the effect was apparent within 1 hr and lasted for at least 4 hr after removal to fresh, drug-free medium. Whether treated in the host or in vitro the reduction in secretion was balanced by an equivalent rise in acetylcholinesterase activity within the parasites. It is suggested that the inhibition of protein secretion may be a specific manifestation of a general effect of these compounds on microtubule function.