N-Acetylneuraminate lyase produced by Escherichia coli was purified and crystallized from a genetically engineered strain (E. coli SF8/pNAL1). The enzyme showed apparent molecular masses of 105,000 Da on gel filtration and 35,000 Da on SDS/PAGE, suggesting that the enzyme is a trimer. The apparent optimum pH and temperature were found to be 6.5-7.0 and 80 degrees C respectively. The Km values for N-acetylneuraminate and N-glycollylneuraminate were 3.3 and 3.3 mM respectively. The enzyme was inhibited by reduction with NaBH4 in the presence of the substrate, indicating that the enzyme belongs to the Schiff-base-forming Class I aldolases. The enzyme was strongly inhibited by Cu2+ ions, p-chloromercuribenzoate and N-bromosuccinimide, and also inhibited competitively by the reaction product, pyruvate, and its structurally related compounds, dihydroxyacetone and DL-glyceraldehyde.

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Benzodiazepines (BZs) suppress ventilation possibly by augmenting the GABA(A) receptor activity in the respiratory control system, but precise sites of action are not well understood. The goals of this study were: (1) to identify GABA(A) receptor subunits in the carotid body (CB) and petrosal ganglion (PG); (2) to test if BZs exert their effects through the GABA(A) receptor in the CB chemosensory unit. Tissues were taken from euthanized adult cats. RNA was extracted from the brain, and cDNA sequences of several GABA(A) receptor subunits were determined. Subsequent RT-PCR analysis demonstrated the gene expression of alpha2, alpha3, beta3, and gamma2 subunits in the CB and the PG. Immunoreactivity for GABA and for GABA(A) receptor beta3 and gamma2 subunits was detected in chemosensory glomus cells (GCs) in the CB and neurons in the PG. The functional aspects of the GABA-GABA(A) receptor system in the CB was studied by measuring CB neural output using in vitro perfusion setup. Two BZs, midazolam and diazepam, decreased the CB neural response to hypoxia. With continuous application of bicuculline, a GABA(A) receptor antagonist, the effects of BZs were abolished. In conclusion, the GABA-GABA(A) receptor system is functioning in the CB chemosensory system. BZs inhibit CB neural response to hypoxia by enhancing GABA(A) receptor activity.

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