1. 2,4-Dichlorophenol hydroxylase has been purified 13-fold from Acinetobacter grown on 2,4-dichloro-2. The enzyme has a relative molecular mass of about 240000 and consists of four subunits of identical size. 3. The enzyme cont,ains FAD as the prosthetic group. FAD could not be replaced by riboflavin or FMN in reconstituting active enzyme from apoenzyme.4. The reaction catalysed is an NADPH-dependent hydroxylation of 2,4-dichlorophenol with the formation of 3,5-dichlorocatechol as product. The reaction stoichiometry is typical of a monooxygenase with an external electron donor. NADPH is the preferred reduced pyridine nucleotide substrate but the enzyme can function with NADH. 5.The enzyme possesses broad effector specificity. In addition to 2,4-dichlorophenol, 4-chlorophenol and 4-chloro-2-methylphenol are true substrates for the enzyme. A number of 'non-substrate effectors' has been found.6. The enzyme is sensitive to thiol-inhibiting reagents.phenoxyacetic acid as sole carbon source. The enzyme was estimated to be 80-90 % pure by electrophoresis.The bacterial degradation of 2,4-dichlorophenoxyacetic acid has been the subject of study for almost as long as the herbicide has been in use. Evans et al. [l] identified the intermediates of the 2,4-dichlorophenoxyacetate breakdown pathway in Pseudomonas and demonstrated that 2,4-dichlorophenoxyacetate is metabolised via 2,4-dichlorophenol and 3,5-dichlorocatechol prior to 1 : 2-ring fission. The same pathway is followed in Arthrobacter [2-131.

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Burkholderia cepacia 2a inducibly degraded 2,4-dichlorophenoxyacetate (2,4-D) sequentially via 2,4-dichlorophenol, 3,5-dichlorocatechol, 2,4-dichloromuconate, 2-chloromuconolactone and 2-chloromaleylacetate. Cells grown on nutrient agar or broth grew on 2,4-D-salts only if first passaged on 4-hydroxybenzoate- or succinate-salts agar. Buffered suspensions of 4-hydroxybenzoate-grown cells did not adapt to 2,4-D or 3,5-dichlorocatechol, but responded to 2,4-dichlorophenol at concentrations <0.4 mM. Uptake of 2,4-dichlorophenol by non-induced cells displayed a type S (cooperative uptake) uptake isotherm in which the accelerated uptake of the phenol began before the equivalent of a surface monolayer had been adsorbed, and growth inhibition corresponded with the acquisition of 2.2-fold excess of phenol required for the establishment of the monolayer. No evidence of saturation was seen even at 2 mM 2,4-dichlorophenol, possibly due to absorption by intracellular poly-beta-hydroxybutyrate inclusions. With increasing concentration, 2,4-dichlorophenol caused progressive cell membrane damage and, sequentially, leakage of intracellular K(+), P(i), ribose and material absorbing light at 260 nm (presumed nucleotide cofactors), until at 0.4 mM, protein synthesis and enzyme induction were forestalled. Growth of non-adapted cells was inhibited by 0.35 mM 2,4-dichlorophenol and 0.25 mM 3,5-dichlorocatechol; the corresponding minimum bacteriocidal concentrations were 0.45 and 0.35 mM. Strain 2a grew in chemostat culture on carbon-limited media containing 2,4-D, with an apparent growth yield coefficient of 0.23, and on 2,4-dichlorophenol. Growth on 3,5-dichlorocatechol did not occur without a supplement of succinate, probably due to accumulation of toxic quantities of quinonoid and polymerisation products. Cells grown on these compounds were active towards all three, but not when grown on other substrates. The enzymes of the pathway therefore appeared to be induced by 3,5-dichlorocatechol or some later metabolite. A possible reason is offered for the environmental persistence of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T).

Gamma-aminobutyric acid (GABA) at 10−1 moll−1 inhibited spontaneous activity and produced conductance changes in 60% of cultured cockroach neurones tested. The reversal potential for the GABA-evoked response was between −65 mV and −75 mV. Under whole-cell voltage-clamp conditions, with 114mmoll−1 potassium chloride in the electrode, the reversal potential had a similar value to that predicted for a chloride current. The response was blocked by 10−5 moll−1 picrotoxin but was not affected by 10−5 moll−1 bicuculline. In the whole-cell voltage-clamp conditions, 50 μmoll−1 GABA evoked an inward current that was accompanied by an increase in current noise. Fluctuation analysis of the noise gave a mean channel opening time of 11.8 ms for GABA and 6.5 ms for muscimol. The single-channel conductance was 18.6 pS for GABA and 15.2 pS for muscimol. When 50 μmoll−1 GABA was applied in the presence of the benzodiazepine, flunitrazepam, there was an increase in both the evoked current and the accompanying current noise. Analysis of this noise gave values of 14.3 ms for the mean channel opening time and 18.3 pS for the singlechannel conductance. The variance of the noise was increased by approximately 60% in the presence of flunitrazepam, suggesting that this drug potentiates the GABA responses of cockroach neurones by increasing the frequency of channel events. Note: Present address: Pesticide Research Department, Wellcome Research Laboratories, Berkhamsted, Herts, UK. Present address: Department of Biology, Oxford Polytechnic, Gipsy Lane, Headington, Oxford.