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Notes: The kinetic constants for 4-aminobutyrate: 2-oxoglutarate aminotransferase (GABA-trans-aminase) and succinate-semialdehyde: NAD+ oxidoreductase (SSA-DH) have been determined using rat brain homogenate.The Michaelis constants for GABA-T at saturated substrate concentrations were calculated to be Kgaba= 1.5 mM, K2-OG= 0.25 mM, KGLU= 620 μM, and KSSA= 87 μm. The Vmax for the reaction using GABA and 2-oxoglutarate (2-OG) as substrates (forward reaction) was found to be 35.2 μmol/ These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/gh and 167 pmol/g These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/g/h and 167 pmol/g/h in the brain and spinal cord respectively were calculated.The kinetics of GABA-T have been shown to be consistent with a Ping Pong Bi Bi mechanism. Substrate inhibition of the forward reaction, through formation of a dead-end complex, was found to occur with 2-OG (Ki 3.3 mM), whereas GABA was found to be a product inhibitor of the reverse reaction (Ki= 0.6 mM). Using the appropriate Haldane relationship, a Keq of 0.04 for GGBA-T was found, indicating that the reaction was strongly biased towards GABA.For SSA-DH, the Km of SSA was determined (9.1 μM) and the Vmax was 27.5 μmol/ These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/g/h and 167 pmol/g These results indicate that MOPEG is a measure for changes in central noradrenaline turnover and that drugs affect MOPEG in the brain and spinal cord similarly. Fractional rate constants of MOPEG in the rat brain and spinal cord were estimated with the exponential decline curves produced by treatment with pargyline. Turnover rates of 193 pmol/g/h and 167 pmol/g/h in the brain and spinal cord respectively were calculated. h. The effect of di-n-propylacetate (DPA) on both GABA-T and SSA-DH was measured. DPA inhibited SSA-DH competitively with respect to SSA, giving a Ki of 0.5 mM. GABA-T was only slightly inhibited. The Ki of DPA for the forward reaction was 23.2 mM with respect to GABA, which was 40-50 times higher than that for SSA-DH. For the reverse reaction the Ki of DPA was found to be nearly the same (15.2 mM with respect to Glu and 22.9 mM with respect to SSA). These results suggest that GABA accumulation in the brain, after administration of DPA in vivo, is caused by SSA-DH inhibition. Two mechanisms are indicated by the data. (1) The higher level of SSA, which results from inhibition of SSA-DH, initiates the reverse reaction of GABA-T, thus increasing the level of GABA via conversion of SSA. (2) The degradation of GABA is inhibited by SSA, since SSA has a strong inhibitory effect on the forward reaction, as calculated from the present data.

Notes: A procedure is described for the transfer of protein crystals from an ammonium sulfate-containing mother liquor to a solution with another precipitant, such as polyethylene glycol. The suitable concentration of the alternative precipitant is established via a novel protocol, using a hanging-drop equilibration method. This crystal transfer procedure is illustrated by experiments with crystals of trypanosomal triosephosphate isomerase and bacterial p-hydroxybenzoate hydroxylase, but it might have more general applicability.