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Enzyme assays.
Enzyme assays were carried out according to the method described by Nakatani et al. (1970)Down. The temperature for the assay was set to 45 °C to avoid non-enzymic oxidation of NADH. The enzyme reaction was initiated by adding 10 µl of the enzyme solution (0·25 mg ml–1) to 1 ml reaction buffer (100 mM potassium phosphate buffer, pH 7·5, 50 mM NH4Cl, 10–120 µM 2-OA, 2·0 mM L-glutamate, 0·15 mM NADH, 10 µM pyridoxal 5'-phosphate, 11·9 U glutamate dehydrogenase ml–1), which was preincubated at 45 °C for 5 min. For measuring the activity of 2-oxoisocaproate (2-OIC), 2-oxoisovalerate (2-OIV) and 2-oxo-3-methylvalerate (2-OMV), 10–200 µM 2-OIC, 10–120 µM 2-OIV or 0·1–5 mM 2-OMV was added to the reaction buffer instead of 2-OA. The reaction was monitored at 45 °C by measuring the decrease in absorption at 340 nm. Kinetic parameters were calculated by using an initial velocity program, HYPER (Cleland, 1979Down). Specific activity was determined by using 20 mM L-glutamate as the amino donor, and 1 mM 2-OA, 2-OIC, 2-OIV or phenylpyruvate, or 10 mM 2-OMV, pyruvate or oxaloacetate as the amino acceptor. One unit of enzyme activity was defined as the amount of enzyme that reduced 1 µmol NADH min–1 at 45 °C in the reaction.

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