Catalytic efficiencies, percentages of rates of product formation per NADPH oxidized, and rates of product formation per O2 consumed of ionic mutants of cytochrome P450 1A2 (P450 1A2) were studied. Efficiencies of Lys99Glu, Lys453Glu, and Arg455Glu mutants for the hydroxylation reaction toward 7-ethoxycoumarin in the reconstituted system were much lower than that of the wild type (less than 17%), which corresponds to lower turnover numbers for these mutants. In contrast, the catalytic efficiencies for the hydroxylation reaction toward methanol of the three mutants were more than 45% that of the wild type in spite of these mutants' lower turnover numbers. Turnover numbers and catalytic efficiencies of Arg137Leu and Lys401 Glu mutants toward both substrates were comparable to those of the wild type. The electron-transfer rate from the reductase to the heme of P450 1A2 was decreased by 30% upon addition of excess methanol, while it was not influenced by addition of excess 7-ethoxycoumarin. The turnover numbers toward both 7-ethoxycoumarin and methanol as well as the rate constant of electron transfer were decreased by 25-40% by raising the concentration of KCl from 0 to 300 mM in the reconstituted system containing 50 mM potassium phosphate buffer. The turnover numbers toward both substrates of the above-mentioned five ionic mutants caused by tert-butyl hydroperoxide in the absence of the reductase and NADPH were comparable to those of the wild type. The effect of phospholipid constituents on the catalytic activity toward 7-ethoxycoumarin of the wild type was also studied.(ABSTRACT TRUNCATED AT 250 WORDS)

The synergistic stimulating effect of combined intake of carbohydrate and protein on plasma insulin concentration has been reported previously. However, it remains unclear whether the amount of protein ingested after exercise affects the concentrations of plasma insulin and amino acids. This study of trained men compared the effects of post-exercise co-ingestion of carbohydrate plus different amounts of whey protein hydrolysates (WPHs) with carbohydrate alone on (1) blood biochemical parameters of carbohydrate metabolism during the post-exercise phase, and (2) endurance performance. Eight trained men exercised continuously for 70 min. Immediately after exercise and 30, 60, 90, and 120 min later, the participants received supplements containing: (1) 17.5 g carbohydrate, (2) 3.0 g WPHs and 17.5 g carbohydrate (L-WPH), or (3) 8.0 g WPHs and 17.5 g carbohydrate (H-WPH). After a 2-h recovery period, the participants performed an endurance performance test. The concentrations of blood glucose were lower and plasma insulin significantly higher in the H-WPH trial compared with the carbohydrate trial. The concentrations of plasma amino acids were increased in a dosedependent manner following ingestion of different amounts of WPHs with carbohydrate. Endurance performance was not significantly different between the three trials. Co-ingestion of carbohydrate and H-WPH was more effective than ingestion of carbohydrate alone for stimulating insulin secretion and increasing the availability of plasma amino acids. These results suggest that plasma concentrations of amino acids during the recovery period are determined by the amount of dietary protein ingested, and that it is necessary to increase the concentration of plasma amino acids above a certain level to stimulate insulin secretion.

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