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KMID : 0351619750160010245
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Kyungpook Medical Journal
1975 Volume.16 No. 1 p.245 ~ p.249
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A Simplified Assay for Phosphoenolpyruvate Carboxykinase Activity
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Abstract
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This study is carried out to simplify the determing method of phosphoenolpyruvate carboxykinase activity. The activity is assayed through the determination of 1~C-oxaloacetate, as a form of 2, 4-dint,rophenylhydrazone, induced during reactions of oxaloacetate-Ht~CO3 exchange and carbcxyla*ion. In t:he conventional method, oxaloacetate is changed enzymatically malate to determine the yielding of 1´C-malate o~ declining rate of NADH. The simplified :method allows a direct chemical change of oxaloacetate into 2, 4-dinitrophenylhydrazone. This method is summerized as follows;
The reaction mixture far the exchange (or carboxylation) assay contains the following compounds in total volume of 1.0 ml: lOQ ,umoles of imidazole-HCl butter, pH 6.9; 1.0 mole each of binClz, GSH, GTP(oi GDP for the carboxylation), oxaloacetate(or PEP for the carboxylation); 25 umoles of NaH1~COs: and 0.1 ml of enzyme. The final pH is to be 7.0. The mixture is incubated for 2 min (or 10 min for the carboxylation) at 30, and reaction stopps when added with 1 ml of 6N HCl saturated by 2, 4-dinitrophenylhydrazine. The hydrazone of oxaloacetate is extracted with 2.0 ml of ethylacetate. And 1.0 ml of the extract is placed on a plarchet, dried, and measured wiih a gasflow counter. The activity is expressed as a !emole of ^14C-compound formed per min.
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KEYWORD
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